EP0815244A1 - Thermitase variants having decreased adsorption and increased hydrolysis - Google Patents

Abstract

The present invention relates to Thermitase variants having a modified amino acid sequence of wild-type Thermitase amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type Thermitase (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase. The present invention also relates to DNA sequences encoding such Thermitase variants. The present invention also relates to compositions comprising such Thermitase variants for cleaning a variety of surfaces.

Description

THERMITASE VARIANTS HAVING DECREASED ADSORPTION AND INCREASED HYDROLYSIS

TECHNICAL FIELD

The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and DNA sequences encoding such enzyme variants

BACKGROUND

Enzymes make up the largest class of naturally occurring proteins Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains

Unfortunately, the efficacy level of these proteins in their natural bacterial environment, frequently does not translate into the relatively unnatural wash environment Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme

The ammo acid sequence of the protease determines the characteristics of the protease A change of the ammo acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the ammo acid sequence Several approaches have been taken to alter the wild-type ammo acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment These approaches include altering the am o acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions

Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces

Obiects of the Present Invention

It is an object of the present invention to provide Thermitase enzyme variants having improved hydrolysis versus the wild-type of the enzyme

It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants.

SUMMARY

The present invention relates to Thermitase variants having a modified ammo acid sequence of wild-type Thermitase am o acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region, wherein the modified am o acid sequence comprises different ammo acids than that occurring in wild-type Thermitase (i.e , substitution) at specifically identified positions in one or more of the loop regions whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase The present invention also relates to DNA sequences encoding such Thermitase variants The present invention also relates to compositions comprising such Thermitase variants for cleaning a variety of surfaces.

DESCRIPTION I. Thermitase Variants

This invention pertains to subtilisin enzymes, in particular Thermitase, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, "Thermitase variants") of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to DNA sequences encoding for such Thermitase variants.

The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds One type of protease is a seπne protease. A serme protease is distinguished by the fact that there is an essential serme residue at the active site

The observation that an enzyme's rate of hydrolysis of soluble substrates increases with enzyme concentration is well documente It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case (Brode, P.F. Ill and D. S. Rauch, LANGMUIR, "Subtilisin BPN': Activity on an Immobilized Substrate", Vol. 8, pp. 1325-1329 (1992)) In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varie (Rubingh, D N. and M. D. Bauer, "Catalysis of Hydrolysis by Proteases at the Protein-Solution Interface." in POLYMER SOLUTIONS, BLENDS AND INTERFACES. Ed. by I. Noda and D. N. Rubingh. Elsevier, p. 464 (1992)) Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).

While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface

The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils In Thermitase, certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 66- 73 form the first loop region; positions 103-115 form the second loop region; positions 134-141 form the third loop region; positions 162-171 form the fourth loop region; positions 191-195 form the fifth loop region; and positions 204-224 form the sixth loop region (position numbering analogous to positions in the amino acid sequence for wild-type subtilisin Thermitase (SEQ ID N0.1 )). It is believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the Thermitase molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the Thermitase molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).

As used herein, "variant" means an enzyme having an amino acid sequence which differs from that of wild-type.

As used herein, "mutant Thermitase DNA" means a DNA sequence coding for a Thermitase variant.

As used herein, "wild-type Thermitase" refers to an enzyme represented by SEQ ID NO:1. The amino acid sequence for Thermitase is further described by Meloun, B., Baudys, M., Kostka, V., Hausdorf, G., Frommel, C, and Hohne, W.E., FEBS LETT., Vol. 183, pp. 195-200 (1985), incorporated herein by reference.

As used herein, the term "Thermitase wild-type amino acid sequence" encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 66, 67, 68, 69, 70, 72, 73, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 134, 135, 136, 137, 138, 139, 140, 141 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 191 , 192,193,194, 195, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223 or 224.

As used herein, "more hydrophilic amino acid" refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1 ) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T.P., and Woods, K.R., "Prediction of Protein Antigenic Determinants from Amino Acid Sequences", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981 , incorporated herein by reference). TABLE 1

Amino Acid Hydrophilicity Value

Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9). The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).

Therefore, for example, the statement "substitute Gin with an equally or more hydrophilic amino acid which is neutral or has a negative charge" means Gin would be substituted with Asn (which is equally hydrophilic to Gin), or Ser, Glu or Asp (which are more hydrophilic than Gin); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin. Likewise, the statement "substitute Pro with a more hydrophilic amino acid which is neutral or has a negative charge" means Pro would be substituted with Gin, Asn, Ser, Glu or Asp.

In one embodiment of the present invention, the Thermitase variant has a modified amino acid sequence of Thermitase wild-type amino acid sequence, wherein the wild-type amino acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region; whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type Thermitase

In a preferred embodiment of the present invention, the substituting ammo acid for one or more of the positions in one or more of the loop regions is with reference to Table 1 neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the ammo acid at the subject position in the wild-type ammo acid sequence A Substitutions in the First Loop Region

When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 66, 67, 68, 69, 70, 72 or 73

When a substitution occurs at position 66, the substituting ammo acid

When a substitution occurs at position 67, the substituting ammo acid

When a substitution occurs at position 68, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 69, the substituting ammo acid

When a substitution occurs at position 70, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 72, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 73, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser B Substitutions in the Second LOOP Region

When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114 or 115

When a substitution occurs at position 103, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.

When a substitution occurs at position 104, the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser , Thr or Val

When a substitution occurs at position 105, the substituting ammo

When a substitution occurs at position 106, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 107, the substituting ammo acid is Asp or Glu.

When a substitution occurs at position 108, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 109, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 110, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 111 , the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 112, the substituting amino acid is Ala. Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val.

When a substitution occurs at position 113, the substituting amino acid is Asn, Asp, Gin. Glu, Gly, Pro or Ser.

When a substitution occurs at position 114, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 115, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr. C. Substitutions in the Third Loop Region

When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 134, 135, 136, 137, 138, 139, 140 or 141. wherein

When a substitution occurs at position 134, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 135, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 136, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 137, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 138, the substituting amino acid is Ala, Asn, Asp, cys, Gin, Glu, Gly, His, Met, Pro, Ser or Thr.

When a substitution occurs at position 139, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 140, the substituting amino acid is Asp, gin, Glu or Ser. and

When a substitution occurs at position 141 , the substituting amino

D Substitutions in the Fourth Loop Region

When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 162, 163, 164, 165, 166, 167, 168, 169, 170 or 171

When a substitution occurs at position 162, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 163, the substituting ammo acid is Asp, Gin, Glu or Ser.

When a substitution occurs at position 164, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 165, the substituting am o acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 166, the substituting ammo acid is Asp, Gin, Glu or Ser

When a substitution occurs at position 167, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 168, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 169, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly or Ser

When a substitution occurs at position 170, the substituting am o acid is Asp, Gin, Glu or Ser. and

When a substitution occurs at position 171 , the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val. E Substitutions in the Fifth LOOP Region

When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 191 , 192, 193, 194 or 195.

When a substitution occurs at position 191 , the substituting ammo

When a substitution occurs at position 192, the substituting ammo

When a substitution occurs at position 193, the substituting am o acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr, Tyr or Val.

When a substitution occurs at position 194, the substituting ammo acid is Asp or Glu.

When a substitution occurs at position 195, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser. F Substitutions in the Sixth Loop Region

When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218 or 219, 220, 221 , 223 or 224.

When a substitution occurs at position 204, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr.

When a substitution occurs at position 205, the substituting amino acid is Asn, Asp, Gin, Glu, Gly or Ser.

When a substitution occurs at position 206, the substituting amino acid is Asn, Asp, Gin, Glu, Pro or Ser.

When a substitution occurs at position 207, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, Tyr or Val.

When a substitution occurs at position 209, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 210, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Pro, Ser, Thr or Val.

When a substitution occurs at position 211 , the substituting amino acid is Asp or Glu.

When a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr or Val.

When a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, or Ser.

When a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser.

When a substitution occurs at position 216, the substituting amino acid is Asp or Glu.

When a substitution occurs at position 217, the substituting amino acid is Asn Asp, Gin, Glu, Gly, Pro or Ser

When a substitution occurs at position 218, the substituting ammo acid is Ala Asn Asp, Cys Gin, Glu, Gly, His, lie, Leu, Met, Pro Ser, Thr or Val

When a substitution occurs at position 219, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, His, Pro, Ser or Thr

When a substitution occurs at position 220, the substituting ammo

When a substitution occurs at position 221 , the substituting ammo acid is Ala, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Met, Pro, Ser, Thr or Val

When a substitution occurs at position 222, the substituting am o

When a substitution occurs at position 223, the substituting ammo acid is Asn, Asp, Gin, Glu, Pro or Ser

When a substitution occurs at position 224, the substituting ammo acid is Asn, Asp, Gin, Glu, Gly, Pro or Ser G Preparation of enzyme variants

Example 1 Mutant Thermitase DNA

A phagemid ("TP") containing the wild type Thermitase gene is constructed The 2 8 Kbp Pvu II restriction enzyme fragment of plasmid pUC119, (Vieira, J and Messing, J , "Production of Single-Stranded Plasmid DNA", 153 METHODS IN ENZYMOLOGY 3-11 (1989)) is cloned into the Pvu II site of plasmid pUB110 (Bacillus Genetic Stock Center, Columbus OH 1 E9) The pUC119-pUB110 hybrid plasmid is named pJMA601 Into the 8amH1 restriction site of PJMA601 is cloned the polymerase chain reaction-amplified Thermitase gene gene giving TP Phagemid TP is transformed into Escherichia coli Ung" strain CJ236 and a single stranded uracil-contaming DNA template is produced using the VCSM13 helper phage (Kunkel, T A , J D Roberts and R A Zakour, "Rapid and efficient site-specific mutagenesis without phenotypic selection", METHODS IN ENZYMOLOGY, Vol 154, pp 367-382, (1987), as modified by Yuckenberg, P D , F Witney, J Geisselsoder and J McClary, "Site-directed in vitro mutagenesis using uracil-contaming DNA and phagemid vectors", DIRECTED MUTAGENESIS - A PRACTICAL APPROACH, ed M.J McPherson, pp. 27-48, (1991 ), both of which are incorporated herein by reference) A single primer site-directed mutagenesis modification of the method of Zoller and Smith (Zoller, M J and M Smith, "Oligonucleotide-directed mutagenesis using M13-derιved vectors an efficient and general procedure for the production of point mutations in any fragment of DNA", NUCLEIC ACIDS RESEARCH, Vol 10, pp 6487-6500, (1982), incorporated herein by reference) is used to produce all mutants (basically as presented by Yuckenberg, et al , 1991 , above). Oligonucleotides are made using an Applied Biosystem Inc 380B DNA synthesizer Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625) All mutants are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain BG2036 (Yang, M Y., E Ferrari and D. J. Henner, (1984), "Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Create an In Vitro-deπved Deletion Mutation", JOURNAL OF BACTERIOLOGY, Vol 160, pp 15-21 ) For some of the mutants a modified TP with a frameshift-stop codon mutation in the corresponding loop is used to produce the uracil template Oligonucleotides are designed to restore the proper reading frame and to encode for random substitutions at positions 66, 67, 68, 69, 70, 72, 73, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 134, 135, 136, 137, 138, 139, 140, 141 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 191 , 192,193,194, 195, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223 or 224 (equimolar and/or variable mixtures of all four nucleotides for all three bases at these codons) Mutations that correct for the frameshift-stop and produce a functional enzyme are identified by their ability to digest casein. The random substitutions are determined by DNA sequencing

