KR102700187B1 - Modified soluble RSV F protein antigen - Google Patents

Abstract

The present invention relates to available respiratory syncytial virus (RSV) fusion (F) protein, including immunogenic compositions such as vaccines for treating and/or preventing RSV infection.

Description

Modified soluble RSV F protein antigen

The present invention relates to a respiratory syncytial virus (RSV) protein antigen and/or an RSV immunogenic composition for treating and/or preventing RSV infection. More specifically, it relates to a soluble RSV F protein antigen, an RSV immunogenic composition comprising the same, an RSV vaccine, and a method for inducing RSV immunity using the same.

Respiratory syncytial virus (RSV) is a virus that is prevalent worldwide, causing respiratory disease and is the main cause of death from severe respiratory infection in infants and young children. Almost all infants (more than 95%) are infected within the first 2 years of life, and in the United States, it is known to cause infection in close to 100% of infants aged 2-3 years, and hundreds of infants and thousands of infants die directly and indirectly from RSV-related causes. In addition, although infants and young children are the main targets of infection, it is known to cause fatal respiratory diseases by infecting patients with weakened immune systems and the elderly. It is the second most common cause of respiratory disease after influenza, but the annual mortality rate due to RSV per 100,000 infants under the age of 1 is known to be about 1.3 to 2.5 times higher than that of influenza. According to a 2002 WHO report, 64 million people are infected with RSV each year, and 160,000 of them die.

In particular, RSV is strong in the external environment, surviving for 6 hours on hard surfaces such as telephones and doorknobs, which allows for continuous infection and rapid transmission, making it very likely to cause an outbreak in hospitals and places with group living. Symptoms appear about 4-6 days after exposure to the RSV virus, starting with a runny nose and loss of appetite, and symptoms such as fever, cough, and wheezing appear about 3 days after the onset of the first symptom. Clinical manifestations vary greatly, from relatively mild symptoms to otitis media, apnea of premature infants, asthma, pneumonia, and bronchiolitis.

RSV belongs to the Orthopneumovirus genus of the Pneumoviridae family of the Mononegavirales order. Similar viruses to RSV include measles virus, mumps virus, and parainfluenza types 1, 2, and 3. RSV is a medium-sized virus, about 120-200 nm in size, with a genome of linear negative-strand RNA of about 15,000 nucleotides. RSV is divided into serotypes A and B based on the G protein. The RSV genome encodes 10 proteins, which are divided into nonstructural and structural protein encoding regions. Nonstructural proteins include Nonstructural (NS)1, Nonstructural (NS)2, nucleocapsid (N) protein, phosphoprotein (P), and viral polymerase (L), and these proteins play an important role in virus replication. Among the structural proteins, matrix (M) protein, fusion (F) protein, and glyco (G) protein form the viral envelope, and among these, F protein and G protein form the spike.

The RSV F protein is an important component involved in the initial stages of virus entry and fusion with the cell membrane, and is known as a major target of vaccines and antiviral drugs due to its high antigenicity. The RSV F protein changes its structure from the "pre-fusion" type to the "post-fusion" type during the infection process and entry into cells. The F protein consists of approximately 574 amino acids, and when the entire F protein is referred to as F0, it can be divided into F2 (approximately 20 kDa) and F1 (approximately 50 kDa) subunits. There is a furin cleavage portion between subunits F2 and F1, which is cleaved into F2 and F1 by furin-family proteases. Furin cleavage recognizes the amino acid sequences of two parts (sites) "RARR" and "KKRKRR", and among them, Arginine (R) acts as an important core amino acid. The F1 subunit is named HRA (fusion domain) at the N-terminal side and HRB (trans-membrane domain) at the C-terminal side, and it was confirmed that two or more heptad forms are formed in the HRA and HRB parts when it becomes a post-fusion type. The transformation of the F protein and cell-virus fusion occur when the hinge part of the bent form of HRA becomes unfolded. In addition, it was revealed that trimer formation is induced when the p27 part is removed by furin cleavage.

As mentioned above, RSV is widely prevalent in many countries around the world and is a particularly dangerous virus for infants and immunocompromised people, but there is no available preventive vaccine. Therefore, it is thought that the soluble RSV F protein obtainable by the present invention can provide the immunogenic substance required for this need.
(Patent Document 1) US 2014/0271699 A
(Patent Document 2) US 2014/0024076 A

The present invention provides a recombinant soluble respiratory syncytial virus (RSV) fusion (F) polypeptide. The soluble RSV F polypeptide of the present invention comprises at least one epitope specific for the RSV F protein. In certain embodiments, the polypeptide is a modified form of the fusion peptide region. In certain embodiments, the polypeptide comprises one, two, or more amino acid modifications.

The present invention also provides soluble RSV F polypeptides, nucleic acid molecules and/or compositions that induce immunogenicity, and their use in inducing an immune response in animals and humans against soluble RSV F protein, particularly as vaccines. The present invention also relates to a method for inducing neutralizing anti-respiratory syncytial virus (RSV) F protein antibodies in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule, and/or a polypeptide transcribed from the nucleic acid molecule, in particular for use as a vaccine. In a particular aspect, the present invention relates to a method for inducing neutralizing anti-respiratory syncytial virus (RSV) F protein antibodies in a subject, the method comprising administering to the subject an effective amount of a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule, and/or a polypeptide transcribed from the nucleic acid molecule.

Definition of common terms

The term "protein" or "polypeptide" as used herein means a collection of amino acids encoded by a specific nucleic acid. Here, the collection means a unit composed of two, three, four, or more amino acids. Here, "protein" or "polypeptide" may be understood in the art to mean "antigen" or "immunogen."

The term "antigen" as used herein refers to any substance that induces immunogenicity. The term "antigen" includes a large protein molecule composed of a protein or a collection of proteins. A large protein refers to a plurality of protein arrays and protein collections, and includes polymeric antigens and VLPs (virus-like particles). Here, induction of immunogenicity refers to both cellular immunity and humoral immunity. The term "antigen" includes, for example, all or part of a foreign substance that has penetrated the body, and induction of cellular immunity by the term "antigen" may also refer to the production of antigen-specific antibodies. Among antigen-specific antibodies, neutralizing antibodies are included that defend against and neutralize re-penetrating foreign substances.

As used herein, the terms "mutated", "mutated", "altered", "mutation" or "modification" refer to any alteration of a nucleic acid and/or a polypeptide that forms a modified nucleic acid or polypeptide. A mutation includes, for example, point mutations, deletions or insertions of single or multiple residues in a polynucleotide, and includes changes occurring within the protein-coding region of a gene as well as changes outside the protein-coding sequence, such as but not limited to, control or promoter sequences. A genetic change can be any form of mutation. For example, a mutation can constitute a point mutation, a frame-shift mutation, an insertion or deletion of all or part of a gene. In some embodiments, a mutation occurs naturally. In other embodiments, a mutation is the result of artificial mutational pressure.

As used herein, the term "vaccine" means a preparation of killed or weakened pathogens or induced antigenic determinants used to induce the formation of antibodies or immunity against a pathogen. Vaccines are given to provide immunity against diseases caused by various types of viruses, for example, influenza. The term "vaccine" also means a suspension or solution of an immunogen (e.g., a modified or mutated RSV F protein) that is administered to a vertebrate to induce protective immunity, i.e., immunity that prevents or reduces the severity of a disease associated with an infection. The present invention provides vaccine compositions that are immunogenic and capable of providing protection against diseases associated with an infection.

In other embodiments, mutations in RSV F proteins are the result of genetic engineering.

The term "includes" as expressed in this document means that the content and contents are present together. The term "includes" also means the addition or inclusion of the content and contents. However, it should be noted that the term "includes" can also be used independently or solely for the content and contents.

"About" includes all values that provide the same effect and result as the reference value. However, "about" only gives meaning to the reference value. In addition, the range included by the term "about" may vary depending on the range or characteristics of the content (reference value) that the term includes. Therefore, depending on the content, "about" may mean, for example, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or less than 1%.

All numerical ranges described herein include all numerical values within the numerical range. For example, it should be understood that a numerical range of "1000-3000" includes all numerical values greater than or equal to 1000 and less than or equal to 3000. In addition, all numerical values described herein may be recognized as independent numerical ranges, or may be recognized as connected-composite ranges between each numerical value.

Specific contents of the present invention

In order to solve the above problems, the present invention provides recombinant RSV F proteins comprising an amino acid sequence that has been modified compared to a wild-type RSV F protein. The inventors of the present invention have found that the modified RSV F proteins of the present invention increase the expression of RSV F proteins and improve immunogenicity compared to wild-type RSV F proteins. In a specific embodiment, the RSV F proteins are human RSV F proteins.

In one embodiment of the present invention, a recombinant respiratory syncytial virus (RSV) F protein antigen is provided, wherein the protein antigen is an RSV F protein antigen having a deletion of positions 525-574 of the amino acid sequence of SEQ ID NO: 1.

The above RSV F protein antigen consists of sequence number 2.

The above RSV F protein antigen may further comprise one or more amino acid substitutions at the fusion peptide position of the amino acid sequence of SEQ ID NO: 2.

The above RSV F protein antigen may substitute one or more hydrophobic amino acids at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 with hydrophilic amino acids, and preferably, the hydrophobic amino acid may be substituted with amino acid “A”. That is, the RSV F protein antigen may be an RSV F protein antigen in which the amino acid sequence FLGFLLGVG at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 is modified to AAGAAAGAG.

The above RSV F protein antigen may have one or more polar amino acids substituted with nonpolar amino acids at positions 137-145 of the amino acid sequence of SEQ ID NO: 2.

The above RSV F protein antigen may be an RSV F protein antigen in which the amino acid sequence FLGFLLGVG at positions 137-145 of the amino acid sequence of sequence number 2 is modified to QNGQNNGSG.

The above RSV F protein antigen may be an RSV F protein antigen in which the amino acid sequence FLGFLLGVG at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 is modified to NSGNSSGGG.

The above RSV F protein antigen may be an RSV F protein antigen in which the amino acid sequence FLGFLLGVG at positions 137-145 of the amino acid sequence of sequence number 2 is modified to TLSKKRKRR.

The present invention provides a recombinant soluble respiratory syncytial virus (RSV) fusion (F) polypeptide comprising a mutated or altered amino acid sequence compared to a wild-type RSV F protein. The present invention specifically provides the use of a novel recombinant RSV F protein obtained by mutating or altering one or more amino acid sequences of a wild-type RSV F protein as an RSV immunogen. The present invention provides an immunogen for a novel form of RSV infection.

The inventors of the present invention have confirmed that excellent immunogenicity can be provided by modifying the amino acid sequence of a specific region of the F protein among RSV structural proteins, and have completed the present invention.