Example 2 Fermentation The Bacillus subtilis cells (BG2036) containing a subtilisin mutant of interest are grown to mid-log phase in a one liter culture of LB-glucose broth and inoculated into a Biostat ED fermenter (B Braun Biotech, Inc., Allentown, Pennsylvania) in a total volume of 10 liters The fermentation media contains Yeast Extract, starch, antifoam, buffers and trace minerals (see FERMENTATION A PRACTICAL APPROACH, Ed. B McNeil and L M Harvey, 1990) The broth is kept at a constant pH of 7.0 during the fermentation run. Chloramphenical is added for antibiotic selection of mutagenized plasmid The cells are grown overnight at 37°C to an AQQQ of about 60 and harvested Example 3 Purification

The fermentation broth is taken through the following steps to obtain pure enzyme The broth is cleared of Bacillus subtilis cells by centnfugation, and clarified by removing fine particulates with a 100K cutoff membrane This is followed by concentration on a 10K cutoff membrane, and flow dialysis to reduce the ionic strength and adjust the pH to 5 5 using 0 025M MES buffer (2-(Λ -morpholιno)ethanesulfonιc acid) The enzyme is further purified by loading it onto either a cation exchange chromatography column or an affinity adsorption chromatography column and elutmg it from the column with a NaCI or a propylene glycol gradient (see Scopes, R K , PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, Spπnger-Verlag, New York (1984), incorporated herein by reference)

The pNA assay (DelMar, E G , C Largman, J W Brodπck and M C Geokas, ANAL BIOCHEM , Vol 99, pp 316-320, (1979), incorporated herein by reference) is used to determine the active enzyme concentration for fractions collected during gradient elution This assay measures the rate at which p-nitroani ne is released as the enzyme hydrolyzes the soluble synthetic substrate, succinyl-alanine-alanine-prolme-phenylalanine-p- nitroani de (sAAPF-pNA) The rate of production of yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration In addition, absorbance measurements at 280 nm are used to determine the total protein concentration The active enzyme/total-protein ratio gives the enzyme purity, and is used to identify fractions to be pooled for the stock solution

To avoid autolysis of the enzyme during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column Upon completion of the purification procedure the purity of the stock enzyme solution is checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined via an active site titration method using trypsm inhibitor type ll-T turkey egg white purchased from Sigma Chemical Company (St. Louis, Missouri) The measured conversion factors will show which changes made in the enzyme molecule at the various positions result in the enzyme variant having increased activity over the wild-type, against the soluble substrate pNA

In preparation for use, the enzyme stock solution is eluted through a Sephadex-G25 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.1 M Tris buffer (Tris(hydroxymethyl-aminomethane) containing 0.01 M CaCl2 and pH adjusted to 8.6 with HCI. All experiments are carried out at pH 8.6 in Tris buffer thermostated at 25°C. H. Characterization of enzyme variants

Example 4 Model Surface Preparation

Aminopropyl controlled pore glass (CPG) purchased from CPG Inc. (Fairfield, New Jersey) is used as a support for covalently attaching the sAAPF-pNA substrate purchased from Bachem, Inc. (Torrence, California). The reaction is carried out in dimethyl sulfoxide and (1-ethyl-3-[3- (dimethylamino)propyl] carbodiimide hydrochloride) (EDC) is used as a coupling agent. Upon completion (monitored by pNA assay), the excess solvent is removed, and the CPG:sAAPF-pNA is rinsed with dimethyl sulfoxide (DMSO) and doubly-distilled water. This is followed by oven drying with a N2 purge at about 70°C. The reaction scheme and preparation of the immobilized substrate are conducted as described by Brode, P.F. Ill, and D.S. Rauch, "Subtilisin BPN': Activity on an Immobilized Substrate," LANGMUIR, Vol. 8, p. 1325-1329, (1992), incorporated herein by reference.

The CPG surface will have 62,000 ± 7,000 pNA molecules/μm². The surface area will remain unchanged from the value of 50.0m2/g reported by CPG Inc. for the CPG as received. This suggests that the procedure used to add sAAPF-pNA to CPG does not damage the porous structure (mean diameter is 486 A).

Example 5 Surface Hydrolysis Assay

Using CPG:sAAPF-pNA, adsorption of an enzyme variant and hydrolysis of a CPG-bound peptide can be measured in a single experiment. A small volume of enzyme variant stock solution is added to a flask containing Tris buffer and CPG:sAAPF-pNA which has been degassed. The flask is shaken on a wrist-action shaker for a period of 90 minutes during which the shaker is stopped at various time intervals (for example, every 2 minutes during the early stages of adsorption hydrolysis - e.g., the first 20 minutes - and every 10 minutes towards the end of the experiment) The CPG:sAAPF-pNA is allowed to settle and the solution is sampled Both the experimental procedure and the calculation of the adsorption and hydrolysis are conducted as described by Brode et al . 1992. above

All enzymes are monitored for stability against autolysis and should show no appreciable autolytic loss over the time course of this experiment Therefore, enzyme adsorption can be determined by measuring solution depletion The difference between the initial enzyme variant concentration and the concentration measured at each individual time point gives the amount of enzyme variant adsorbed The amount of pNA hydrolyzed from the surface is measured by taking an absorbance reading on an aliquot of the sample at 410 nm The total amount of pNA hydrolyzed is calculated by adding the amount sampled and the amount remaining in the flask This value is corrected by subtracting the amount of pNA that is hydrolyzed by Tris buffer at pH 8.6 when no enzyme is present. This base-hydrolysis ranges from 7-29% of the total hydrolysis depending on the efficiency of the enzyme

Example 6 Soluble Substrate Kinetic Analysis

The rates of hydrolysis of the soluble substrate sAAPF-pNA are monitored by measuring the adsorbance increase as a function of time at 410 nm on a DU-70 spectrophotometer. The enzyme concentration is held constant and is prepared to be in the range of 6-10 nanomolar while the substrate concentration is varied from 90-700 μM sAAPF-pNA for each kinetic determination. An adsorbance data point is taken each second over a period of 900 seconds and the data are transferred to a LOTUS™ spreadsheet (Lotus Development Corporation, Cambridge, Massachusetts). Analysis for kinetic parameters is conducted by the standard Lineweaver Burk analysis in which the data in the initial part of the run (generally the first minute) are fit to a linear regression curve to give v₀. The v₀ and s₀ data are plotted in the standard inverse fashion to give KM and k_cat. 58

15

I. Example Thermitase variants

Thermitase variants of the present invention which have decreased adsorption to and increased hydrolysis of surface bound substrates are exemplified in Tables 2-36, below. In describing the specific mutations, the original amino acid occurring in wild-type is given first, the position number second, and the substituted amino acid third. TABLE 2 Loop 1 - Single Mutation Variants

GlnββAsn GlnββAsp GlnββGlu GlnββSer Asn67Asp Asn67Gln Asn67Glu Asn67Ser GlyβθAsn GlyβθGln Gly68Glu GlyβδPro Gly68Ser Asnβ9Asp Asn69Gln Asn69Glu Asn69Ser Gly70Asn Gly70Asp Gly70Gln Gly70Glu Gly70Pro Gly70Ser Gly72Asn Gly72Asp Gly72Gln Gly72Glu Gly72Pro Gly72Ser Thr73Asn Thr73Asp Thr73Gln Thr73Glu Thr73Gly Thr73Pro Thr73Ser 16

TABLE 3

TABLE 4

Loop 1 - Triple Mutation Variants

Ser70Asp + Gly72Gln + Thr^'73Gly

Glyβ7Ser + AsnβδGln + Ser70Asp

AsnββSer + Gly67Asn + Thr73Gln

AsnββGln + Ser70Asp + Gly72Asn

Ser70Glu + Gly72Gln + Thr73Ser i v e ^"7 Pre — AsnβδGlu - Thr73Ser As r. βSe r - Gly69Gln + Gly72Gln

Asr.< i βS e r - Ser70Asp - T r73Gln Giy69Asp - T r73Gin

9Prc Gly72Giu - Thr73Ser

Giv 7Gln Giy69Asn -•- Thr73Asn Asnc5Ser Gly69Asp -r Thr73Asn Gly69Asp Gly72Ser -*^■ Thr73Ser Asn66Asp Gly72Gln + Thr73Ser AsnββGin AsnβδSer + Thr73Asn AsncSSer Giyβ9Ser ^■*- Ser70Asp Asn68Gln Gly69Ser ^■*^■ Thr73Gln Give7Ser Glyβ9Pro + Gly72Glu Gly67Asn Gly69Ser + Thr73Asn Give7Gin Gl ^"?2Asn + Thr73Gln Give^"7Pro AsnβδGln + Gly69Pro Give9Asn Ser70Glu - Thr73Ser AsnββSer Ser70Glu - Gly72Asn Give9Gln Ser70Asp * Giy72Asn Give7Ser Ser70Glu + Thr73Gln Asnβ6Glu Gly67Gln + Thr73Pro Give7Ser Gly72Glu + Thr73Ser Glyό7Pro Giy69Gln + Gly72Asp AsnδβSer Gly67Glu ~ Thr73Pro Asn6βSer Giyβ7Glu T^* AsnβδSer Glyβ7Asn AsnβδGlu + Gly69Asp Asnβ8Glu Gly69Glu + Gly72Pro Asnβ8Glu Glyβ9Glu + Thr73Asn Glyβ7Glu Asn68Asp + Gly69Gln Glyβ7Glu AsnβδAsp + Thr73Gln Asnβ^βAsp Glyβ7Asp + Gly72Ser AsnββGl^'u Glv^β7Glu ~ Thr73Pro

TABLE 5

Loop 1 - Quadruple Mutation Variants

GlylOSSer

Serl09Asp

Serl09Glu

GlyllOAsn

GlyllOAsp

GlyllOGln

GlyllOGlu

GlyllOPro

GlyllOSer

ThrlllAsn

ThrlllAsp

ThrlllGln

ThrlllGlu

ThrlllGly

ThrlllPro

ThrlllSer

Trpll2Ala

Trpll2Asn

Trpll2Asp

Trpll2Cys

Trpll2Gln

Trpll2Glu

Trpll2Gly

Trpll2His

Trpll2Ile

Trpll2Leu

Trpll2Met

Trpll2Phe

Trpll2Pro

Trpll2Ser

Trpll2Thr

Trpll2Tyr

Trpll2Val

Thrll3Asn

Thrll3Asp

Thrll3Gln

Thrll3Glu

Thrll3Gly

Thrll3Pro

Thrll3Ser

Alall4Asn

Alall4Asp

Alall4Gln

Alall4Glu

Alall4Gly

Alall4His

Alall4Pro

Alall4Ser

Alall4Thr

Valll5Ala

Valll5Asn

Valll5Asp

Valll5Cys 20

Vail 15Gln

Vail 15Glu Vail15Gly Vail15Hιs Vail15Met Vail15Pro Vail15Ser Vail15Thr

TABLE 7

Loop 2 - Double Mutation Variants

Seri09Glu GlylOδGln GlyllOAsn GlyllOPro ThrlllGln GlylOδAsp Serl07Glu Alall4Ser Serl09Asp Glyl08Pro Valll5Gly Leul04Gln Thrll3Asn Alall4Gly Valil5Gln ThrlllGln Alall4Glu ValllSGly Thrll3Gln Serl07Asp Alall4Gln Thrll3Ser Alall4Gln Trpll2Ile Thrll3Asp ThrlllGlu Trpll2Tyr Thrll3Gln Valll5Asn AsnlOβSer ThrlllPro Valll5Met AsnlOβGln ThrlllGln Thrll3Gly Aspl05Glu Thrll3Gly Thrll3Glu Trpll2Met Serl09Asp 21