In one embodiment of the present invention, a part of the fusion peptide (F protein) of a wild-type respiratory syncytial virus (RSV) having sequence number 1 is deleted, and an RSV F protein having sequence number 2 can be provided as an RSV immunogen or an antigen for inducing RSV immunity in the body.

Preferably, the RSV F protein of sequence number 2 comprises a form in which amino acid sequence positions 525-574 of the wild-type RSV F protein of sequence number 1 are deleted. Amino acid sequence positions 525-574 of the sequence number 2 can be understood as a region including the transmembrane domain and the cytosol region of the F protein.

The RSV immunogen of the present invention can provide excellent immunogenicity (i.e., high antibody titer) and stability in the body when the RSV F protein of SEQ ID NO: 2, which is a form in which amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1 are deleted, has undergone one or more sequence mutations (amino acid or nucleic acid).

In one embodiment of the present invention, the RSV immunogen can provide soluble F protein when the amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1 are deleted.

In one embodiment of the present invention, the RSV immunogen can provide soluble F protein when the RSV F protein of SEQ ID NO: 2, which is a form in which amino acid sequence positions 525-574 of the wild-type RSV F protein of SEQ ID NO: 1 are deleted, has undergone one or more sequence mutations (amino acid or nucleic acid).

Preferably, the position at which the RSV F protein of the above sequence number 2 undergoes one or more sequence mutations can be understood as a fusion peptide portion of the RSV F protein. The mutation of the above fusion peptide portion confirmed that the recombinant RSV F protein can act with excellent immunogenicity. Preferably, the fusion peptide portion is position 137-145 of the above sequence number 2, and more preferably, position 137-144 of the above sequence number 2.

The inventors of the present invention confirmed that the deletion of positions 137-144 of the above sequence number 2 can contribute to the stabilization of the RSV F protein, but the antibody synergy effect may be minimal. In addition, it was confirmed that there was a difference in the immune response when the amino acid of the fusion peptide was replaced (substituted) with another amino acid, and that it can act as an immunity with excellent effect.

The inventors of the present invention, through long-term research, have developed a recombinant RSV F protein immunogen capable of providing excellent antibody titers through one or more sequence mutations in the fusion peptide of the RSV F protein.

In one embodiment of the present invention, the RSV F protein immunogen can be used for the purpose of preventing, treating, etc., RSV infection, and preferably can be provided as a vaccine for preventing RSV infection.

Specifically, one embodiment of the present invention provides an RSV immunogen in which a portion of the sequence of a fusion peptide of the RSV F protein is mutated or modified. One embodiment of the present invention provides a recombinant soluble RSV F protein immunogen comprising one or more mutations in amino acids at positions 137-145 of the RSV F protein of SEQ ID NO: 2 and/or one or more mutations in the nucleic acid sequence of SEQ ID NO: 24 corresponding to the amino acid sequence at positions 137-145 of the RSV F protein of SEQ ID NO: 2. More specifically, it is an object to provide a recombinant soluble respiratory syncytial virus (RSV) fusion (F) polypeptide. The soluble RSV F polypeptide of one embodiment of the present invention comprises at least one epitope specific for the RSV F protein. In a specific embodiment, the polypeptide is in a form in which the fusion peptide region is modified. In a specific embodiment, the polypeptide comprises one, two, or more amino acid modifications.

The present invention also provides soluble RSV F polypeptides, nucleic acid molecules and/or compositions that induce immunogenicity, and their use in inducing an immune response in animals and humans against soluble RSV F protein, in particular as vaccines. The present invention also relates to a method for inducing neutralizing anti-respiratory syncytial virus (RSV) F protein antibodies in a subject, comprising administering to the subject an effective amount of a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule, and/or a polypeptide transcribed from the nucleic acid molecule, in particular for use as a vaccine. In a particular aspect, the present invention relates to a method for inducing neutralizing anti-respiratory syncytial virus (RSV) F protein antibodies in a subject, the method comprising administering to the subject an effective amount of a nucleic acid molecule encoding said RSV F polypeptide, and/or a vector comprising said nucleic acid molecule, and/or a polypeptide transcribed from the nucleic acid molecule.

In one embodiment, a variant in which amino acid sequence positions 525-574 of the wild-type RSV F protein are deleted can be understood as an RSV F protein having the sequence number 2.

The inventors of the present invention have confirmed that the fusion peptide of the RSV F protein consisting of the above sequence number 2, specifically the mutation of amino acid positions 137-145, can increase antibody titer. In addition, it can reduce repeat infection and decrease the number of vaccinations.

The inventors of the present invention confirmed that the RSV F protein antigen consisting of the above sequence number 2 and a modification or mutation induced at a specific amino acid position of the antigen increase the expression of the RSV F protein in a host cell and significantly increase immunogenicity.

In one embodiment, the RSV F protein antigen may comprise a fusion peptide of SEQ ID NO: 2, more preferably a form in which one or more amino acids are substituted at positions 137-145 of the amino acid sequence of SEQ ID NO: 2. The positions 137-145 of the amino acid sequence of SEQ ID NO: 2 may be understood as a fusion peptide portion of the RSV F protein. In one embodiment, the substitution of an amino acid at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 may comprise one or more modifications or mutations of the amino acids corresponding to the positions. In one embodiment, RSV F proteins may comprise three or more modifications or mutations in the amino acids corresponding to the positions.

The above substitution may be understood to mean a modification or mutation of an amino acid. That is, the above substitution may be understood to mean all cases in which a part of the amino acid sequence of SEQ ID NO: 2 is changed. For example, it may include all cases in which one or more amino acid sequences of the amino acid sequence of SEQ ID NO: 2 are changed.

In one embodiment, one or more hydrophobic amino acids at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 may be substituted with hydrophilic amino acids. More specifically, the RSV F protein of SEQ ID NO: 2 is a mutant in which one or more hydrophobic portions of the fusion peptide portion are modified into hydrophilic portions. More specifically, the present invention includes an RSV F protein antigen (SEQ ID NO: 3, FP1) in which the hydrophobic portion of the FLGFLLGVG sequence at positions 137-145 of SEQ ID NO: 2 is substituted with A to modify it into AAGAAAGAG.

In one embodiment, one or more polar amino acids at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 may be substituted with apolar amino acids. More specifically, the RSV F protein of SEQ ID NO: 2 is a mutant in which the polar portion of the fusion peptide portion is modified into apolar side chain. More specifically, the present invention includes an RSV F protein antigen (SEQ ID NO: 4, FP3) in which the FLGFLLGVG sequence at positions 137-145 of SEQ ID NO: 2 is modified to QNGQNNGSG. In another embodiment, the present invention includes an RSV F protein antigen (SEQ ID NO: 5, FP4) in which the FLGFLLGVG sequence at positions 137-145 of the amino acid sequence of SEQ ID NO: 2 is modified to NSGNSSGGG.

The inventors of the present invention have found that RSV F proteins having a mutated amino acid sequence at positions 137-145 of SEQ ID NO: 2, preferably FP1 (SEQ ID NO: 3), FP3 (SEQ ID NO: 4) and FP4 (SEQ ID NO: 5), can serve as excellent immunogens for the purpose of the present invention. Although mutations may occur in the amino acid sequence at positions 137-145 of SEQ ID NO: 2, as is well known to those skilled in the art, substitution, deletion or insertion of amino acids constituting a protein may result in completely different functions and effects of the protein.

The present invention provides an optimized recombinant RSV F protein, which is expected to have the best immunogenicity, particularly among mutations induced at positions 137-145 of sequence number 2.

According to one embodiment of the present invention, the RSV F protein antigen is a soluble protein, and in one embodiment of the present invention, a soluble RSV F protein antigen and/or an immunogenic composition comprising the RSV F protein antigen is provided.

In one embodiment of the present invention, the RSV F protein antigen may include at least one protein antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. Preferably, in one embodiment, the RSV F protein antigen may include at least one protein antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. More preferably, in one embodiment, the RSV F protein antigen may include at least one protein antigen selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

In one embodiment of the present invention, the RSV F protein antigen may be a protein antigen encoded by any one or more base sequences selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. Preferably, the RSV F protein antigen may be a protein antigen encoded by any one or more base sequences selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. More preferably, the RSV F protein antigen may be a protein antigen encoded by any one or more base sequences selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

In one embodiment of the present invention, the present invention comprises an additional mutation that maintains the stabilization of the F1 portion of the wild RSV F protein. The additional mutation associated with stabilization further comprises at least one additional mutation selected from the group consisting of (a) to (g) based on SEQ ID NO: 2.

(a) mutation of the amino acid residue at position 140;

(b) mutation of amino acid residue at position 163;

(c) mutation of amino acid residue at position 179;

(d) mutation of amino acid residue at position 188;

(e) mutation of amino acid residue at position 189;

(f) mutation of the amino acid residue at position 487; and

(g) Mutation of amino acid residue at position 505.

More specifically, the RSV F protein antigen of the present invention is based on sequence number 2.

(a) mutation of amino acid residue F to W at position 140 (SEQ ID NO: 7);

(b) mutation of amino acid residue E to Q at position 163 (SEQ ID NO: 8);

(c) mutation of amino acid residue V to L at position 179 (SEQ ID NO: 9);

(d) mutation of amino acid residue L to Q at position 188 (SEQ ID NO: 10);

(e) mutation of amino acid residue T to L at position 189 (SEQ ID NO: 11);

(f) mutation of amino acid residue E to L at position 487 (SEQ ID NO: 12); and

(g) comprises at least one mutation selected from the group consisting of a mutation of amino acid residue F to W at position 505 (SEQ ID NO: 13).

In another embodiment, the RSV F protein antigen is

The amino acid corresponding to position 140 of sequence number 2 is substituted with W, and the amino acid corresponding to position 163 is substituted with Q (SEQ ID NO: 14);

The amino acid corresponding to position 140 of sequence number 2 is replaced with W, the amino acid corresponding to position 163 is replaced with Q, the amino acid corresponding to position 188 is replaced with Q, and the amino acid corresponding to position 189 is replaced with L (SEQ ID NO: 15);

The amino acid corresponding to position 488 of sequence number 2 is substituted with W, and the amino acid corresponding to position 163 is substituted with Q (SEQ ID NO: 16);

The amino acid corresponding to position 488 of sequence number 2 is substituted with W, and the amino acid corresponding to position 179 is substituted with L (SEQ ID NO: 17);

The amino acid corresponding to position 140 of sequence number 2 is substituted with W, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 18);

The amino acid corresponding to position 487 of sequence number 2 is substituted with L, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 19);

The amino acid corresponding to position 163 of sequence number 2 is substituted with Q, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 20);

The amino acid corresponding to position 188 of sequence number 2 is substituted with Q, the amino acid corresponding to position 189 is substituted with L, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 21);

The amino acid corresponding to position 163 of sequence number 2 may be substituted with Q, and the amino acid corresponding to position 487 may be substituted with L (SEQ ID NO: 22).

The present invention provides an RSV immunogenic composition and/or vaccine comprising the RSV F protein.

Additionally included within the scope of the present invention are RSV F proteins other than human RSV F protein (SEQ ID NO: 1) comprising a modification corresponding to the above. Such RSV F proteins may include, but are not limited to, RSV F proteins from human RSV strain A, human RSV strain B, strains of bovine RSV, and strains of avian RSV.

In some embodiments, the use of known methods of protein engineering and recombinant DNA technology to improve or modify the properties of the mutant RSV F proteins mentioned above is included. The genes encoding the proteins are modified by codon adaptation, and the modification of the nucleotides is well known to those skilled in the art. Various forms of mutagenesis can be used to produce and/or isolate mutant nucleic acids encoding the protein molecules and/or to further modify/mutate the proteins of the invention. These include, but are not limited to, site-directed mutagenesis, random site mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, gapped-duplex DNA, and the like. Other suitable methods include mutagenesis by point mismatch repair, repair-deficient host strains, mutagenesis using restriction-selection and restriction-purification, deletion mutagenesis, whole gene synthesis, double-strand break repair, etc. For example, mutagenesis involving chimeric constructs is also encompassed by the present invention. In one embodiment, mutagenesis can be guided by known information about the molecule, either innately occurring or naturally occurring by acquisition or mutation, such as sequence, sequence comparison, physical properties, crystal structure, etc.

In another embodiment, the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the vaccine formulations of the present invention.

In another embodiment, the invention provides a method of formulating a vaccine or antigen composition for inducing immunity against an infection or at least one disease symptom thereof in a mammal, comprising the step of adding to said formulation an effective dose of a modified or mutated RSV F protein. In one preferred embodiment, the infection is RSV infection.

The modified or mutant RSV F proteins of the present invention are useful in preparing compositions that stimulate an immune response that provides immunity or substantial immunity to an infectious agent. Accordingly, in one embodiment, the invention provides a method of inducing immunity to an infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a modified or mutant RSV F protein.

In another aspect, the invention provides a method of inducing substantial immunity to RSV virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a modified or mutant RSV F protein.

The compositions of the present invention can induce substantial immunity in a vertebrate (e.g., a human) when administered to the vertebrate. Accordingly, in one embodiment, the present invention provides a method of inducing substantial immunity to an RSV virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a modified or mutant RSV F protein.

RSV F gene introduction recombination of baculovirus production

In one embodiment of the present invention, a method for producing an RSV F protein antigen is provided, comprising the steps of (a) producing a recombinant viral vector by introducing at least one RSV F protein encoding gene selected from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 44, and (b) inoculating the recombinant viral vector into a host cell and culturing the host cell to obtain a culture in which an RSV F protein antigen is expressed.

The method may further comprise a step of purifying the expressed RSV F protein or a fragment thereof.

The above recombinant viral vector may be, for example, a phage, plasmid, virus or retroviral vector, and a viral vector is preferably used.

In one embodiment, the vector is a recombinant baculovirus vector. The construct and/or vector encoding the gene must be operably linked to a suitable promoter, such as the AcMNPV polyhedrin promoter (or other baculovirus), the phage lambda PL promoter, the E. coli lac, phoA and tac promoters, and preferably can be overexpressed by the polyhedrin promoter.

The expression constructs will further comprise sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably comprise a translation initiation codon initially and a termination codon appropriately positioned at the end of the polypeptide to be translated. The expression vectors will preferably comprise at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance genes for eukaryotic cell cultures and tetracycline, kanamycin or impicillin resistance genes for E. coli and other bacterial cultures. Among the vectors, at least one viral vector selected from the group consisting of Baculoviridae (e.g., Autographa californica nucleopolyhedrovirus), Adenoviridae (e.g., canine adenovirus), Hepadnaviridae (e.g., avihepadnavirus), Vacciniaviridae (e.g., modified vaccinia Ankara virus), and Parvoviridae (e.g., Autonomous Parvovirus) is preferred. The baculovirus is an Autographa californica nucleopolyhedrovirus strain or a modified virus strain thereof; Or it may comprise a Bombyx mori nuclear polyhedron virus strain or a modified virus strain thereof. Bacterial vectors may also be used. Exemplary bacterial vectors include pQE70, pQE60, and pQE-9, pBluescript vector, phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among the eukaryotic vectors, pFastBac1 pWINEO, pSV2CAT, pOG44, pXTl, pSG, pSVK3, pBPV, pMSG, and pSVL are preferred.

Recombinant vectors such as those described above can be used to transfect, infect or transform and express proteins in eukaryotic cells and/or prokaryotic cells. Eukaryotic host cells can include yeast, insect, algae, plants, Caenorhabditis elegans (or nematodes) and mammalian host cells. Non-limiting examples of insect cells include Spodoptera frugiperda (Sf) cells such as Sf9, Sf21, Trichoplusiani cells such as High Five cells and Drosophila S2 cells. Examples of fungal (including yeast) host cells include species of Candida including S. cerevisiae, Kluyveromyces lactis (K. lactis), C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells include the human embryonic kidney lineage (293 cell lineage), the Chinese hamster ovary cell lineage (CHO cell lineage), the African green monkey lineage (Vero cell lineage), the human lung fibroblast cell lineage (MRC cell lineage), and the madin-darby canine kidney cell lineage (MDCK cell lineage). Xenopus laevis oocyte or other cells from amphibian sources may also be used. Prokaryotic host cells include bacterial cells, such as, for example, E. coli, B. subtilis, and mycobacteria.

Pharmaceutical or vaccine preparations and administration

One embodiment of the present invention provides an RSV preventive vaccine comprising at least one protein selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. Preferably, one embodiment of the present invention provides an RSV preventive vaccine comprising at least one protein selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

One embodiment of the present invention provides an RSV vaccine comprising a protein encoded by at least one nucleic acid selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. Preferably, one embodiment of the present invention provides an RSV vaccine comprising a protein encoded by at least one nucleic acid selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. More preferably, one embodiment of the present invention provides an RSV vaccine comprising a protein encoded by at least one nucleic acid selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.

The above vaccine may further contain an adjuvant or immunostimulant. For example, it may contain an aluminum adjuvant.

The immunogenic composition of the present invention comprises any pharmaceutical agent that does not by itself induce an immune response deleterious to the vertebrate receiving the composition, and comprises a pharmaceutically acceptable carrier comprising any suitable diluent or excipient that can be administered without undue toxicity together with the RSV F polypeptide. As used herein, the term "pharmaceutically acceptable" means listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeia for use in vertebrates and more particularly in humans. The RSV F immunogen of the present invention is administered in an effective amount or amount (as defined above) sufficient to stimulate an immune response against one or more strains of RSV virus. Such compositions can be used as vaccines and/or immunogenic compositions for inducing a protective immune response in vertebrates. The compositions can contain other RSV F proteins or fragments thereof.

In one non-limiting embodiment, the concentration of the immunogen is at least about 10 μg/mL, about 20 μg/mL, about 30 μg/mL, about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 100 μg/mL, about 200 μg/mL, or about 500 μg/mL. In certain embodiments, the concentration of the immunogen is from about 10 μg/mL to about 1 mg/mL, or from about 20 μg/mL to about 500 μg/mL, or from about 30 μg/mL to about 100 μg/mL, or from about 30 μg/mL to about 50 μg/mL. In other embodiments, the concentration of the immunogen can range from 10 μg/mL to 200 μg/mL.

In one embodiment, the pharmaceutical formulation disclosed herein may comprise RSV F protein, preferably the spike protein; and a pharmaceutically acceptable carrier or excipient.

In another embodiment, the pharmaceutical formulation comprises purified, high affinity antibodies produced in an animal to which the immunogen has been administered. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof. Pharmaceutically acceptable carriers, diluents, and other excipients are provided in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition). The formulation should be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for human administration, preferably sterile, non-particulate, and/or non-pyrogenic. If desired, the composition may contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition may be in a solid form, such as a lyophilized powder suitable for recombinant administration, a liquid solution, a suspension, an emulsion, a tablet, a pill, a capsule, a sustained-release formulation, or a powder. The oral formulations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the immunogenic vaccine formulations. In a preferred embodiment, the kit comprises two containers, one containing RSV F immunogen and the other containing an adjuvant. A notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products may be associated with such container(s), said notice indicating approval by the agency for manufacture, use, or sale for human administration. The formulation may be packaged in a sealed container such as an ampoule or sachette indicating the quantity of the composition. In one embodiment, the composition is supplied as a liquid, and in another embodiment, it is supplied as a dried sterilized lyophilized powder or water-removed concentrate in a sealed container, which may be reconstituted, for example, with water or saline, to an appropriate concentration for administration to a subject. Preferably, the composition is supplied as a dried sterilized lyophilized powder in an airtight container, preferably in a unit dose of about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 50 μg, about 100 μg, about 125 μg, about 150 μg, or about 200 μg. Optionally, the unit dosage of the composition is about 1 μg (e.g., about 0.08 μg, about 0.04 μg, about 0.2 μg, about 0.4 μg, about 0.8 μg, about 0.5 μg or less, about 0.25 μg or less, or about 0.1 μg or less) or greater than about 125 μg (e.g., greater than or equal to about 150 μg, greater than or equal to about 250 μg, or greater than or equal to about 500 μg). Such dosages can be measured as μg of total RSV F protein (e.g., spike protein or a fragment thereof). The immunogen of the present invention should be administered within about 12 hours, preferably within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour, of being reconstituted from a lyophilized powder. In another embodiment, the RSV F protein immunogenic composition is supplied in liquid form in a sealed container indicating the amount and concentration of the RSV F protein composition. Preferably, the liquid form of the immunogenic composition of the present invention is supplied in a sealed container at at least about 50 μg/ml, more preferably at least about 100 μg/ml, at least about 200 μg/ml, at least 500 μg/ml, or at least 1 mg/ml.