GlylOSGln + Aiall4Gly Leul04 et + ValllSGlu Leul04Gly + Alall4Ser Thrll3Ser + Valll5Ala Aspl05Glu + AsnlOβGln Vali03Thr + Aspl05Glu GlyllOGlu + Valll5Met Vall03Gly + GlyllOGlu Seri09Asp + GlyllOGln ThrlllAsp + Alall4Ser AsnlOβGlu + Alall4Pro Serl09Glu + Thrll3Gly GlylOδAsn + Trpll2Glu Vall03Pro + AsnlOβGlu Aspl05Glu + Trpll2Leu Leul04Cys + Aspl05Glu Leul04Cys + ThrlllSer Leul04Thr + Serl09Asp Vall03Asn + Valll5Glu Vall03Ser + Glyl08Glu

TABLE8

Loop 2 - Triple Mutation Variants

Vall03Asn - Trpll2Pro + Alall4Asp GlylOδAsn + GlyllOSer + Trpll2Gly GlyllOAsp + Trpll2Leu + Valll5Hιs Vail03Gly + Leul04Gly + GlylOδAsp Leul04Ala + ThrlllSer + Valll5Ala ThrlllSer + Thrll3Asn + Valll5Hιs Aspl05Glu + GlylOβGln + ValllδGly Leul04Gly + Alall4Ser + Valll5Ala Vail03His + Leul04Gly + AsnlOβGlu Vall03Ser + GlyllOGlu + Thrll3Pro Vall03Pro + GlyllOAsn + Alall4Glu Leul04Met + GlyllOAsp + ValllδPro Leul04Asp + ThrlllGln + Trpll2Ala Serl09Asp + GlyllOPro + Trpll2Asn Leul04Asp + Aspl05Glu + Trpll2Cys GlylOδGlu + Serl09Glu + Alall4Thr GiylOδAsp + Serl09Asp + ThrlllPro GlylOδGlu + Serl09Glu + Alall4Asn AsnlOβSer + Serl09Glu + GlyllOAsp AsnlOβGln + Serl09Asp + GlyllOAsp Vall03Asp + Leul04Asp + GlylOδPro Leul04Thr + Aspl05Glu + AsnlOβGlu Aspl05Glu + AsnlOβAsp + Thrll3Asn GlylOδGln + Thrll3Glu + Alall4Glu Leul04Ser + ThrlllGlu + Trpll2Glu AsnlOβAsp + Serl07Asp + GlylOδAsp Aspl05Glu + Serl09Asp + GlyllOGlu Trpll2Ala + Thrll3Asp + ValllδAsp Vall03Gln + Aspl05Glu + GlylOδAsp Leul04Gln + Aspl05Glu + Serl07Glu Serl07Asp + Seri09Asp + ThrlllGly Vall03Cys + Serl07Asp + Serl09Glu

TABLE9

Loop 2 - Quadruple Mutation Variants

VallC3Asp -r Leul04Asn + Thrll3Asn + Valll5Ala Vail03Asn -r GlyllOAsp + ThrlllAsn + Valll5Cys Vall03Ser - AsnlOβGlu + GlyllOGln i- ValilδMet e l04Val - ThrlllGln + Trpll2His + Alall4Ser Le l04Ser - Aspl05Glu + GlyllOGln + ThrlllAsn Vall03Ala + AsplOSGlu + AsnlOoSer + GlyllOAsn Vall03Glu - GlyllOSer + Trpll2Phe + Valll5Gly AsnlO^βGlu + Thrll3Asn + Alall4Pro + Valll5Asn Leul04Glu Aspl05Glu + GlylOδSer + GlyllOAsn Glyl08Glu + Serl09Glu + ThrlllAsn + Alall4Pro GlylOδGlu + Serl09Glu + ThrlllGln + Alall4Thr Leul04Ile + GlylOδPro + Serl09Glu + GlyllOAsp Vall03Glu + Leul04Asp + Trpll2Cys + Thrll3Ser Aspl05Glu + AsnlOβGlu + GlyllOPro + Thrll3Gly GlyllOGln - ThrlllGlu + Trpll2Glu + Thrll3Gly Serl07Asp + GlylOδAsp + Serl09Asp + Trpll2Val AsnlO^βAsp - Serl07Asp + GlylOδGlu + ValllδPro Aspl05Glu + Serl07Glu + GlylOδAsp + GlyllOSer Aspl05Glu + GlylOδAsp + Serl09Glu + ThrlllGln Aspl05Glu + GlylOδGlu + Serl09Glu + ThrlllPro Leul04Asp + Serl09Glu + Trpll2Asn + Alall4Gly Vall03Ser + Leul04Glu + GlylOδAsn + Serl09Glu Aspl05Glu + Serl09Glu + GlyllOGlu + Trpll2Gln Vall03Ser + Aspl05Glu + GlylOδGlu + Valll5Pro Aspl05Glu + Serl07Asp + Trpll2Leu + Thrll3Gly Leul04His + Aspl05Glu + AsnlOβSer + Serl07Asp Aspl05Glu + Serl07Glu + Serl09Asp + ValllδGln Aspl05Glu + Serl07Glu + Serl09Glu + Thrll3Gly Serl07Glu + Serl09Glu + ThrlllGln + Thrll3Gln Serl07Glu + Serl09Glu + Trpll2Val + ValllδPro Serl07Glu + Serl09Glu + Trpll2Ile + ValllδGln Aspl05Glu + Serl09Glu + GlyllOGln + ValllδPro Leul04Glu + Aspl05Glu + Serl07Glu + Alall4Gly Aspl05Glu + AsnlOβGlu + GlylOδPro + Serl09Asp eul04Asn + Aspl05Glu + GlyllOAsp + Alall4Gly Serl07Glu + GlylOδGln + Serl09Asp + GlyllOAsp GlylOδGln + GlyllOGlu + Trpll2Asp + Valll5Glu Vall03Asp + Leul04Ser + Aspl05Glu + ValllδGln Leul04Glu + AsnlOβGlu + GlyllOGlu + ValllδAsn Leul04Asp + GlyllOGlu + ThrlllSer + Trpll2Asp Serl09Asp + GlyllOGlu + Thrll3Asn + ValllδGlu Vall03Ser + GlylOδAsp + GlyllOGlu + ThrlllGlu Leul04Gly + AsnlOβGlu + GlylOδAsn + Serl09Glu Vall03Asp + Leul04Gly + ThrlllGlu + Valll5Met

TABLE 10

Loop 3 - Single Mutation Variants

Leul34Ala Leul34Asn Leul34Asp Leul34Cys Leul34Gln Leul34Glu Leul34Gly Leul34His Leul34Ile Leul34Met Leul34Pro Leul34Ser Leul34Thr Leul34Val Glyl35Asn Glyl35Asp Glyl35Gln Glyl35Glu Glyl35Pro Glyl35Ser Glyl36Asn Glyl36Asp Glyl36Gln Glyl36Glu Glyl36Pro Glyl36Ser Thrl37Asn Thrl37Asp Thrl37Gln Thrl37Glu Thrl37Gly Thrl37Pro Thrl37Ser Vall36Ala Vall36Asn Vall36Asp Vall3δCys Vall36Gln Vall36Glu Vall36Gly Vall3δHis Vall38Met Vall38Pro Vall38Ser Vall38Thr Glyl39Asn Glyl39Asp Glyl39Gln Glyl39Glu Glyl39Pro Glyl39Ser Asnl40Asp Asnl40Gln Asnl40Glu Asnl40Ser Serl41Asp Serl41Glu TABLE 11

Loop 3 - Double Mutation Variants

26

TABLE 12

Loop 3 - Triple Mutation Variants

Leul34Val + Thrl37Asp Glyl39Gln Leul34His + Glyl35Pro Glyl39Gln Thrl37Pro + Vail38Gln Asnl40Asp Thrl37Gln + Vail38Cys Glyl39Glu Thrl37Ser + Glyl39Glu Asnl40Gln Glyl35Gln + Thrl37Gln Asnl40Asp Glyl35Gln + Thrl37Gln Vall36Asp Leul34His + Glyl39Gln Asnl40Asp Leul34Ala + Thrl37Asn Glyl39Glu Vall38Pro + Glyl39Pro Asnl40Gln Glyl35Asn + Asnl40Ser Serl41Glu Leul34Ile + Glyl35Asp Vall36Cys Thrl37Gln + Glyl39Ser Asnl40Ser Leul34Ala + Glyl36Pro Asnl40Asp Glyl35Ser + Glyl39Asp Asnl40Ser Leul34Thr + Glyl35Asn Serl41Glu Leul34Gly + Thrl37Ser Vall36Ala Leul34Thr + Glyl35Gln Vall38Pro Glyl35Gln + Glyl36Asp Thrl37Ser Glyl35Ser + Vail36Thr Asnl40Asp Leul34Cys + Thrl37Ser Vall38Glu Glyl36Ser + Thrl37Asn Serl41Asp Leul34Thr + Glyl35Glu Glyl39Gln Leul34Thr + Glyl35Asp Thrl37Ser