The vaccine or immunogenic composition of the present invention can be administered to an animal to induce an immune response against RSV. In one embodiment, the animal is susceptible to RSV infection. In one embodiment, the animal is a human. Preferably, the administration of the immunogenic induces substantial immunity against at least one RSV strain, isolate, clade and/or species. In one embodiment, the administration of the immunogenic induces substantial immunity against at least two RSV strains, isolates, clades and/or species. Typically, the dosage can be adjusted within this range based on, for example, age, physical condition, weight, age, food, time of administration and other clinical factors. Accordingly, the present invention includes a method of formulating a vaccine or immunogenic composition that induces substantial immunity against an infection or at least one symptom thereof in a subject, comprising adding an effective amount of an immunogenic to the formulation. Although stimulation of substantial immunity by a single dose is preferred, additional doses may be administered by the same or different route to achieve the desired effect. In neonates and infants, for example, multiple administrations may be required to induce sufficient levels of immunity. Administration may be spaced and continued throughout childhood, as needed to maintain sufficient levels of protection against infection. Similarly, adults who are particularly susceptible to repeated or severe influenza infections, such as health care workers, daycare teachers, family members of children, the elderly, and individuals with impaired cardiopulmonary function, may require multiple immunizations to induce and/or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing glands and serum antibodies and the amount of adjusted doses or repeated vaccinations, as needed to induce and maintain the desired level of protection. Thus, in one embodiment, a method of inducing substantial immunity to a viral infection or at least one symptom thereof in a subject comprises administering at least one effective amount of RSV F protein or a fragment or aggregate thereof. Methods of administering the vaccine and/or immunogenic compositions include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous, and subcutaneous), epidural, and mucosal (e.g., nasal and oral or pulmonary routes or suppositories). In certain embodiments, the compositions are administered intramuscularly, intravenously, subcutaneously, orally, or intradermally. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, absorption through an epithelium or mucosal lining (e.g., oral mucosa, colon, conjunctiva, nasopharynx, oropharynx, vagina, urinary tract, bladder, intestinal mucosa, and the like), and may be administered together with other biologically active substances. In some embodiments, administration via the nasal or other mucosal route can induce substantially higher antibody or other immune responses than other routes of administration. In other embodiments, the nasal or other mucosal route of administration of the immunogenic composition and/or vaccine can induce antibody or other immune responses that will induce cross-protection against other strains of the virus. Administration may be systemic or local. The prophylactic vaccine formulation is administered systemically by subcutaneous or intramuscular injection using a needle and syringe or by needle-free injection devices. Alternatively, the vaccine formulation is administered nasally by droplets, large particle aerosols (greater than about 10 microns) or spray into the upper respiratory tract. While any of the above routes of delivery will elicit an immune response, nasal administration provides an enhanced effect of inducing mucosal immunity at the site of viral entry. In another embodiment, the vaccine and/or immunogenic formulation is administered in a manner that targets mucosal tissue to elicit an immune response at the site of immunization. For example, mucosal tissues such as gut associated lymphoid tissue (GALT) can be targeted for immunization by using oral administration of a composition containing an immunogenic adjuvant with specific targeting properties. Other mucosal tissues may also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT). The vaccine and/or immunogenic formulations may be administered according to a dosing schedule, such as administering an initial vaccine composition followed by a booster dose. In certain embodiments, a second dose of the composition is administered any time from about 2 weeks to about 1 year after the initial administration, preferably about 1, about 2, about 3, about 4, about 5 to about 6 months. Additionally, a third dose may be administered after the second dose and at least about 3 months to about 2 years, preferably about 4, about 5, or about 6 months or about 7 months to about 1 year after the initial administration. The third dose may be administered orally when no or low amounts of the specific immunoglobulin are detected in the subject's serum and/or urine or mucosal secretions after the second dose. In a preferred embodiment, the second dose is administered about 1 month after the first dose and the third dose is administered about 6 months after the first dose. In another embodiment, the second dose is administered about 6 months after the first dose. In another embodiment, the immunogen comprising the RSV F protein may be administered as part of a combination therapy. For example, the RSV F protein or a fragment thereof or a complex thereof may be formulated with another immunogenic composition and/or an antiviral agent. The dosage of the pharmaceutical formulation can be readily determined by one of ordinary skill in the art by first identifying a dose that is effective to elicit a prophylactic or therapeutic immune response, for example, by measuring serum titers of virus-specific immunoglobulins or measuring the percentage of inhibition of antibodies in serum samples or urine samples or mucosal secretions. Additionally, human clinical studies can be performed by those skilled in the art to determine the desired effective dose for humans. Such clinical studies are routine and well known in the art. The exact dosage to be used will depend on the route of administration. The effective dose can be estimated from dose-response curves derived from in vitro or animal test systems. As is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants have been used experimentally to enhance the general enhancement of immunity against unknown immunogens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and adjuvants are well known to those skilled in the art. Some adjuvants affect the way in which the antigens are presented. For example, the immune response is enhanced when protein antigens are soaked in alum. Emulsification of the antigen extends the duration of antigen presentation. Adjuvants may be included. Suitable adjuvants include those described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), which is incorporated by reference in its entirety for all purposes. Other exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, montanide ISA 206, and monophosphoryl lipid A (MPL). A composition containing three components extracted from the cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion, MPL, trehalose dimycolate (TDM), and (CWS) RIBI is considered. MF-59, Novasome ®, MHC antigens may also be used. For example, the adjuvant is a paucilamellar lipid vesicle having 2 to 10 bilayers arranged in a substantially spherical envelope separated by an aqueous layer surrounding a large amorphous central cavity where the lipid bilayer has been eliminated.

In one embodiment, the adjuvant effect is achieved by the use of a substance such as alum, used in a solution of about 0.05 to about 0.1% in phosphate buffered saline. Alternatively, the immunogen may be prepared as a premix with a synthetic polymer of sugar (Carbopol®), used in a solution of about 0.25%. Some adjuvants, for example, certain organic molecules obtained from bacteria, act on the host rather than the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. In other embodiments, hemocyanins and hemoerythrins may be used. Although molluscan and arthropod hemocyanins and hemoerythrins may be used, hemocyanins from keyhole limpet (KLH) are preferred in certain embodiments. A variety of polysaccharide adjuvants may be used. For example, the use of a pneumococcal polysaccharide adjuvant for antibody responses in mice is disclosed (Yin et al, 1989). Doses that elicit an optimal response or do not elicit suppression should be used as directed (Yin et al, 1989). Polyamine variants of the polysaccharide are particularly preferred, such as chitin and chitosan, including deacetylated chitin. In another embodiment, a muramyl dipeptide lipophilic disaccharide-tripeptide derivative is disclosed for use in artificial liposomes, formed from phosphatidyl choline and phosphatidyl glycerol. Other suitable adjuvants include amphiphilic surfactants, such as saponins and derivatives such as QS21 (Cambridge Biotech). Saponin-based adjuvants include those containing substrate A and substrate C, alone or in combination. Nonionic block copolymer surfactants (Rabinovich et al, 1994) may be used. Oligonucleotides are another useful group of adjuvants (Yamamoto et al, 1988). Another group of adjuvants are detoxified endotoxins, such as the purified detoxified endotoxins of U.S. Pat. No. 4,866,034. Such purified detoxified endotoxins are effective in eliciting adjuvant responses in vertebrates. Of course, the detoxified endotoxins may be combined with other adjuvants to produce multivalent adjuvant preparations. For example, the combination of detoxified endotoxins with trehalose dimycolate, as disclosed in U.S. Pat. No. 4,435,386, is particularly contemplated. Also contemplated are combinations of detoxified endotoxin with trehalose dimycolate and endotoxin glycolipids (U.S. Pat. No. 4,505,899), and combinations of detoxified endotoxin with cell wall skeleton (CWS) or CWS and trehalose dimycolate are contemplated as disclosed in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. In the absence of detoxified endotoxin, combinations of CWS and trehalose dimycolate are also thought to be effective as disclosed in U.S. Pat. No. 4,520,019. Other types of adjuvants that can be conjugated to the vaccine are further contemplated, including alkyl lysophospholipids (ALP); BCG; and biotin (including biotinylated derivatives). One particular adjuvant contemplated for use is teichoic acid derived from Gram cells. These include lipoteichoic acid (LTA), ribitol teichoic acid (RTA) and glycerol teichoic acid (GTA). Active forms of these synthetic counterparts may also be used (Takada et al, 1995).

Various adjuvants that are not commonly used in humans can still be used in other vertebrates, for example, when it is desired to generate antibodies or subsequently obtain activated T cells. Toxicity or other adverse effects that may arise from the adjuvant or from the cells, such as might occur using, for example, non-irradiated tumor cells, are irrelevant in this setting. Another way to induce an immune response can be accomplished by formulating the immunogens of the invention with "immunogenic agents." These are the body's own chemical messengers (cytokines) that are intended to increase the response of the immune system. Immunogenic agents include various cytokines, lymphokines and chemokines that have immunostimulatory, immunopotentiating and proinflammatory activities, such as the interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); Growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF); and other immunostimulatory molecules such as macrophage inflammatory factors, Flt3 ligand, B7.1, B7.2, etc. The immunostimulatory molecules may be administered in the same formulation as the immunogen or may be administered separately. The protein or an expression vector encoding the protein may be administered to produce an immunostimulatory effect. Alum may be present in a range having the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about About 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, or about 150 μg. The alum can be present in a range having the following upper limits: about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, or about 200 μg. In certain embodiments, the alum range is from about 80 μg to about 120 μg or about 100 μg to about 120 μg. The saponin-based adjuvant can be present in a range having the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg. The saponin-based adjuvant can be present in a range having the following upper limits: about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, About 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg or about 200 μg. In certain embodiments, the saponin-based adjuvant is about 5 μg to about 20 μg or about 1 μg to about 10 μg. Such dosages are particularly appropriate in mice and can be adjusted for human use based on a typical mouse body weight of 20 g versus a human body weight of about 60 kg.

In one embodiment of the present invention, a method for inducing prophylactic immunity against RSV F infection is provided, comprising administering an RSV F immunogenic composition comprising RSV F protein, a fragment thereof, or a composite thereof, more preferably at least one protein selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, or a vaccine comprising the same.

Preferably, in one embodiment of the present invention, a method for inducing prophylactic immunity against RSV F infection is provided, comprising administering an RSV F immunogenic composition comprising at least one protein selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, or a vaccine comprising the same.

In one embodiment of the present invention, a method of inducing an immune response against RSV is provided, comprising administering to a subject a composition comprising the RSV F protein antigen.

The above method can reduce or prevent RSV infection.

The present invention provides an immunogenic composition for preventing and/or treating RSV infection.

The present invention can achieve a more improved RSV F protein expression rate in a host cell than the wild-type RSV F protein by mutating the RSV F protein.

The present invention can achieve high immunogenicity of RSV F protein. That is, an antigen completed through sequence mutation of RSV F protein of the present invention (and/or RSV F protein expression base sequence mutation) can achieve excellent immunogenicity by providing excellent neutralizing antibody.