TABLE 13

Loop 3 - Quadruple Mutation Variants

Leul34Met + Thrl37Pro + Asnl40Ser Serl 41Glu Leul34Asn + Glyl36Pro + Thrl37Ser Vail38Gln Leul34Gln + Glyl36Gln + Vall36Ser Glyl39Ser Glyl35Glu + Glyl36Asn + Thrl37Pro Asnl40Gln Leul34Ser + Glyl36Asn + Glyl39Gln Serl41G1U Leul34Gln + Vall36Pro + Glyl39Ser Serl41Asp Leul34Pro + Glyl35Asn + Thrl37Glu Asnl40Gln Glyl35Asp + Thrl37Gly + Vall3δGln Glyl39Ser Leul34Val + Glyl35Asp + Thrl37Asn Asnl40Ser Leul34Thr + Glyl35Glu + Glyl36Ser Thrl37Ser Glyl36Pro + Thrl37Gln + Glyl39Gln Asnl40Glu Leul34His + Glyl35Glu + Vall38Asn Asnl40Gln Leul34Gln + Glyl35Ser + Thrl37Asn Serl41Glu Glyl35Gln + Glyl39Asn + Asnl40Gln Serl41Asp Thrl37Gln + Vall38His + Glyl39Asn Serl41Glu Leul34Thr + Glyl35Gln + Vall38Thr Asnl40Asp Leul34Met + Glyl35Glu + Thrl37Ser Vail38Ser Vall36Thr + Glyl39Ser + Asnl40Ser Serl41Glu Leul34Met + Glyl36Pro + Vall3δCys Glyl39Pro Leul34His + Glyl36Pro + Thrl37Gln Glyl39Glu Glyl35Asn + Glyl36Gln + Vall3δCys Serl41Glu Leul34Asn + Thrl37Ser + Vall38Gln Serl41Asp Glyl35Gln + Glyl36Pro + Thrl37Asn Asnl40Ser Glyl35Asn + Glyl39Ser + Asnl40Gln Serl41Asp eul 3 Glyl3^βPro + Vail38Met Serl41Glu Leui3 Thrl37Gln + Vail3δPro Serl 1Asp Glyi3 Thrl37Gln + Glyl39Asn Asnl40Ser Leul3 Thrl37Ser + Glyl39Ser Asnl40Asp Glyl3 Glyl3βAsp + Vall3δMet Asnl40Ser Glyl3 Glyl3βAsp + Vall3δHis Glyl39Gln Leul3 Glyl3βGlu + Thrl37Glu Vail36Thr Leul3 Vall3δGln + Asnl40Glu Serl41Glu Glvl3 Thrl37Asn + Asnl40Glu Serl41Glu Glyl3 Thrl37Gly + Vall38Asp Glyl39Asp Leul3 Glyl35Glu + Glyl36Asp Thrl37Asp Vall3 Glyl39Asp + Asnl40Glu Serl41Asp Leul3 Glyl39Glu + Asnl40Asp Serl41Asp Glyl3 Glyl39Glu + Asnl40Glu Serl41Asp Leul3 Glyl39Asp + Asnl40Asp Serl41Glu Leul3 Glyl36Glu + Vall38Asp Asnl40Gln Glyl3 Thrl37Gln + Vall36Asp Glyl39Asn Glyl3 Thrl37Glu + Vall3δHis Glyl39Asp Leui3 Thrl37Gln + Vall36Glu Asnl40Glu Leui3 Glyl35Ser + Thrl37Glu Glyl39Glu Leul3 Glyl35Pro + Thrl37Asp Glyl39Asp Leul3 Glyl35Asn + Thrl37Asp Glyl39Glu Leul3 Glyl35Glu + Thrl37Asp Glyl39Gln Glyl3 Thrl37Ser + Glyl39Asp Serl41Asp Leul3 Thrl37Asn + Glyl39Glu Serl41Asp Leul3 Thrl37Pro + Glyl39Asp Serl 1Asp Leul3 Vall36Asn + Glyl39Glu Serl41Asp Glyl3 Thrl37Gly + Glyl39Glu Serl41Asp Glyl3 Vall3δAla + Glyl39Glu Serl41Glu Leul3 Glyl39Asp + Asnl40Ser Serl41Asp Glyl3 Thrl37Gly + Glyl39Glu Serl41Asp Leul3 Glyl35Asn + Glyl39Asp Serl41Glu Thrl3 Vall36Gly + Glyl39Asp Serl41Asp Glyl3 Thrl37Pro + Vall36Gln Glyl39Asp Thrl3 Vall38Glu + Glyl39Asp Serl41Asp Leul3 Vall38Glu + Glyl39Glu Serl41Glu

TABLE 14

Loop 4 - Single Mutation Variants

Glyl62Asn Glyl62Asp Glyl62Gln Glyl62Glu Glyl62Pro Glyl62Ser Asnl63Asp Asnl63Gln Asnl63Glu Asnl63Ser Alal64Asn Alal64Asp Alal64Gln

Alal64Glu

Alal64Gly

Alal64His

Alal64Pro

Alal64Ser

Alal64Thr

GlylβδAsn

Glyl65Asp

Glyl65Gln

Glyl65Glu

Glyl65Pro

Glyl65Ser

AsnlββAsp

AsnlββGln

AsnlββGlu

AsnlββSer

Thrlβ7Asn

Thrl67Asp

Thrl67Gln

Thrl67Glu

Thrl67Gly

Thrl67Pro

Thrl67Ser

AlalβδAsn

AlalβδAsp

AlalβδGln

AlalβδGlu

AlalβδGly

AlalβδHis

AlalβδPro

AlalβδSer

AlalβδThr

Prol69Asn

Prol69Asp

Prolβ9Gln

Prol69Glu

Prol69Gly

Prolβ9Ser

Asnl70Asp

Asnl70Gln

Asnl70Glu

Asnl70Ser

Tyrl71Ala

Tyrl71Asn

Tyrl71Asp

Tyrl71Cys

Tyrl71Gln

Tyrl71Glu

Tyrl71Gly

Tyrl71His

Tyrl71Ile

Tyrl71Leu Tyrl71Met Tyrl71Pro Tyrl7lSer Tyrl71Thr Tv l71Vai

TABLE 15

Loop 4 - Double Mutation Variants

TABLE 16

Loop 4 - Triple Mutation Variants

AlalβδGlu + Prol Tyrl71Ile Glyl62Pro + Alal Tyrl71Pro Glyl62Pro + Asnl Prol69Glu Alal64Gly + Alal Tyrl71Ser Alal 64Thr + Asnl Tyrl71Leu As l β3Gln + Thrl Prol69Ser AsnlόβAsp + Alal Tyrl71Pro Glyl 62Asn + Alal Tyrl71Ala Asnl όβGlu -r Thrl AlalβδPro Alal 64Pro + Alal Asnl70Asp Alal 64Thr + Glyl AlalβδAsp Alal 64Ser + Alal Asnl70Glu Alal 64His + Glyl Tyrl71Met Alal64Gln + Glyl AsnlββGln Glyl62Gln + Thrl AlalβδAsn AlalβδHis + Prol Tyrl71Glu Alal64Gln + Asnl Tyrl71Val Glyl β2Pro + Alal Asnl70Gln Thrl67Glu + Alal Asnl70Ser Thrl 67Ser + Prol Asnl70Ser Asnl 63Glu + Asnl AlalβδSer Asnl63Gln + Alal Tyrl71Ala Thrl67Ser + Alal Asnl70Asp Glyl65Asp + Asnl Tyrl71Pro Asnl63Asp + Alal Thrl67Ser Alal64Asp + Glyl AsnlββGln Glylβ2Ser + Alal Glyl65Asp Glyl65Asn + Asnl Tyrl71Glu Glyl 65Pro + Asnl Tyrl71Glu

TABLE 17

Loop 4 - Quadruple Mutation Variants

Alal64Pro + Thrl67Glu + Prol69Ser + Tyrl71Val Glyl65Asn + AsnlββGln + Prol69Gly + Asnl70Asp AsnlββSer + Thrl67Pro + AlalβδAsn + Asnl70Glu Alal^β4Gly + AlalβδAsn + Prol69Asp + Tyrl71Ala Alal64Gln + GlylβSGln + Thrl67Ser + Prol69Glu Asnl63Glu + Glyl65Ser + Thrl67Gln + AlalβδThr Giyl62Pro + Alal64Thr + Thrl67Ser + Prol69Gin Glyl62Gln + Asnl63Glu + GlylβSGln + Thrl67Pro Asnl63Ser + Prolβ9Gly + Asήl70Glu + Tyrl71Ser Asnlβ3Gln + Glyl65Ser + Asnl70Asp + Tyrl71Leu Alal64His + Glyl65Pro + Prol69Ser + Asnl70Gln Glylβ2Pro + AsnlββGln + AlalβδAsn + Asnl70Glu Asnlβ3Gln + Glyl65Pro + Thrl67Ser + Tyrl71Gly Asnl63Gln + Glyl65Asn + AsnlββSer + Tyrl71Cys Asnl63Asp + Glyl65Pro + AsnlββSer + Tyrl71Gly Alal64Pro + Thrl67Pro + Prol69Glu + Asnl70Ser Asnl63Asp + Alal64Glu + Prol69Gln + Tyrl71His Asnl63Glu + Alal64Glu + Alal68Gly + Tyrl71His Thrl67Asp + AlalβδGlu + Prolβ9Gln + Asnl70Ser Alal64Asp + GlylβδAsp + AlalβδAsn + Prol69Gln Glyl62Ser + Alal64Gly + Asnl70Asp + Tyrl71Glu Asnl63Gln + Thrl67Asn + AlalβδAsp + Prol69Glu Asnl63Ser + GlylβδPro + AlalβδAsp + Prol69Glu Glyl65Ser + Alalβ8Asp + Prol69Glu + Asnl70Ser AsnlββGln + AlalβδGln + Prol69Asp + Asnl70Asp Alal64Gly + Thrlβ7Gly + Prol69Glu + Asnl70Asp Glyl62Pro + Alal64Gly + Prolβ9Asp + Asnl70Glu Alal64Pro + Thrl67Glu + AlalβδAsp + Prol69Asp Glylβ2Asp + Asnl63Gln + AlalβδHis + Asnl70Asp Glyl62Glu + Alal64Ser + AsnlββSer + Asnl70Glu Glyl62Glu + Alal64Ser + AsnlββGln + Asnl70Glu Thrl67Asp + AlalβδHis + Prol69Glu + Tyrl71Pro Asnl63Glu + Alal64His + GlylβδAsp + AsnlββGln Asnlβ3Asp + Alal64Gln + GlylβδAsp + AsnlββSer Asnl63Glu + Alal64Asp + GlylβδGln + AlalβδAsp Glyl62Glu + Alal64Glu + AsnlββSer + Prol69Asp Alal64Glu + AsnlββAsp + Asnl70Ser + Tyrl71Gly Alal64Glu + GlylβδGln + AsnlββGlu + Tyrl71Cys Alal64Asp + AsnlββGlu + Thrl67Asn + Prol69Asn Thrl67Asp + Prol69Glu + Asnl70Glu + Tyrl71Ser AsnlββAsp + Thrlβ7Ser + Alal68Asp + Tyrl71His

TABLE 18

Loop 5 - Single Mutation Variants

Serl91Asp Serl91Glu Serl92Asp Serl92Glu Phel93Ala Phel93Asn Phel93Asp Phel93Cys Phel93Gln Phel93Glu Phel93Gly Phel93His Phel93Ile Phel93Leu Phel93Met Phel93Pro Phel93Ser Phel93Thr Phel93Tyr Phel93Val Serl94Asp Serl94Glu Thrl95Asn Thrl95Asp Thrl95Gln Thrl95Glu Thrl95Gly Thrl95Pro Thrl95Ser

TABLE 19

Loop 5 - Double Mutation Variants

Serl92Asp + Phel93Ser Serl92Asp + Phel93Met Serl91Glu + Phel93Asn Serl91Asp + Phel93Ile Serl91Glu + Phel93Gln Serl91Asp + Thrl95Ser Phel93Gly + Thrl95Asp Serl92Asp + Phel93Cys Serl91Glu + Thrl95Pro Serl91Glu + Phel93Met Phel93Thr + Thrl95Glu Serl92Asp + Thrl95Gln Phel93Asp + Thrl95Asn Serl91Asp + Phel93Gln Phel93Gly + Thrl95Pro Serl92Asp + Thrl95Ser Serl91Glu + Phel93Ile Phel93Gln + Thrl95Asn Serl91Glu + Thrl95Gly Serl91Glu + Phel93His Serl91Asp + Phel93Leu Serl92Glu + Phel93Pro Phel93His + Thrl95Glu Serl92Asp + Thrl95Gly Serl92Glu + Phel93Ala Serl92Glu + Phel93Val Serl __Asr - P . l93Le Pr.el93Tyr - T.-.rl95Ser Phel93Kιs + T.~.rl95Asr_ Serl92Giu - Tr.rl95Ser Pnel93Iie + Tr_rl95Glu Pnel93Vai - Tr.rl95Asn Pnel93Me + Tr.rl95Gln Serl91Asp ^•■- Tr.rl95Pro

Serl91Asp * Pπel93Ser Serl92Asp - Pnel93Ile Serl92Asp - Pπel93Hιs Serl92Asp ^■*^• Pnel93Gly Serl91Glu + Pnel93Ser Serl92Glu - Tr.rl95Gln Serl91Giu - Tr.rl95Ser Serl91Asp - Tr.ri95Asπ 5erl91Glu - Pnel93Vai Phel93Tyr - Th l95Asp Phel93Hιs - Thrl95Ser Serl92Glu - Phel93Ile Phel93Met * Tnrl95Gly Serl91Glu + Phel93Tyr Serl91Asp + Thrl95Gln Serl92Asp + Pnel93Asn Serl92Asp ^■*^■ Phel93Thr Serl92Glu + Phel93Cys Phel93Asp - Tr.rl95Gln Phel93Val - Tnrl95Gln 5erl92Glu - Pr.el93Thr Pnel93Leu - Tr_rl95Giu

TABLE20

Loop 5 - Triple Mutation Variants

Serl92Glu + Phel93Thr + Tnrl95Gly Serl91Glu + Phel93Hιs + Thrl95Ser Serl92Asp + Phel93Leu + Thrl95Asn Serl91Asp + Phel93Ala - Thrl95Ser Serl92Asp - Phel93Val + Thrl95Gln Serl91Asp + Phel93Ser - Thrl95Pro Serl91Asp - Phel93Iie + Thrl95Gly Serl92Glu + Phel93Hιs + Thrl95Gly Serl92Glu + Phel93Leu + Thrl95Asn Serl92Asp + Phel93Gly + Thrl95Gln Serl92Asp + Phel9311e + Thrl95Pro Serl91Glu + Phel93Asn - Thrl95Ser Serl92Asp + Phel93Met + Thrl95Asn Serl91Glu + phel93Gly + Thrl95Asn 5erl92Glu -t- ?hel93Kιs + Thrl95Asn Serl92Asp - Phel93Met + Thrl95Pro Serl91Glu + Phel93Leu + Tnrl95Asn Serl91Glu + Phel93Met - Thrl95Asn Serl92Glu + Phel93Ala + Thrl95Gln Serl91Glu + Phel93Tyr + Thrl95Gly Serl92Asp + Phel93Ala + Thrl95Gly Serl91Glu + Phel93Met + Thrl95Gly Serl92Glu - Phel93Thr + Thrl95Gln Serl91Asp 4^* Phel93Asn + Thrl95Gly Serl92Glu + Phel93Asn + Thrl95Ser Serl92Asp + Phel93Ser + Thrl95Gly Serl92Glu + Phel93Leu + Thrl95Pro Serl91Asp + Phel93Cys + Thrl95Gly Serl91Asp + Serl92Asp 4- Phel93Tyr Serl91Asp 4^* Serl92Asp + Phel93Gly Serl91Asp + Serl92Glu 4- Phel93Met Serl91Asp + Serl92Glu + Thrl95Gln Serl91Glu + Serl92Asp + Phel93Val Serl91Glu -r 3erl92Glu + Phel93Thr Serl91Glu + Serl92Asp + Phel93Thr Serl91Asp + Serl92Asp + Thrl95Gly Serl91Glu 4* Serl92Glu + Phel93Asn Serl91Asp + Serl92Glu + Phel93Ala Serl91Asp 4* Serl92Asp + Phel93Asn Serl91Asp -J- Serl92Asp +^• Phel93Pro Serl91Asp 4- Serl92Glu + Phel93Thr Serl91Asp 4* Serl92Asp + Thrl95Asn Serl91Asp + Serl92Asp + Thrl95Gln Serl91Glu + Serl92Asp + Phel93Asn Serl91Glu 4^* Serl92Glu + Phel93Gln Serl91Glu 4^* Serl92Asp + Phel93Leu Serl91Asp + Serl92Asp + Thrl95Ser Serl91Asp + Serl92Glu 4^* Phel93Ser Serl91Glu + Serl92Asp + Phel93Tyr Serl91Asp 4^* Serl92Asp 4- Phel93Thr Serl91Asp 4* Serl92Glu 4- Phel93Pro Serl91Glu + Serl92Asp + Phel93Ile Serl91Asp + Serl92Glu 4^* Phel93Tyr Serl91Glu + Serl92Glu + Phel93Tyr Serl91Asp + Serl92Asp 4- Phel93Ser Serl91Glu + Serl92Glu + Thrl95Pro Serl91Asp + Serl92Glu + Thrl95Ser Serl91Glu + Serl92Asp + Phel93Met Serl91Asp + Serl92Glu + Thrl95Pro Serl91Glu + Serl92Asp + Phel93Hιs

TABLE21

Loop 5 - Quadruple Mutation Variants

Serl91Asp + Serl92Glu + Phel93Gly + Thrl95Gln Serl91Glu + Serl92Asp + Phel93His + Thrl95Gly Serl91Glu + Serl92Glu + Phel93Cys + Thrl95Pro Serl91Asp + Serl92Glu + Phel93Cys + Thrl95Gln Serl91Asp + Serl92Glu + Phel93Thr + Thrl95Pro Seri91Glu f^* 3eri92Glu + Phel93Val + Thrl95Asn Ξerl91Asp - Serl92Glu + Phel93Val + Thrl95Asn Serl91Glu + Serl92Glu + Phel93Leu + Thrl95Ser Serl91Glu --- Serl92Asp + Phel93Cys -^■- Thrl95Gln Serl91Glu + Serl92Asp + Phel93Pro + Thrl95Pro Serl91Glu + Serl92Asp 4^* Phel93Ser + Thrl95Gly Serl91Glu + Serl92Asp + Phel93Pro + Thrl95Asn Serl91Glu + Serl92Glu + Phel93Ser 4^* Thrl95Pro Serl91Glu + Serl92Asp + Phel93Ala 4^* Thrl95Gly Serl91Asp + Serl92Glu + Phel93His 4- Thrl95Asn Serl91Asp + Serl92Glu + Phel93Asn + Thrl95Asn Serl91Glu + Serl92Glu + Phel93Gln + Thrl95Ser Serl91Glu + Serl92Glu + Phel93Thr + Thrl95Gln Serl91Glu + Serl92Glu + Phel93Asn + Thrl95Gln Serl91Glu + Serl92Glu + Phel93Tyr ^• Thrl95Ser Serl91Asp + Serl92Glu + Phel93Ile 4^* Thrl95Pro Serl91Glu + Serl92Asp + Phel93Val + Thrl95Gln Serl91Glu + Serl92Asp + Phel93Val 4^* Thrl95Asn Serl91Asp + Serl92Glu + Phel93Tyr 4^* Thrl95Ser Serl91Asp + Serl92Glu + Phel93Ala + Thrl95Ser Serl91Glu + Serl92Glu + Phel93His 4^* Thrl95Asn Serl91Asp + Serl92Asp + Phel93Pro 4^* Thrl95Asn Serl91Asp + Serl92Asp + Phel93Gln + Thrl95Ser Serl91Asp + Serl92Asp + Phel93Leu + Thrl95Asn Serl91Asp 4^* Serl92Glu + Phel93Val + Thrl95Gln Serl91Glu + Serl92Glu + Phel93Gly + Thrl95Gly Serl91Glu + Serl92Glu + Phel93Thr + Thrl95Pro Serl91Glu + Serl92Glu + Phel93Thr 4^* Thrl95Gly Serl91Glu + Serl92Asp + Phel93Ile 4- Thrl95Ser Serl91Glu + Serl92Glu + Phel93Ile + Thrl95Gly Serl91Asp + Serl92Asp + Phel93Pro 4^* Thrl95Pro Serl91Asp + Serl92Asp + Phel93Ile + Thrl95Gly Serl91Glu + Serl92Asp + Phel93Thr + Thrl95Gly Serl91Asp Serl92Glu + Phel93Ser 4^* Thrl95Gly Serl91Glu + Serl92Asp + Phel93Tyr + Thrl95Ser Serl91Asp ^■+^■ Serl92Asp + Phel93Thr + Thrl95Gln Serl91Asp + Serl92Glu + Phel93Ser 4- Thrl95Gln Serl91Glu + Serl92Glu + Phel93His 4^* Thrl95Ser Serl91Glu + Serl92Asp + Phel93Leu 4- Thrl95Asn Serl91Asp ^■+- Serl92Glu + Phel93Leu 4^* Thrl95Pro Serl91Asp + Serl92Asp + Phel93Ile 4^* Thrl95Pro Serl91Asp 4^* Serl92Asp + Phel93Tyr - Thrl95Ser Serl91Asp + Serl92Glu + Phel93Ile 4- Thrl95Gln Serl91Asp + Serl92Glu + Phel93Pro -t- Thrl95Ser Serl91Asp + Serl92Glu + Phel93Leu + Thrl95Gln Serl91Glu + Serl92Asp + Phel93Asn + Thrl95Ser Serl91Glu + Serl92Glu + Phel93Ser 4^* Thrl95Asn Serl91Asp + Serl92Asp + Phel93Glu + Thrl95Gln Serl91Asp + Serl92Glu + Phel93Glu -+^• Thrl95Pro Serl91Glu + Serl92Asp + Phel93Glu 4^* Thrl95Gln