The present invention can provide a stabilized and available RSV F protein.

The present invention can be applied to various cell lines and can provide a safe RSV immunogenic composition, more preferably a vaccine.

The RSV immunogen completed through the mutation of the present invention can achieve a long-term immune maintenance period in the human body. The RSV immunogen completed through the mutation of the present invention has less concern about repeated infection.

The RSV immunogen completed through the mutation of the present invention can induce excellent immune development with less interference by blood antibodies in the body.

Figure 1 is a schematic diagram of the mutation of the RSV F protein mainly implemented in the present invention.
Figure 2 is an experiment confirming the expression level of RSV F protein mutations performed in the present invention. This experiment measured the reactivity with palivizumab, which binds to the site II epitope. It was confirmed that the mutations attempted in the present invention had a superior expression rate than previously known mutation forms.
Figure 3 shows the results of Western blot and Coomassie staining of proteins expressed and purified through mutations implemented in the present invention. 3-a is the result of confirming F0 and F1 using βME. 3-b is the result of confirming FP1 and FP4 among the final purified mutant proteins.
Figure 4 is a transmission electron microscopy image of the RSV F protein produced by the present invention.
FIGS. 5A and 5B are graphs showing the results of a total antibody titer measurement experiment to determine the induction of mouse immunogenicity of the RSV F protein produced by the present invention. Here, the total antibody titer is a value confirmed through ELISA analysis. Here, GMT is Geometric Mean Titer. Here, "1ug+Adjuvant", "10ug+Adjuvant", and "30ug+Adjuvant" are substances in which 1ug, 10ug, and 30ug of RSV F protein are adsorbed to aluminum adjuvant, respectively. The PBS treatment was considered as 1.
Figure 6 is a schematic diagram of the results of an experiment to confirm the concentration of a specific antibody capable of competing with palivizumab binding to the site II epitope in mouse serum induced with immunity using the RSV F protein produced by the present invention.
Figures 7A and 7B are graphs showing the results of an experiment to determine the concentration of specific antibodies capable of competing with palivizumab binding to the site II epitope in mouse serum induced with the RSV F protein produced by the present invention. The results represent the GMT value that inhibits palivizumab by 50%.
Figures 8A and 8B are experiments to confirm that the RSV F protein produced by the present invention inhibits infection of wild-type RSV virus in mouse serum induced with immunity. The wild-type virus used here is RSV A2 (ATCC. VR-1540). Through a neutralization immune response experiment, it was confirmed that the RSV F protein produced by the present invention inhibits infection of wild-type RSV A2.
Figure 9 shows the screening results for soluble F protein expression of FP1, 3, 4, and 6.

Hereinafter, in order to help understand the present invention, examples and the like will be described in detail. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

Example 1. RSV F gene mutation

In order to obtain a stabilized and available RSV F protein in the present invention, several kinds of amino acid sequence mutations were performed. It is a fact known to those skilled in the art that the mutations are applicable to all RSV subtypes, including wild RSV-A2 and RSV-B, and also RSV-A long.

First, a mutation was performed on wild RSV-B (SEQ ID No. 1). The mutation removed the transmembrane domain. The amino acid sequence of the deleted transmembrane domain is amino acids at positions 525-574 of SEQ ID No. 2.

Specific experiments were conducted to confirm that mutations in the fusion peptide positions (positions 137-145 of SEQ ID NO: 2) of the present invention, more preferably amino acids mutated to FP1, FP3, FP4, and FP6, can act as excellent RSV immunogenic agents.

In one embodiment, the mutation mutates the fusion peptide portion (amino acid sequence 137-145). In one embodiment, specifically, the RSV F protein is a mutation in which the hydrophobic portion of the fusion peptide portion is modified into a hydrophilic portion. Specifically, the hydrophobic portion in the sequence FLGFLLGVG of the fusion peptide is modified to AAGAAAGAG by substituting it with A (SEQ ID NO: 3). For the purpose of the present invention, the optimal effect can be obtained as a result of substituting it with A, among hydrophilic amino acids.

In addition, in one embodiment, the polar portion of FLGFLLGVG in the fusion peptide portion was modified to a nonpolar side chain, and the modified fusion peptide portion was modified to QNGQNNGSG and NSGNSSGGG (SEQ ID NO: 4 and SEQ ID NO: 5, respectively). In another embodiment, the FLGFLLGVG portion in the fusion peptide portion was modified to TLSKKRKRR (SEQ ID NO: 6).

In one embodiment of the present invention, the present invention comprises an additional mutation that maintains the stabilization of the F1 portion of the wild RSV F protein. The additional mutation associated with stabilization further comprises at least one additional mutation selected from the group consisting of (a) to (g) based on SEQ ID NO: 2.

(a) mutation of the amino acid residue at position 140;

(b) mutation of amino acid residue at position 163;

(c) mutation of amino acid residue at position 179;

(d) mutation of amino acid residue at position 188;

(e) mutation of amino acid residue at position 189;

(f) mutation of amino acid residue at position 487;

(g) Mutation of amino acid residue at position 505.

More specifically, the RSV F protein antigen of the present invention is based on sequence number 2.

(a) mutation of amino acid residue F to W at position 140 (SEQ ID NO: 7);

(b) mutation of amino acid residue E to Q at position 163 (SEQ ID NO: 8);

(c) mutation of amino acid residue V to L at position 179 (SEQ ID NO: 9);

(d) mutation of amino acid residue L to Q at position 188 (SEQ ID NO: 10);

(e) mutation of amino acid residue T to L at position 189 (SEQ ID NO: 11);

(f) mutation of amino acid residue E to L at position 487 (SEQ ID NO: 12); and

(g) comprises at least one mutation selected from the group consisting of a mutation of amino acid residue F to W at position 505 (SEQ ID NO: 13).

In another embodiment, the RSV F protein antigen is

The amino acid corresponding to position 140 of sequence number 2 is substituted with W, and the amino acid corresponding to position 163 is substituted with Q (SEQ ID NO: 14);

The amino acid corresponding to position 140 of sequence number 2 is replaced with W, the amino acid corresponding to position 163 is replaced with Q, the amino acid corresponding to position 188 is replaced with Q, and the amino acid corresponding to position 189 is replaced with L (SEQ ID NO: 15);

The amino acid corresponding to position 488 of sequence number 2 is substituted with W, and the amino acid corresponding to position 163 is substituted with Q (SEQ ID NO: 16);

The amino acid corresponding to position 488 of sequence number 2 is substituted with W, and the amino acid corresponding to position 179 is substituted with L (SEQ ID NO: 17);

The amino acid corresponding to position 140 of sequence number 2 is substituted with W, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 18);

The amino acid corresponding to position 487 of sequence number 2 is substituted with L, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 19);

The amino acid corresponding to position 163 of sequence number 2 is substituted with Q, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 20);

The amino acid corresponding to position 188 of sequence number 2 is substituted with Q, the amino acid corresponding to position 189 is substituted with L, and the amino acid corresponding to position 505 is substituted with W (SEQ ID NO: 21);

The amino acid corresponding to position 163 of sequence number 2 may be substituted with Q, and the amino acid corresponding to position 487 may be substituted with L (SEQ ID NO: 22).

In some embodiments, the use of known methods of protein engineering and recombinant DNA technology to improve or modify the properties of the mutant RSV F proteins mentioned above is included. The genes encoding the proteins are modified by codon adaptation, and the modification of the nucleotides is well known to those skilled in the art. Various forms of mutagenesis can be used to produce and/or isolate mutant nucleic acids encoding the protein molecules and/or to further modify/mutate the proteins of the invention. These include, but are not limited to, site-directed mutagenesis, random site mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, gapped-duplex DNA, and the like. Other suitable methods include mutagenesis by point mismatch repair, repair-deficient host strains, mutagenesis using restriction-selection and restriction-purification, deletion mutagenesis, whole gene synthesis, double-strand break repair, etc. For example, mutagenesis involving chimeric constructs is also encompassed by the present invention. In one embodiment, mutagenesis can be guided by known information about the molecule, either innately occurring or naturally occurring by acquisition or mutation, such as sequence, sequence comparison, physical properties, crystal structure, etc.

Example 2. RSV F gene Expression protein for mutation Screening

The genetic mutation for the F protein described in the previous example was confirmed in terms of expression level through a protein expression test using 293FT cells. The candidate material plasmid to which each mutation was applied was inserted into a vector containing a eukaryotic promoter, and the expression level was confirmed. The vector used in one example is pcDNA3.1. It is well known to those skilled in the art that not only pcDNA3.1 but also other eukaryotic expression vectors can be used. The ELISA method was used to evaluate the expression level, and the antibody used at this time was palivizumab, which binds to RSV site II. As can be confirmed in Fig. 2, the screening result of the expressed protein for the applied mutation showed a higher expression rate than the mutation of the already known patent.

Screening results for expression of available F proteins of FP1, 3, 4 and 6

Samples were harvested 5 days after transfection. Samples were harvested in two ways: lysis (Medium & Pellet) and no lysis (Medium). First, in the case of no lysis, only the supernatant from the transfected well was harvested. In the case of lysis, 10 μl of 10% Triton-X 100 was added to each 96-well culture medium (approximately 100 μl), rocked at 4˚C for 30 minutes, spun down for 1 minute to settle the debris, and then the clear lysate was harvested.

After adding 50 μl of PBS to a nickel-coated plate, 50 μl of the lysed sample and the non-lysed sample were added each and incubated at room temperature for one hour. Synagis strain was used as the primary antibody. Polyclonal rabbit anti-human IgG/HRP was used as the secondary antibody. TMB acts as an electron donor that reduces hydrogen peroxide, etc. by peroxidase such as horseradish peroxidase (HRP). When the HRP enzyme acts, TMB exhibits blue light in the 650 nm wavelength range, and when the stop reagent, sulfuric acid, is added, it changes to yellow in the 450 nm wavelength range. The difference between the two wavelength values (450 nm - 650 nm) was measured to select a construct with good expression.

　 Medium Cell & Medium 　 1st 2nd mean STDEV %CV 1st 2nd mean STDEV %CV FP1 0.462 0.47 0.466 0.006 1.2 0.488 0.53 0.509 0.030 5.8 FP3 0.437 0.432 0.435 0.004 0.8 0.478 0.48 0.479 0.001 0.3 FP4 0.467 0.465 0.466 0.001 0.3 0.468 0.484 0.476 0.011 2.4 FP6 0.423 0.379 0.401 0.031 7.8 0.421 0.415 0.418 0.004 1.0 Mock 0.017 0.017 0.017 0.000 0.0 0.016 0.016 0.016 0.000 0.0

* STDEV: standard deviation %CV: the percent coefficient of Variation

As can be seen in Table 1 and Figure 9 above, it was confirmed that the expression rates of FP1, FP3, FP4 and FP6 of the present invention were excellent, and it was found that they could be used as antigens.