TABLE 22

Loop 6 - Single Mutation Variants

Ala204Asn Ala204Asp Ala204Gln Ala204Glu Ala204Gly Aia204Hιs Ala204Pro Ala204Ser Ala204Thr Pro205Asn Pro205Asp Pro205Gln Pro205Glu Pro205Gly Pro205Ser Gly206Asn Gly206Asp Gly206Gln Gly206Glu Gly206Pro Gly206Ser Ser207Asp Ser207Glu Trp208Ala Trp208Asn Trp208Asp Trp208Cys Trp208Gln Trp208Glu Trp208Gly Trp208His Trp208Ile Trp208Leu Trp208Met Trp208Phe Trp208Pro Trp208Ser Trp208Thr Trp208Tyr Trp208Val Ile209Ala Ile209Asn Ile209AsD Ile209Cys Ile209Gln Ile209Glu Ile209Gly Ile209His Ile209Leu Ile209Met Ile209Pro Ile209Ser Ile209Thr Ile209Val Tyr210Ala Tyr210Asn Tyr210Asp Tyr210Cys Tyr210Gln Tyr210Glu Tyr210Gly Tyr210His Tyr210Ile Tyr210Leu Tyr210Met Tyr210Pro Tyr210Ser Tyr210Thr Tyr210Val Ser211Asp Ser211Glu Thr212Asn Thr212Asp Thr212Gln Thr212Glu Thr212Gly Thr212Pro Thr212Ser Tyr213Ala Tyr213Asn Tyr213Asp Tyr213Cys Tyr213Gln Tyr213Glu Tyr213Gly Tyr213His Tyr213Ile Tyr213Leu Tyr213Met Tyr213Pro Tyr213Ser Tyr213Thr Tyr213Val Pro214Asn Pro214Asp Pro214Gln Pro214Glu Pro214Gly Pro214Ser Thr215Asn Thr215Asp Thr215Gln Thr215Glu Thr215Gly Thr215Pro Thr215Ser Ser216Asp Ser216Glu Thr217Asn Thr217Asp Thr217Gln Thr217Glu Thr217Gly Thr217Pro Thr217Ser Tyr218Ala Tyr218Asn Tyr218Asp Tyr218Cys Tyr218Gln Tyr218Glu Tyr218Gly Tyr218His Tyr218Ile Tyr218Leu Tyr218Met Tyr218Pro Tyr218Ser Tyr218Thr Tyr218Val Ala219Asn Ala219Asp Ala219Gln Ala219Glu Ala219Gly Ala219His Ala219Pro Ala219Ser Ala219Thr Ser220Asp Ser220Glu Leu221Ala Leu221Asn eu221Asp Leu221Cys Leu221Gln Leu221Glu Leu221Gly Leu221His Leu221Met Leu221Pro Leu221Ser Leu221Thr Leu221Val Ser222Asp Ser222Glu Gly223Asn Gly223ASD Gly223Gln Glv223Glu Gly223Pro Gly223Ser Thr224Asn Thr224Asp Thr224Gln Thr224Glu Thr224Gly Thr224Pro Thr224Ser

TABLE23

Loop 6 - Double Mutation Variants

42

Thr212Gln - Ala219Glu Ala204Gly + Trp20SGln Ala204Thr - 3er21όAsp Thr212Gln ^■*^■ Tyr218Gln Ser207Glu + Leu221Met Thr215Pro + Gly223Gln Ala204Gly + Ser216Asp Tyr210Asp + Ala219His Ile209Gln + Tyr218Ala Ile209Ala + Ala219Pro Ala204Asn + Ser222Glu Tyr210Asp + Tyr213Ala Tyr218Leu + Thr224Glu Tyr218Asn + Ser220Glu Trp208Met + Ala219Pro Pro205Gly + Ser222Asp Leu221Val + Thr224Gln Trp208Ile + Pro214Ser Trp208Asp + Thr212Pro Ala204Thr + Gly223Gln

Pro214Asp + Thr217Pro Ser207Glu + Ala219His Thr217Asp + Tyr218His Trp208Ala + Thr217Ser

Thr215Gly + Tyr218Gln Thr217Ser + Leu221Ala Ala219Pro + Leu221His Pro205Gln + Leu221Met Tyr213Met + Leu221Ile Trp208Asn 4- Thr212Ser Ser207Glu + Thr224Gly Pro205Ser + Leu221Cys Ala204Gly + Tyr210Val Trp208Phe + Ser222Asp Ile209Thr + Ser222Glu Leu221Ile + Ser222Glu Tyr213Pro + Thr217Glu Ile209Thr + Ser216Glu Tyr210Cys - Ser222Asp Leu221Glu + Thr224Ser Tyr213Ala + Thr217Pr^'o Trp208Cys + Thr215Glu Ala204Ser + Thr217Ser Ile209His + Gly223Ser Ala204Asn + Trp208Tyr Gly206Pro + Ser222Glu Ala204Thr + Ser220Asp Ala219Thr + Thr224Pro Trp208Ile + Ala219Glu Ile209Gly + Thr215Ser Gly206Gln + Tyr210Glu Pro214Gln + Ser220Glu Ser207Glu + Thr217Asn Ala204Pro + Gly223Asp Ile209Cys + Ser222Asp Pro214Gly + Gly223Pro Ile209Met + Thr224Pro Pro214Glu 4- Thr217Ser Pro205Ser + Ser207Asp Tyr210Thr + Thr212Gln Gly206Gln + Tyr218Pro Tyr218Val + Ser222Glu Leu221Asn + Gly223Glu Pro205Gln + Tyr218Val Pro205Gln + Thr217Asn Trp208His + Ile209Ala Pro205Ser + Gly223Gln Pro214Ser + Thr217Gly Tyr218His + Ser222Asp Thr212Gln + Gly223Ser Thr215Glu + Ala219Thr Tyr213Gly + Tyr218Asp Thr212Gln + Tyr213Gln Tyr210Ile + Thr215Asp Ala204Asn + Gly223Pro Ser207Glu + Ile209Asn Thr215Gln + Ala219Asp Ser220Asp + Gly223Pro Pro205Ser + Tyr213Ala Trp208Gly + Ser222Glu Thr215Glu + Tyr218Ala Ala204Gln + Ala219Gln Pro205Asn + Tyr213Thr Tyr218Thr + Thr224Gln Ser222Asp + Thr224Ser Ala204Pro + Tyr213Cys Gly206Gln + Leu221Gly Ser216Glu 4- Thr224Ser Ser207Asp 4- Ala219Asn Trp208Leu + Ile209His Pro205Ser + Tyr213Val Thr212Gly + Thr215Asp

TABLE24

Loop 6 - Triple Mutation Variants

Gly206Ser + Thr212Ser + Ala219Glu Ala204Gln Tyr210Hιs + Thr224Glu Pro205Ser Thr212Pro + Ala219Ser Pro205Gln Tyr210Met + Thr217Pro Ala204Gly Ile209His + Ser21^βGlu Thr212Asn Thr217Ser + Thr224Asn Gly2O^βPro Tyr210Gly + Gly223Gln Pro205Gln Thr217Asp + Ala219Thr Pro205Ser Ile209Met + Ser216Asp Thr215Asn Ser216Glu -⁽- Thr224Ser Pro205Gln Leu221Thr + Ser222Glu Tyr2lOCys Ala219Asn + Leu221Thr Ala204Ser Gly206Ser + Ala219Gln Ile209Pro Ser216Asp + Leu221Cys Trp208Thr Tyr210Val + Thr212Ser Thr212Gly Pro214Asp + Gly223Gln Pro214Gln Thr215Ser + Thr224Glu Thr212Ser Pro214Gln + Leu221His Trp208Gly Ser220Glu + Gly223Ser Pro205Ser Ser220Asp + Leu221Thr Gly206Asn Thr215Pro + Leu221Ala Tyr213Leu Leu221His + Gly223Asp Ser207Asp Thr212Gly + Tyr218Cys Gly2OβGln Ile209Val + Ala219Thr Trp208Ile Tyr218Ile + Gly223Asn Ala204His Ile209Gly -t- Ser216Asp Trp208Leu Thr212Gly 4- Ala219Ser Thr212Gln Pro214Glu 4- Ala219Thr Pro205Gln Ser207Asp -J- Thr212Pro Pro205Ser Tyr210Pro + Tyr218Ile Leu221Ala Gly223Asn + Thr224Glu Ala204Thr Gly206Gln 4- Ser207Asp Ser2lβGlu Ala219Gln + Gly223Gln Pro205Gly Trp208Leu + Leu221Gln Thr217Gln Gly223Gln + Thr224Glu Thr212Gly Thr217Ser + Tyr218Ala Gly206Asn Gly223Asp + Thr224Asn Ala204Gly Tyr210Cys + Tyr213Thr Gly2OβSer Thr212Asn + Thr215Gly Ala204Asn Thr212Asn + Ser216Glu Gly2OβGln Tyr218Cys + Gly223Glu Ala204Ser Pro205Ser + Gly206Asn Ile209Gly Leu221Val + Thr224Pro Gly2OβAsn Trp208His + Thr217Gly Gly2OβSer Trp208Leu -t- Ser222Asp Pro205Gly Thr212Gln 4- Ser222Asp Ala204Thr Tyr213Gln 4- Thr224Pro Thr212Ser Thr217Ser 4- Gly223Asp Pro205Ser Tyr213Ile 4- Ser222Glu Ile209Pro Tyr210Asp + Ala219Pro Gly2OβGln Thr215Pro + Ser220Glu Trp208Cys Tyr210Leu + Thr215Gly Trp208Tyr Thr212Gln + Ser222Glu Gly2OβAsn Ser222Asp + Gly223Asn Pro205Gly + Gly206Asn + Leu221Cys Pro205Gln + Tyr213lle -t- Ser216GΪu Ala204His + Tyr210His + Ser220Asp Ala204Thr + Thr212Asn + Ala219Asp Ser207Glu + Tyr210Ala + Thr212Gly Tyr210Hιs + Thr217Gly + Leu221Met Ala204Thr + Ile209Met + Ser222Glu Trp208Pro + Ala219Pro + Ser222Asp Ala204His + Ile209Ser 4- Ser220Glu Pro214Ser + Leu221Glu 4- Gly223Gln Gly206Pro 4- Leu221Met 4- Gly223Asp Ala204His + Tyr213Thr 4- Ser222Asp Tyr213Ala 4- Leu221Met - Ser222Asp Trp208Leu 4- Thr212Ser 4- Pro214Asp Tyr213Ile 4- Ser216Glu + Thr217Pro Pro205Gln 4- Pro214Gln 4- Thr224Gly Tyr213Cys 4- Thr217Gly + Ser220Glu Gly2OβAsn - Thr212Gly 4- Tyr218Pro Gly206Pro + Ser21βGlu + Gly223Pro Thr212Gln + Thr215Gly + Ser222Glu Ala204Pro 4- Thr217Asp + Thr224Ser Pro205Ser + Thr212Pro -t- Ala219Asp Ile209Cys 4- Pro214Ser + Thr215Asp Ile209Cys 4- Ser220Asp 4- Gly223Gln Thr217Gly -J- Ser220Asp 4- Leu221Gln Ala204His 4- Pro205Asn + Trp208Glu Ala204Gln + Thr212Ser 4- Thr215Asp Pro214Asn + Ser220Glu 4- Leu221Gly Ile209Gly 4- Thr215Asn -t- Ala219Pro Ser207Glu + Thr217Gln 4- Thr224Ser Ala204Gly -t- Thr215Asn + Gly223Pro Ile209Ser 4- Tyr213Ile + Leu221Met Tyr210Thr 4- Pro214Ser - Ser222Asp Tyr210Leu + Thr212Ser 4- Leu221Asn Gly206Asn 4- Pro214Asp 4- Tyr218Met Gly2OβAsn 4- Pro214Gln 4- Ala219Asn Ser207Glu + Pro214Ser 4- Ala219His Ala204Asn + Trp208Ala + Ile209Gln Thr217Glu + Tyr218Leu + Gly223Asn Trp208Pro 4- Pro214Gly + Leu221Ala Gly206Pro + Thr215Ser + Leu221Cys Ala204Ser + Ile209Val 4- Thr224Asp Trp208Ile + Tyr210Pro ^■ Thr224Asp Thr212Gln 4- Thr215Asn + Thr217Asp Tyr210Ser + Tyr213Cys 4- Thr217Asn Thr212Gln + Ser222Asp -t- Gly223Gln Tyr210Asn + Thr217Glu 4- Tyr218Met Pro214Asn 4- Ala219Gln + Ser220Glu Pro205Ser + Trp208Met 4- Leu221Asp Ala204Asn 4- Tyr210Cys + Tyr213Asn Tyr218Leu + Leu221His + Ser222Glu Ala204His -t- Tyr218Ser + Ser222Asp Gly206Pro + Tyr213Asn 4- Pro214Asn

TABLE 25

Loop 6 - Quadruple Mutation Variants

Gly20 6Asn + Thr21 5Gln + Tyr218Ile + Leu221Ile Tyr21 OGly + Thr212Gln + Tyr213Gly + Thr224Gly Pro20 5Gly -r Gly206Pro + Trp208Asn + Thr224Ser Ile20 9Thr + Pro214Asp + Thr217Asn + Tyr218Cys Ala20 4Ser + Tyr218His + Ala219Gln + Leu221Asn Ala20 4Ser + Pro205Ser + Tyr213Ser + Ala219Ser Ala20 4Gly + Thr212Gln 4- Ser216Asp + Gly223Ser Ser20 7Glu + Thr215Pro + Tyr218Gln + Leu221Met Ala20 4Gly Ser216Asp + Ala219His + Gly223Gln Ile20 9Gln Tyr21OAsp + Tyr218Ala + Ala219Pro Trp20 8Met Tyr218Leu + Ala219Pro + Ser222Glu Pro20 5Gly Leu22lVal + Ser222Asp 4- Thr224Gln Ala20 4Thr Thr212Pro + Ser222Glu + Gly223Gln Gly20 6Gln Ile209Gln Thr212Gln + Leu221His Ala20 4Ser Gly206Pro Ser220Asp 4- Leu221Ser Ala20 4Gly Thr212Gln Tyr213Thr + Thr224Gln Tyr21 OAla Pro214Ser Tyr218Val + Ser222Asp Ala20 4Gln + Pro205Ser Gly206Ser + Ser222Glu Trp20 8Cys + Pro214Gln Ala219Thr + Thr224Asp Ala20 4Gly + Gly20βSer Tyr210Asp + Leu221Gly Ala20 4Gln + Gly20βAsn Tyr210Gln + Ala219Asp Pro20 SSer Gly206Pro Ile209Val + Tyr213Pro Gly20 6Pro Pro214Glu Thr215Pro + Leu221Met Ser20 7Asp Trp208Met Tyr218Val - Leu221Pro Tyr21 OGly Thr212Pro Pro214Gly + Ser222Asp Gly20 6Pro Thr212Gln Thr217Ser + Ser222Asp Ala20 4Pro Trp208Met Ile209Val + Ser216Glu Trp20 8Ser Tyr218His Ala219His + Gly223Pro Aia204Gln + Trp208Ala + Tyr210Asn Ser222Glu Gly206Gln + Ser220Asp + Leu221Ala Thr224Ser Ile209Gln + Thr212Ser + Ala219Gln Ser220Glu Pro205Asn + Thr212Gly + Tyr218Ala Ser222Glu Pro205Gln + Trp208Cys + Tyr213Cys Pro214Asp Pro205Gly + Thr215Gln 4- Thr217Pro Gly223Ser Ala204His 4- Gly20βPro + Ser2lβGlu Leu221Ser Ser207Asp + Leu221Gly + Gly223Gln Thr224Asn Tyr21OHis Thr212Ser + Leu221Glu Gly223Ser Pro205Gly Thr215Asn + Gly223Asp Thr224Gly Trp208Phe Tyr213Met + Thr215Pro Ser220Glu Gly20βSer Trp208Phe 4- He209Ser Tyr213Gln Ala204Gln Pro205Ser + Thr212Pro Ala219Ser Ala204Gly + Tyr210Met + Ser2lβGlu Thr217Pro Gly206Pro + Thr212Asn + Thr217Gln Thr224Asn Pro205Ser + Ile209Met -J- Thr215Asn Thr224Ser Tyr21OThr + Thr212Pro + Tyr218Asn Ser220Glu Trp208Thr + Tyr210Val + Thr212Ser Leu221Cys Thr212Gly + Pro214Asp + Thr215Ser Gly223Gln Ser207Glu + Thr212Ser + Pro214Gln Leu221Pro Pro20 5Asn + Gly206Gln -t- Ser207Asp Leu221Thr Pro205Ser + Thr215Pro 4- Ser220Asp Leu221Cys Gly20 6Asn + Tyr213Leu + Leu221Ala Gly223Asp Ser207Asp + Thr212Gly + Pro214Asn Thr224Asn Ala204Pro Ser207Glu + Ile209Val Ala219Thr Gly206Gln Trp208Ile + Tyr218Ile Gly223Asn Ala204His Trp208Leu + Ile209Gly Ser216Asp Ala204Gln Thr212Gly 4- Pro214Glu Ala219Ser Ala204Asn Pro205Gln -J- Ser207Asp Thr212Pro Pro205Gly Trp208Leu 4- Ser2IβGlu Ala219Gln Trp208Tyr + Thr217Gln 4- Leu221Gln Thr224Glu Thr212Gly + Thr217Ser + Tyr218Ala + Gly223Asp Ala204Gly + Gly206Asn + Thr215Asn + Gly223Asp Gly20βSer 4- Tyr210Cys + Tyr213Thr + Thr215Gly Ala204Gln + Pro205Ser + Gly2OβAsn + Ala219Asp Ala204Ser Ile209Gly + Leu221Val + Thr224Pro Gly20β sn Ser207Glu 4- Trp208His + Thr217Gly Gly20βSer Trp208Pro 4- Ala219Thr + Ser222Asp Ala204Thr Tyr213Gln + Thr217Ser + Thr224Pro Pro205Ser Thr212Ser + Tyr213Ile + Gly223Asp Gly206Gln Ile209Asn 4- Thr215Pro + Ser220Glu Trp208Cys Thr212Gln + Thr215Gly + Ser222Glu Gly20βAsn Trp208Tyr 4- Ser222Glu + Gly223Asn Pro205Gly Gly2OβAsn 4- Leu221Cys + Ser222Asp Pro205Gln Tyr210His 4- Tyr213Ile + Ser220Asp Ala204His Trp208Asn + Thr212Asn + Ala219Asp Ala204His Gly206Pro 4- Leu221Met + Gly223Asp Ala204Ser Tyr213Thr 4- Leu221Met + Ser222Asp Tyr213Ile Pro214Gln 4- Ser2lβGlu + Thr217Pro Pro205Gln Tyr213Cys 4- Ser220Glu + Thr224Gly Gly20βAsn Tyr213Asn 4- Tyr218Pro + Ser220Glu Pro205Ser Gly206Pro + Tyr218Asp + Gly223Pro Ala204Pro Pro205Ser + Thr217Asp + Thr224Ser Pro205Asn - Trp208Ile + Thr212Gln Thr217Gly Ile209Cys - Pro214Ser + Ser220Asp Gly223Gln Ala204His - Pro205Asn + Trp208Glu Tyr210His Ile209Gly - Thr215Asn + Thr217Asn Ala219Pro Pro205Gly - Pro214Asn + Tyr218Ile Ser222Asp Ala204Ser * Pro205Ser + Thr212Asn Tyr213Ile Pro205Gln - Ser207Glu + Tyr2lOPro Thr215Ser Pro205Gln - Ser207Glu + Thr212Gln Tyr213Ser Ala204Ser - Ile209Asn + Tyr213Cys Ser216Asp Trp208Ala -r Ile209Gln + Thr217Glu Gly223Asn Trp208Pro + Pro214Gly + Tyr218Leu Leu221Ala Gly206Pro + Thr215Ser -t- Ser2lβGlu Leu221Cys Ala204Ser + Trρ208Ile + He209Val Thr224Asp Tyr21OPro + Thr212Gln + Thr215Asn Thr217Asp Tyr21OSer + Tyr213Cys + Pro214Asn Thr217Asn Thr212Gln + Tyr218Met + Ser222Asp Gly223Gln Pro205Ser - Trp208Met + Ala219Gln Leu221Asp Ala204Asn -t- Tyr210Cys + Tyr213Asn Tyr218Leu Ala204His - Pro205Gly + Tyr218Ser Ser222Asp Gly206Pro Tyr213Asn + Pro214Asn Tyr218Ser Ala204Pro Ile209His + Thr215Gln Leu221Thr Ala204Asn Gly206Gln + Thr212Gly Gly223Glu Thr215Gly Tyr218Ala + Gly223Glu Thr224Pro Pro205Gln Tyr213Gly + Ser220Asp Leu221Thr Pro205Gly Trp208His + Tyr2lOHis Leu221Gln Tyr21OLeu - Thr212Pro 4- Tyr218Gln Ala219Glu Gly206Gln - Thr212Gln + Tyr218His Gly223Asp Trp208Ile + Tyr213His + Thr215Ser Ser222Glu Trp208Val f Pro214Asn + Thr215Gly Leu221Asp Pro205Asn + Tyr213Met 4- Pro214Ser Thr224Ser Pro205Gly + Trp208Met + Ile209Leu Tyr213Met Pro205Gly + Thr217Glu + Tyr218Leu Ala219His Trp208Val + Tyr213Gly + Ser2lβAsp Leu221Thr Tyr21OGln Thr212Pro 4- Tyr218Gly Gly223Glu Pro205Gly Trp208Asp + Ile209His Tyr210Ala Trp208Ala Ile209Leu + Tyr2lOGlu Tyr213Cys Gly20βAsn Trp208Leu + Tyr213Val Ser2lβAsp Ala204Pro Pro205Gly + Thr217Glu Leu221Ala Trp208Asn Thr212Asn + Tyr213His Ser216Glu Ala204His Thr212Gly + Ala219Gln Leu221Thr Gly206Gln Thr212Pro + Tyr213Met Thr224Pro Trp208Thr Ile209Gln + Tyr213Ser Ser222Asp Ala204Asn Ile209Asn + Tyr213Pro Leu221Pro Pro205Gly + Tyr210His + Tyr218Ile Leu221Asn Ala204Pro + Tyr210Cys + Pro214Gln Thr215Pro Pro205Ser Tyr213Ser + Ser2lβAsp Thr217Gly Pro205Asn Trp208Ser + Ala219Glu Ser220Asp Tyr21O la Tyr218Met + Ala219Glu Ser220Asp Ala204Gly Pro205Gln + Pro214Asp Thr215Asp Tyr21OSer Thr215Asn + Ser222Glu Gly223Glu Gly206Pro Tyr213Val + Ser222Glu Gly223Asp Pro214Ξer Leu221Glu + Ser222Asp Gly223Gln Pro205Gln Ser216Asp + Thr217Glu Ala219Thr Trp208Leu + Thr212Gln + Ser220Glu + Leu221Glu