Example 3. RSV F gene introduction recombination of baculovirus production

The codon of the RSV-B F (SEQ ID NO:1,) protein was optimized, and an antigen obtained through the genetic mutation described above was prepared. The described RSV F gene was cloned to create a RSV F protein recombinant baculovirus (AcMNPV), which was then inoculated into insect cells, Sf9.

The usual cell concentration of Sf9 insect cells should not exceed 3.00E6/ml. Sf9 insect cells are cultured in sterile 125ml/250ml/500ml/1L spinner flasks or 5L/50L/100L bioreactors. In this example, a serum-free insect cell-specific medium that does not contain serum was used for culture. Here, the serum-free insect cell-specific medium can include Insect Express (Lonza), etc. In this example, the RSV F recombinant baculovirus for producing RSV F protein was inoculated at a multiplicity of infection (MOI) between 0.01 and 0.8, and the concentration of Sf9 insect cells was 5.00E5-1.50E6, which is the early stage of the exponential phase of growth. The RSV F recombinant baculovirus used in this example is harvested 60-90 hours after inoculation, and the viability should be between 75-98%. The reason for inoculating low concentrations of virus into low-growth-phase cells and harvesting at relatively high viability in this example is to minimize target protein cleavage by proteases released from cells in the death phase and to minimize the amount of virus inoculum used.

Example 4. RSV Expression, purification, and analysis of F antigen

First, the Sf9 insect cells secured after inoculation with the recombinant baculovirus of Example 3 are centrifuged at 3000-8000xg for 1-50 minutes to separate the cell sedimentation layer and the media supernatant. The separated cell sedimentation layer is removed, and the media supernatant is secured. Infection with the recombinant baculovirus can effectively occur when the cells are in the early-log phase of growth and at a concentration of 1.0E5-7.0E6 cells/ml, preferably 5.00E5-2.50E6 cells/ml.

Further purification of RSV F can be accomplished by ion exchange, affinity chromatography known in the art. In one example, the crude extract was further purified by passing it through anion exchange chromatography, lentil lectin affinity and cation exchange chromatography.

Further purification of RSV F can be accomplished by ion exchange, affinity chromatography known in the art. In one example, the crude extract was further purified by passing it through anion exchange chromatography, lentil lectin affinity and cation exchange chromatography.

In one implementation, the same volume of sample from each purification process was diluted with 2x SDS sample buffer containing βME (beta-mercaptoethanol), 15-20 ㎕ per well of an SDS-gel, electrophoresed, and total proteins were stained by Coomassie staining. In addition, the SDS-gel was transferred to a membrane, and further analyzed by WB (western blotting) using anti-single or multiplexed RSV F-specific antibodies. For WB, the membrane to which the proteins were transferred was incubated with a buffer solution (PBS) containing 5% skim milk (Sigma) at 4°C for 4-24 h, washed with the buffer, and RSV F-specific antibodies diluted 1:2000 were added and incubated at 4°C for 4-24 h. After incubation, wash three times with a buffer solution containing 0.1% Tween20, add a 1:5000 diluted horseradish peroxidase (HRP)-conjugated secondary antibody, and incubate for 1-2 hours at room temperature. After incubation, the membrane is washed three times with a buffer solution containing 0.1% Tween20, develop color with ECL detection reagent (Amersham), and develop by film. (Figure 3)

RSV Animal administration/immunization induction using F antigen

In this example, an animal experiment was performed using a mouse with the antigen obtained. The mouse used in the experiment can be used as is, or can be used with an immune enhancer, such as an aluminum adjuvant. The adjuvant can include, for example, an aluminum or calcium salt, specifically, an inorganic salt such as hydroxide, phosphate, or calcium phosphate.

Aluminum (Aluminum or Alum) adjuvants are the most commonly used adjuvants in current human vaccines, and their safety and effectiveness have been proven to the extent that they are used as standards for developing and evaluating new adjuvants. Representative alum adjuvants are aluminum hydroxide (Al(OH) ₃ ) and aluminum phosphate (AlPO4), and these two alum adjuvants exhibit different physical properties and adjuvant properties. In particular, aluminum hydroxide is the most widely used in aluminum-adsorbed vaccines, and in this example, aluminum hydroxide was used as an immune enhancer, and at this time, aluminum hydroxide was used at a level of 180 ug/administration.

In this example, the concentrations of RSV F antigen administered were 1 ug/ml, 10 ug/ml, and 30 ug/ml, and the number of administrations was 2 times. In this example, the administration schedule was 2 times 2 weeks after the initial administration, and blood collection was performed at 2 and 4 week intervals after the initial administration.

RSV Confirmation of immunogenicity in animals administered F antigen

The immunogenicity-inducing effect of the RSV F antigen obtained in this example was confirmed. The immunogenicity induced in this example was confirmed through total antibody measurement, PCA measurement, and neutralizing antibody measurement.

In this example, total antibody measurement to confirm antigen-specific IgG antibody titer was performed using the ELISA method. 100 ng of RSV F antigen was coated per well on a 96-well plate and incubated overnight at 4℃. After incubation, the plate was washed three times with a buffer solution containing 0.05% Tween20, incubated for 2 hours with a buffer solution containing 5% skim milk, and washed again three times with a buffer solution containing 0.05% Tween20. The obtained mouse blood was centrifuged to obtain a serum sample, and the prepared serum sample was diluted to an initial concentration of 1/20, and then serially diluted 4-fold to obtain a final concentration of 1/327680. After adding 100 µl of the diluted serum sample per well to the coated plate, incubated for 2 hours at room temperature and washed three times with a buffer solution containing 0.05% Tween20. After washing, 100 µl per well of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Invitrogen, US) diluted 1:5000 was added to the plate, incubated for 1 hour at room temperature, and washed three times with a buffer solution containing 0.05% Tween20. 3,3A,5,5A-tetramethylbenzidine substrate (TMB substrate, KPL) was added to each well of the washed plate, reacted with HRP for 10 minutes, and then the reaction was stopped with TMB stop solution (KPL). The absorbance at 450 nm for each well was measured using a microtiter plate reader (Molecular Devices), and the results measured in this example indicate that the expressed/purified RSV F immunogen induces high antibody titers in mice. (Figure 5)

To determine whether the expressed/purified RSV F immunogen in this example induces neutralizing antibodies against the site II epitope of the RSV F protein, a competitive ELISA was performed using the palivizumab antigen that binds to the site II epitope. 200 ng of RSV F antigen per well was coated on a 96-well plate and incubated overnight at 4°C. After incubation, the plate was washed three times with a buffer solution containing 0.05% Tween20, incubated for 2 hours with a buffer solution containing 5% skim milk, and then washed three more times with a buffer solution containing 0.05% Tween20. The collected mouse blood was centrifuged to obtain a serum sample, and the prepared serum sample was diluted to an initial concentration of 1/10, and then serially diluted two-fold to obtain a final concentration of 1/1280. Biotin-conjugated palivizumab was diluted to 50 ng per well, and serial dilutions were performed two-fold to obtain a final concentration of 390 pg per well. The diluted serum samples and biotin-conjugated palivizumab were incubated for 2 hours, and washed three times with a buffer solution containing 0.05% Tween20. After washing, the plate was incubated with 1:8000 diluted horseradish peroxidase (HRP)-conjugated streptavidin for 1 hour, and washed three times with a buffer solution containing 0.05% Tween20. 3,3A,5,5A-tetramethylbenzidine substrate (TMB substrate, KPL) was added to each well of the washed plate, and the reaction was reacted with HRP for 2 minutes, and the reaction was stopped by adding TMB stop solution (KPL). The absorbance value at 450 nm for each well was measured using a microtiter plate reader (Molecular Devices). In this example, the measured antibody titer against palivizumab confirmed that it induced antibodies against site II, a neutralizing epitope of the expressed/purified RSV F antigen. (Figure 6, Figure 7)

In this example, the measurement of antibody titers that induce a neutralizing immune response was performed. Hep2 cells were seeded in a 24-well plate at a number of 3.5E5 per well and then cultured for one day at 37°C, 5% CO2. The serum sample collected from the animal experiment was diluted to an initial concentration of 1/20, and then serially diluted three times to make the final concentration 1/4860 times. 50 ㎕ of the diluted serum sample and 50 ㎕ (40 pfu) of RSV virus were mixed, incubated at room temperature for 1 hour, and then inoculated onto the Hep2 cells on the plate seeded the previous day. After inoculation, the plates were cultured for 5 days at 37°C, 5% CO2. The neutralization value was measured by staining the plates after the culture was completed with neutral red. The neutralization value was calculated as the value that protects 50% of the virus (ND50). (Figure 8)

Amino acid sequence List

1. SK- Seq 1 aa sequence (JN032120)

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPI VNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSD EFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK.

2. SK- Seq 1_ dTM (transmembrane deletion_1-524 aa sequence )

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

3. SK-FP1 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRRAAGAAAGAGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPI VNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

4. SK-FP3 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRQNGQNNGSGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

5. SK-FP4 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRNSGNSSGGGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPI VNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

6. SK-FP6 aa

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRR1SAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQ QSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPI TNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLE GKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

7. SK- Seq 1_ dTM _ F140W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

8. SK- Seq 1_ dTM _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

9. SK- Seq 1_ dTM _ V179L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVLSLSNGVSVLTSKVLDLKNYINNQLLPI VNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

10. SK- Seq 1_ dTM _ L188Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVQTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

11. SK- Seq 1_ dTM _ T189L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLLSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

12. SK- Seq 1_ dTM _ E487L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

13. SK- Seq 1_ dTM _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

14. SK- Seq 1_ dTM _ F140W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINN QLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

15. SK- Seq 1_ dTM _ F140W _ E163Q _ L188Q _ T189L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVQLSKVLDLKNYIN NQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

16. SK- Seq 1_ dTM _ F488W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEWDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

17. SK- Seq 1_ dTM _ F488W _ V179L

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVLSLSNGVSVLTSKVLDLKNYINNQLLPI VNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEWDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

18. SK- Seq 1_ dTM _ F505W _ F140W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGWLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

19. SK- Seq 1_ dTM _ E487L _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLL PIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

20. SK- Seq 1_ dTM _ F505W _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

21. SK- Seq 1_ dTM _ L188Q _ T189L _ F505W

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVQLSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAWIRRSDELLHNVNTGKSTTN

22. SK- Seq 1_ dTM _ E487L _ E163Q

MELLIHRSSAIFLTLAINAFYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGQVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQ LLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLIN DMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYV NKLEGKNLYVKGEPIINYYDPLVFPSDLFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTN

List of base sequences

23. Wild type nt sequence (JN032120)

ATGGAGTTGCTGATCCATAGATCAAGTGCAATCTTCCTAACTCTTGCTATTAATGCATTTTACCTCACCTCAAGTCAGAACATAACTGAGGAGTTTTACCAATCGACATGTAGTGCAGTTAGCAGAGGTTACTTGAGTGCTTTAAGAACAGGTTGGTATACCAGTGTCATAACAATAGAATTAAGTAATATAAAAGAAACCAAATGCAATGGAACTGACACTAAAGTAAAACTTATAAAACAAGAATTAGATAAGTATAAGAATGCAGT AACAGAATTACAGTTACTTATGCAAAACACACCAGCTGCCAACAACCGGGCCAGAAGAGAAGCACCACAGTATATGAACTACACAATCAATACCACTAAAACCTAAATGTATCAATAAGCAAGAAGAGGAAACGAAGATTTCTGGGCTTCTTGTTAGGTGT AGGATCTGCAATAGCAAGTGGTATAGCTGTATCCAAAAGTTCTACACCTTGAAGGAGAAGTGAACAAGATCAAAAATGCTTTGCTGTCTACAAACAAAGCTGTAGTCAGTCTATCAAATGGGGTCAGTGTTTTAACCAGCAAAGTGTTAGATCTCAAGAATTATATAAACAACCAATTATTACCTATAGTAAATCAACAGAGTTGTCGCATTTCCAACATTGAAACAGTTATAGAATTCCAGCAGAAGAACAGCAGATTGTTGGAAA TCACCAGAGAATTTAGTGTCAATGCTGGTGTAACGACACCTTTAAGCACTTACATGTTAACAAACAGTGAGTTACTATCATTAATCAATGATATGCCTATAACAAATGATCAGAAAAAATTAATGTCAAGCAATGTTCAGATAGTAAGGCAACAAAGTTATTCTA TCATGTCTATAATAAAGGAAGAAGTCCTTGCATATGTTGTACAGCTACCTATCTATGGTGTAATTGATACACCTTGCTGGAAATTACACACATCACCTCTGTGCACCACCAACATCAAAGAAGGATCAAATATTTGTTTAACAAGGACTGATAGAGGATGGTACTGTGATAATGCAGGATCAGTATCCTTCTTTCCACAGGCTGACACTTGTAAAGTACAGTCCAATCGAGTATTTTGTGACACCATGAACAGTTTG ACATTACCAAGTGAAGTCAGCCTTTGTAACACTGACATATTCAATTCCAAGTATGACTGCAAAATTATGACATCAAAAACAGACATAAGCAGCTCAGTAATTACTTCTCTAGGAGCTATAGTGTCATGCTATGGTAAAACTAAATGCACTGCATCCAACAAAAATCGTGGAATT ATAAAGACATTTTCTAATGGTTGTGATTATGTGTCAAACAAAGGAGTAGATACTGTATCAGTGGGCAACACTTTATACTATGTCAACAAGCTGGAAGGCAAAAACCTTTATGTAAAAGGGGAACCTATAATAAATTACTATGACCCTCTAGTGTTTCCTTCTGATGAGTTTGATGCATCAATATCTCAAGTCAATGAAAAAAATTAATCAAAGTTTAGCTTTTATTCGTAGATCCGATGAATTATTACATAATGTAAATACTGGAAA ATCTACTACAAATATTATGATAACTGCAATTATTATAGTAATCATTGTAGTATTGTTATCATTAATAGCTATTGGTTTACTGTTGTATTGCAAAGCCAAAAACACACCAGTTACACTAAGCAAAGACCAACTAAGTGGAATCAATAATAATTGCATTCAGCAAATAG

24. SK- Seq 1_ dTM (transmembrane deletion_1- 524aa's nt sequence )

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

25. SK-FP1 nt

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTGCCGCGGGTGCCGCGGCCGGTGCGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

26. SK-FP3 nt

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTCAGAATGGTCAAAATAACGGTTCGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATAATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAACC GTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

27. SK-FP4 nt

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTAACTCGGGTAACTCGTCCGGTGGAGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

28. SK-FP6 nt

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTACCCTGTCTAAAAAGCGCAAAAGAAGATCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATAATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAACC GTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

29. SK- Seq 1_ dTM _ F140W

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTGGCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

30. SK- Seq 1_ dTM _ E163Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

31. SK- Seq 1_ dTM _ V179L

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCTTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

32. SK- Seq 1_ dTM _ L188Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCAGACCTCTAAAGTGCTGGACCTGAAGAACTATAATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

33. SK- Seq 1_ dTM _ T189L

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCCTCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

34. SK- Seq 1_ dTM _ E487L

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

35. SK- Seq 1_ dTM _ F505W

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

36. SK- Seq 1_ dTM _ F140W _ E163Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTGGCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

37. SK- Seq 1_ dTM _ F140W _ E163Q _ L188Q _ T189L

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTGGCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCAGCTCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

38. SK- Seq 1_ dTM _ F488W _ E163Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTGGGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

39. SK- Seq 1_ dTM _ F488W _ V179L

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCTTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTGGGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

40. SK- Seq 1_ dTM _ F505W _ F140W

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTGGCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

41. SK- Seq 1_ dTM _ E487L _ F505W

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

42. SK- Seq 1_ dTM _ F505W _ E163Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

43. SK- Seq 1_ dTM _ L188Q _ T189L _ F505W

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTGAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCAGCTCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATGAGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTGGATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

44. SK- Seq 1_ dTM _ E487L _ E163Q

ATGGAGCTCCTGATCCACCGTTCCTCCGCTATCTTCCTGACCCTGGCTATCAACGCTTTTTACCTGACCAGCTCCCAGAACATCACTGAGGAGTTTTTACCAGTCCACCTGCTCCGCCGTGTCCCGTGGTTACCTCTCCGCTCTGCGCACCGGTTGGTATACCTCCGTGATTACTATTGAGCTGAGCAATATCAAAGAAACCAAACCAAATGCAATGGCACCGACACCAAAGTGAAACTGATTAAGCAAGAGCTCGACAAGTATA AGAATGCTGTCACCGAACTGCAGCTGCTCATGCAAAACACTCCTGCTGCTAACAACCGTGCCCGTCGTGAGGCTCCCCAGTACATGAACTACACCATCAACACCACCAAGAACCTGAATGTGTCCATCTCCAAG AAGCGCAAACGTCGTTTCCTGGGTTTCCTGCTCGGTGTGGGCTCCGCCATCGCTTCTGGTATCGCTGTCTCCAAGGTGCTGCACCTGGAAGGTCAGGTGAACAAGATCAAAAACGCTCTGCTGTCCACCAACAAGGCTGTCGTGTCCCTGTCCAATGGCGTGTCTGTCCTCACCTCTAAAGTGCTGGACCTGAAGAACTATATTAACAACCAGCTGCTCCCCATCGTGAATCAGCAGTCCTGCCGTATTTCCAACATCGAAA CCGTCATTGAATTCCAGCAGAAGAACTCCCGTCTGCTCGAGATCACCCGCGAATTCTCCGTGAACGCTGGTGTGACCACTCCTCTGTCTACCTACATGCTGACCAACTCCGAGCTGCTGTCCCTCATCAAT GATATGCCCATTACCAACGACCAGAAAAAACTGATGTCCTCCAATGTCCAGATCGTGCGTCAGCAGTCCTACTCCATCATGTCCATTATCAAGGAGGAGGTCCTGGCTTATGTGGTGCAGCTGCCCATCTATGGTGTGATTGATACCCCCTGCTGGAAACTGCACACTTCCCCTCTGTGCACCACCAACATCAAGGAAGGCTCCAATATCTGTCTGACTCGTACCGACCGTGGTTGGTACTGTGACAATGCCGGTTCCGTCTCC TTCTTTCCCCAGGCTGACACCTGCAAAGTGCAGTCCAACCGTGTGTTCTGTGACACCATGAACTCCCTCACTCTGCCCTCCGAAGTGTCCCTGTGCAACACCGACATTTTCAATTCCAAGTATGACTGC AAGATCATGACCTCTAAAACCGACATCTCCTCTCCGTGATTACCTCCCTGGGCGCTATTGTGTCCTGCTATGGTAAAACTAAGTGCACCGCCTCCAACAAAAATCGTGGTATCATTAAGACCTTTTCCAATGGTTGTTGATTACGTGTCCAACAAAGGTGTCGATACCGTCTCCGTGGGTAACACTCTCTACTATGTGAACAAGCTGGAGGGCAAGAACCTCTACGTGAAAGGTTGAACCTATCATTAACTACTATGA CCCCCTGGTGTTTCCCTCTGATCTGTTTGACGCTTCCATTTCCCAAGTGAATGAGAAAATCAATCAAAGCCTGGCTTTCATTCGTCGTTCCGATGAACTGCTCCATAACGTGAATACTGGTAAATCCACCACCAAC

<110> SK CHEMICALS CO., LTD. <120> Modified soluble RSV F protein antigen <130> P18-280 <150>KR 10-2017-0128003 <151> 2017-09-29 <160> 44 <170> KoPatentIn 3.0 <210> 1 <211> 574 <212> PRT <213> respiratory syncytial virus <400> 1 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525 Ala Ile Ile Ile Val Ile Ile Val Val Leu Leu Ser Leu Ile Ala Ile 530 535 540 Gly Leu Leu Leu Tyr Cys Lys Ala Lys Asn Thr Pro Val Thr Leu Ser 545 550 555 560 Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Lys 565 570 <210> 2 <211> 524 <212> PRT <213> respiratory syncytial virus <400> 2 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 3 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-FP1 aa <400> 3 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Ala Ala Gly Ala Ala Ala Gly Ala 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 4 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-FP3 aa <400> 4 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Gln Asn Gly Gln Asn Asn Gly Ser 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 5 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-FP4 aa <400> 5 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Asn Ser Gly Asn Ser Ser Gly Gly 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 6 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-FP6 aa <400> 6 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Thr Leu Ser Lys Lys Arg Lys Arg 130 135 140 Arg Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 7 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W <400> 7 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Trp Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 8 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E163Q <400> 8 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 9 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_V179L <400> 9 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Leu Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 10 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_L188Q <400> 10 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Gln Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 11 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_T189L <400> 11 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Leu Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 12 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L <400> 12 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Leu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 13 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F505W <400> 13 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Trp Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 14 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W_E163Q <400> 14 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Trp Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 15 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W_E163Q_L188Q_T189L <400> 15 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Trp Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Gln Leu Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 16 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F488W_E163Q <400> 16 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Trp Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 17 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F488W_V179L <400> 17 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Leu Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Trp Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 18 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F505W_F140W <400> 18 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Trp Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Trp Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 19 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L_F505W <400> 19 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Leu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Trp Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 20 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_ F505W_E163Q <400> 20 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Trp Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 21 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_L188Q_T189L_ F505W <400> 21 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Gln Leu Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Trp Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 22 <211> 524 <212> PRT <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L_E163Q <400> 22 Met Glu Leu Leu Ile His Arg Ser Ser Ala Ile Phe Leu Thr Leu Ala 1 5 10 15 Ile Asn Ala Phe Tyr Leu Thr Ser Ser Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Thr Lys Cys Asn Gly Thr Asp Thr Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Asn Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Ala Pro 100 105 110 Gln Tyr Met Asn Tyr Thr Ile Asn Thr Thr Lys Asn Leu Asn Val Ser 115 120 125 Ile Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly Gln Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn Tyr Ile Asn Asn Gln Leu Leu Pro Ile Val Asn 195 200 205 Gln Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 Gln Lys Asn Ser Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala Gly Val Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270 Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295 300 Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Ile Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Asp Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Ser Leu Cys Asn Thr 370 375 380 Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Ile Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly 450 455 460 Lys Asn Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Leu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Thr Gly Lys Ser Thr Thr Asn 515 520 <210> 23 <211> 1725 <212> DNA <213> respiratory syncytial virus <400> 23 atggagttgc tgatccatag atcaagtgca atcttcctaa ctcttgctat taatgcattt 60 tacctcacct caagtcagaa cataactgag gagttttacc aatcgacatg tagtgcagtt 120 agcagaggtt acttgagtgc tttaagaaca ggttggtata ccagtgtcat aac aatagaa 180 ttaagtaata taaaagaaac caaatgcaat ggaactgaca ctaaagtaaa acttataaaa 240 caagaattag ataagtataa gaatgcagta acagaattac agttacttat gcaaaacaca 300 ccagctgcca acaaccgggc cagaagagaa gcaccacagt atatgaacta cacaatcaat 360 accactaaaa acctaaatgt atcaataagc aagaagagga aacgaagatt tctgggcttc 420 ttgttaggtg taggatctgc aatagcaagt ggtatagctg tatccaaagt tctacacctt 480 gaaggagaag tgaacaagat caaaaatgct ttgctgtcta agc tgtagtcagt 540 ctatcaaatg gggtcagtgt tttaaccagc aaagtgttag atctcaagaa ttatataaac 600 aaccaattat tacctatagt aaatcaacag agttgtcgca tttccaacat tgaaacagtt 660 atagaattcc agcagaagaa cagcagattg ttggaaatca ccagagaatt tagtgtcaat 720 gctggtgtaa cgacaccttt aagcacttac atgttaacaa acagtgagtt actatcatta 780 atcaatgata tgcctataac aaatgatcag aaaaaattaa tgtcaagcaa tgttcagata 840 gtaaggcaac aaagttattc ta tcatgtct ataataaagg aagaagtcct tgcatatgtt 900 gtacagctac ctatctatgg tgtaattgat acaccttgct ggaaattaca cacatcacct 960 ctgtgcacca ccaacatcaa agaaggatca aatatttgtt taacaaggac tgatagagga 1020 tggtactgtg ataatgcagg atcagtatcc ttctttccac aggctgacac ttgtaaagta 1080 cagtccaatc gagtattttg tgacaccatg aacagtttga cattaccaag tgaagtcagc 1140 ctttgtaaca ctgacatatt caattccaag tatgactgca aaattatgac atca aaaaca 1200 gacataagca gctcagtaat tacttctcta ggagctatag tgtcatgcta tggtaaaact 1260 aaatgcactg catccaacaa aaatcgtgga attataaaga cattttctaa tggttgtgat 1320 tatgtgtcaa acaaaggagt agatactgta tcagtgggca acactttata ctatgtcaac 1380 aagctggaag gcaaaaacct ttatgtaaaa ggggaaccta taataaatta ctatgaccct 1440 ctagtgtttc cttctgatga gtttgatgca tcaatatctc aagtcaatga aaaaattaat 1500 caaagtttag ct tttaattcg tagatccgat gaattattac ataatgtaaa tactggaaaa 1560 tctactacaa atattatgat aactgcaatt attatagtaa tcattgtagt attgttatca 1620 ttaatagcta ttggtttact gttgtattgc aaagccaaaa acacaccagt tacactaagc 1680 aaagaccaac taagtggaat caataatatt gcattcagca aatag 1725 <210> 24 <211> 1572 <212> DNA <213> respiratory syncytial virus <400> 24 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 25 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-FP1 nt <400> 25 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgtgc cgcgggtgcc 420 gcggccggtg cgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggt gagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 26 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-FP3 nt <400> 26 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgtca gaatggtcaa 420 aataacggtt cgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggtgagg t gaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa tgtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgcaaca ccgacatt tt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacgct tccatttccc a agtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 27 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-FP4 nt <400> 27 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgtaa ctcgggtaac 420 tcgtccggtg gaggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggtgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 28 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-FP6 nt <400> 28 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgtac cctgtctaaa 420 aagcgcaaaa gaagatccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggtgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa tgtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgcaaca ccgacatt tt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacgct tccatttccc a agtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 29 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W <400> 29 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggttgg 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggt gagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 30 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E163Q <400> 30 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tcagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 31 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_V179L <400> 31 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcttgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 32 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_L188Q <400> 32 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt ccagacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 33 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_T189L <400> 33 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcctctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 34 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L <400> 34 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatct gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 35 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F505W <400> 35 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg cttggattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 36 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W_E163Q <400> 36 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggttgg 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggt cagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 37 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F140W_E163Q_L188Q_T189L <400> 37 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggttgg 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggt cagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt ccagctctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 38 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F488W_E163Q <400> 38 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tcagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtgggacgct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 39 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F488W_V179L <400> 39 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcttgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtgggacgct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 40 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_F505W_F140W <400> 40 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggttgg 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaaggt gagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg cttggattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 41 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L_F505W <400> 41 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatct gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg cttggattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 42 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_ F505W_E163Q <400> 42 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tcagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg cttggattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 43 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_L188Q_T189L_ F505W <400> 43 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tgagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt ccagctctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatga gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg cttggattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572 <210> 44 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> SK-Seq 1_dTM_E487L_E163Q <400> 44 atggagctcc tgatccaccg ttcctccgct atcttcctga ccctggctat caacgctttt 60 tacctgacca gctcccagaa catcactgag gagttttacc agtccacctg ctccgccgtg 120 tcccgtggtt acctctccgc tctgcgcacc ggttggtata cctccgt gat tactattgag 180 ctgagcaata tcaaagaaac caaatgcaat ggcaccgaca ccaaagtgaa actgattaag 240 caagagctcg acaagtataa gaatgctgtc accgaactgc agctgctcat gcaaaacact 300 cctgctgcta acaaccgtgc ccgtcgtgag gctccccagt acatgaacta caccatcaac 360 accaccaaga acctgaatgt gtccatctcc aagaagcgca aacgtcgttt cctgggtttc 420 ctgctcggtg tgggctccgc catcgcttct ggtatcgctg tctccaaggt gctgcacctg 480 gaagg tcagg tgaacaagat caaaaacgct ctgctgtcca ccaacaaggc tgtcgtgtcc 540 ctgtccaatg gcgtgtctgt cctcacctct aaagtgctgg acctgaagaa ctatattaac 600 aaccagctgc tccccatcgt gaatcagcag tcctgccgta tttccaacat cgaaaccgtc 660 attgaattcc agcagaagaa ctcccgtctg ctcgagatca cccgcgaatt ctccgtgaac 720 gctggtgtga ccactcctct gtctacctac atgctgacca actccgagct gctgtccctc 780 atcaatgata tgcccattac caacgaccag aaaaaactga tgtcctccaa t gtccagatc 840 gtgcgtcagc agtcctactc catcatgtcc attatcaagg aggaggtcct ggcttatgtg 900 gtgcagctgc ccatctatgg tgtgattgat accccctgct ggaaactgca cacttcccct 960 ctgtgcacca ccaacatcaa ggaaggctcc aatatctgtc tgactcgtac cgaccgtggt 1020 tggtactgtg acaatgccgg ttccgtctcc ttctttcccc aggctgacac ctgcaaagtg 1080 cagtccaacc gtgtgttctg tgacaccatg aactccctca ctctgccctc cgaagtgtcc 1140 ctgtgca aca ccgacatttt caattccaag tatgactgca agatcatgac ctctaaaacc 1200 gacatctcct cctccgtgat tacctccctg ggcgctattg tgtcctgcta tggtaaaact 1260 aagtgcaccg cctccaaacaa aaatcgtggt atcattaaga ccttttccaa tggttgtgat 1320 tacgtgtcca acaaaggtgt cgataccgtc tccgtgggta acactctcta ctatgtgaac 1380 aagctggagg gcaagaacct ctacgtgaaa ggtgaaccta tcattaacta ctatgacccc 1440 ctggtgtttc cctctgatct gtttgacg ct tccatttccc aagtgaatga gaaaatcaat 1500 caaagcctgg ctttcattcg tcgttccgat gaactgctcc ataacgtgaa tactggtaaa 1560 tccaccacca ac 1572

Claims (18)

delete delete delete delete delete It is a recombinant respiratory syncytial virus (RSV) F protein antigen.
The above RSV F protein antigen consists of the amino acid sequence of sequence number 3, or
An RSV F protein antigen characterized by comprising an amino acid sequence of sequence number 5. delete delete delete delete delete delete In claim 6, the RSV F protein antigen is characterized in that the RSV F protein antigen is encoded by a nucleic acid consisting of the sequence of SEQ ID NO: 25 or SEQ ID NO: 27. An RSV F protein antigen according to claim 6 or 13, characterized in that the RSV F protein is a soluble protein. An immunogenic pharmaceutical composition for treating or preventing respiratory syncytial virus (RSV) infection comprising the RSV F protein antigen of claim 6 or 13. A pharmaceutically acceptable vaccine composition comprising the RSV F protein antigen of claim 6 or 13 and a pharmaceutically acceptable carrier, wherein the RSV F protein is capable of inducing an immune response in a host. A pharmaceutically acceptable vaccine composition for treating or preventing respiratory syncytial virus (RSV) infection. A vaccine composition for treating or preventing respiratory syncytial virus (RSV) infection, further comprising an antigen adjuvant in claim 16. A vaccine composition for treating or preventing respiratory syncytial virus (RSV) infection, wherein the antigen adjuvant in claim 17 is aluminum.