Gly206Gln + Ser220Asp + Leu221Asp + Gly223Asn

Thr217Asp -r Tyr218Asp + Gly223Ser + Thr224Gly

Pro205Gln * Tle209Ala ÷ Thr217Glu + Tyr218Glu

Pro214Gln + Thr215Asp + Ser216Glu + Thr224Pro

Ala204Kis + Ser207Glu + Tyr213Cys + Ser222Asp

Pro205Gly + Ser207Glu + Thr212Gln - Ser222Asp

Ser207Glu + Tyr2lOSer + Tyr218Leu + Ser222Glu

Ser207Glu + Tyr218Cys + Ser222Glu + Thr224Pro

Pro205Ser + Trp208Glu + Leu221Glu + Ser222Glu

Trp208Glu + Ile209Asn + Thr217Gly + Ser222Glu

Ser216Glu + Thr217Asp + Tyr218Glu + Thr224Gly

Trp208Asp 4- Tyr218Leu + Ala219Thr + Leu221Glu

Ala204His + Thr212Gly + Thr215Glu + Thr217Glu

Pro214Asρ + Ser *->IβAsp + Ala219His + Gly223Asn

Tyr210Pro -<- Pro214Asp + Ser216Glu + Tyr218Ile

Pro205Gln + Tyr2lOLeu + Pro214Glu + Thr217Asp

Ala204Ser + Ser207Glu + Pro214Ser + Gly223Asp

Ser207Asp + Thr212Pro + Thr217Gly + Gly223Asp

Ser207Glu + Pro214Asn + Ser220Glu + Ser222Asp

Pro205Ser + Thr217Pro + Ala219Glu + Leu221Glu

Trp208Gln + Tyr2lOGlu + Thr212Asn + Leu221Glu

Trp208Gln 4- Thr215Pro + Ser220Glu + Ser222Glu

Gly20βPro + Thr217Gly + Ser220Asp + Ser222Glu

Gly20βGln + Tyr2lOGly 4- Ser220Glu + Ser222Asp

Ser207Glu + Thr212Asn + Tyr218Pro + Leu221Asp

Ala204Asn + Ser207Glu + Trp208His - Leu221Asp

Tyr213Ile + Tyr218Leu + Ser222Asp + Thr224Glu

Gly206Gln + Tyr2lOMet + Ser222Asp + Thr224Glu

Ile209Ser + Tyr2lOHis + Ser222Glu + Thr224Asp

Ala204Ser 4- Tyr218Asp 4- Ala219Pro + Ser220Glu

Gly206Gln + Thr217Ser + Tyr218Glu + Ser220Glu

Pro214Asp 4- Tyr218Asp 4- Gly223Ser + Thr224Gly

Gly206Gln + Tyr2lOGly 4- Pro214Glu + Tyr218Glu

Trp208Asp + Thr217Gln + Ser220Asp + Gly223Asp

Ser207Glu + Tyr2lOAla + Thr215Asn + Ser220Asp

Ser207Glu + Thr217Gly + Ser220Asp + Leu221Gln

Ser207Glu 4- Thr217Gly 4- Tyr218Asn + Ser220Asp

Pro205Asn 4- Ser207Asp 4- Thr212Gly + Ser220Asp

Ser207Glu 4- Thr212Asn + Ala219Gly + Ser220Asp

Gly206Pro + Ser207Glu 4- Trp208Asp + Thr224Asp

Ser207Asp 4- Trp208Thr + Tyr218Gln + Thr224Glu

Ala204Thr + Ser207Asp + Leu221Ala + Thr224Glu

Ser207Asp 4- Ala219Glu + Ser220Asp + Gly223Ser

Ser207Glu + Tyr2lOGlu + Thr215Gln + Thr224Asn

Ala204Gln + Tyr2lOGlu 4- Thr212Asn + Ser222Asp

TABLE26

Loop 6 - Quintuple Substitution Variants

Gly206Asn + Tyr210Gly 4- Thr212Gln + Tyr218Ile +

Thr224Gly Ala204Asπ - Pro205Gly - Trp208Glu - Thr215Asn +

Gly223Pro Pro205Gln - Gly20βPro + Trp208Asn + Thr212Gln +

Thr224Ser Ala204Ser - Pro205Ser + Tyr213Ser + Thr215Ser +

Ala219Gly Ala204Gly + Gly206Gln + Thr212Pro + Ser220Asp +

Leu221Ser Pro214Ser + Thr215Gln + Tyr218Val 4- Ser222Asp +

Thr224Asn Ala204Pro - Ser207Asp + Tyr210Cys + Thr212Pro +

Thr215Pro Gly206Asn + Pro214Gln + Ala219Gly + Gly223Asn +

Thr224Asp Ala204Gln + Pro205Ser 4- Gly206Ser + Tyr210Gln +

Ala219Asp Gly206Pro + Ser207Asp + Trp208Met + Thr215Pro +

Leu221Pro Ala204Asn --- Pro205Gln + Tyr210Cys + Thr212Asn +

Leu221Ser Ala204Asn + Gly20βGln + Trp208Thr + Ile209Ser +

Ser222Asp Pro205Gln + Tyr213Leu + Thr217Glu 4- Tyr218Met +

Leu221Gln Ala204Pro -J- Trp208Met + Ile209Val + Tyr210Leu +

Ser216Glu Trp208Ser + Ile209Ser + Tyr218His + Ala219His +

Gly223Pro Trp208Pro + Ile209Thr + Thr212Gly + Thr215Gly +

Thr224Glu Ile209Val - Pro214Ser + Ser216Asp + Tyr218Met +

Leu221Met Gly20βGln + Ile209Gln + Ser220Asp + Leu221Gly +

Thr224Ser Pro205Gln + Trp208Cys 4- Thr212Asn + Tyr213Cys ^*

Pro214Asp Pro205Gln + Trp208Gln + Thr217Asp 4- Gly223Ser +

Thr224Gly Ser207Glu 4- Thr212Ser 4- Thr217Gly 4- Tyr218Gln +

Leu221His Tyr210His + Thr212Ser + Leu221Glu 4- Gly223Ser +

Thr224Pro Trp208Phe + Tyr213Met + Thr215Pro -t- Ser220Glu +

Thr224Gly Gly206Ser + Trp208Phe 4- Ile209Ser 4- Tyr213Cys +

Ser216Glu Ala204Gln 4- Pro205Ser + Tyr210His -J- Tyr218Pro +

Thr224Glu Pro205Gln 4- Gly206Gln 4- Leu221His 4- Ser222Asp +

Gly223Gln Ala204Gly - Ile209His 4- Tyr210Met + Ser216Glu +

Thr217Pro Gly20βPro * Ile209Ala + Tyr210Gly + Leu221Asp + Gly223Gln Gly206Asn + Tyr210Met + Thr215Glu + Gly223Asn +

Thr224Gly Gly20βGln + Ile209Pro + Ser216Asp + Leu221Cys +

Gly223Pro Trp208Thr f Tyr210Val + Thr212Ser + Pro214Asp +

Gly223Gln Thr212Gly - Pro214Gln + Thr215Ser + Leu221Gly +

Thr224Glu Ala204Thr + Thr212Ser + Thr217Ser + Gly223Asp +

Thr224Pro Pro205Ser + Ile209Pro + Tyr213Ile + Ala219Pro +

Ser222Glu Trp208Gly + Thr212Gln + Tyr218Gly + Ser222Glu +

Gly223Ser Gly206Pro + Ile209Pro + Tyr213Ser + Thr217Gly +

Tyr218Val Pro205Gln + Tyr213Cys + Pro214Gln + Ser220Glu +

Thr224Gly Gly20^βAsn + Thr212Gly + Tyr213Asn + Tyr218Pro +

Ser220Glu Ala204Pro + Pro205Ser + Thr212Gln + Thr217Asp +

Thr224Ser Pro205Asn + Trp208Ile + Thr212Gln + Pro214Ser +

Thr217Gly Ile209Cys + Thr217Gly 4- Ser220Asp + Leu221Gln +

Gly223Gln Ile209Thr + Thr215Gly + Ser216Glu + Tyr218Leu +

Ala219Pro Ile209Met + Pro214Gly + Thr217Gly + Tyr218Cys +

Leu221Met Pro205Gly + Pro214Asn + Tyr218Ile + Ala219Gly +

Ser222Asp Tyr210Thr 4- Thr212Ser 4- Pro214Ser 4- Leu221Asn +

Ser222Asp Gly206Asn + Tyr210Ile + Pro214Gln + Ala219Asn +

Leu221Val Pro205Asn + Thr215Ser + Ala219Ser + Leu221Ala +

Thr224Asp Gly206Pro + Pro214Asn + Thr215Gln + Tyr218Ser +

Leu221Thr Ala204Pro + Gly206Gln -t- Ile209Hιs + Thr212Gly +

Gly223Glu Pro205Gln + Tyr213Gly + Tyr218Ala 4- Gly223Glu +

Thr224Pro Pro205Ser 4- Pro214Gly + Thr217Pro 4- Ala219Thr +

Leu221Glu Ala204Pro 4- Pro205Asn + Ser207Asp + Tyr210Ala +

Pro214Ser Gly206Ser 4- Tyr213Thr + Ala219Gln 4- Ser220Glu +

Gly223Ser Pro205Gly 4- Thr212Asn 4- Thr215Pro 4- Ser216Glu +

Leu221Asn Ser207Glu - Ile209Ala + Thr212Asn + Thr215Gln +

Tyr218Met Tyr213Hιs - Thr215Gln + Thr217Asn + Ala219Glu +

Leu221Ser

^*V I I r Gin - Tyr213Asn - Thr215Ser + Tyr218Ala +

Leu221Ala Trp208Met + Tyr210Met + Tyr218Gln + Ser220Glu +

Thr224Ser Ala204Thr + Pro205Ser + Ile209Leu + Thr217Asn +

Thr224Pro Pro205Gly + Ile209Leu + Thr215Asn + Ser216Glu +

Gly223Ser Ile209Ser + Tyr213Leu + Thr217Asp + Tyr218Asn +

Ala219His Ala204Ser + Pro205Ser + Ser207Glu + Leu221Val +

Gly223Pro Ala204Hιs + Thr212Gly + Ala219Gln + Leu221Thr +

Gly223Glu Pro205Ser + Gly20βGln + Thr212Pro + Gly223Pro +

Thr224Pro Ala204Asn + Trp208Thr + Tyr213Pro 4- Leu221Pro +

Ser222Asp Pro205Gly + Ile209Met 4- Tyr218Met 4- Leu221Val +

Gly223Asn Thr212Gly + Tyr218Ile + Ala219Asn + Ser220Glu +

Thr224Pro Ala204Gln + Pro205Gln + Gly206Gln -*- Ile209Met +

Ser216Asp Ala204Thr -r Thr212Gly + Ala219Asn + Gly223Asp +

Thr224Asn Ala204Pro + Trp208His + Tyr213Leu 4- Thr217Ser +

Ser222Glu Pro205Ser + Ser207Asp 4- Ile209Asn + Tyr213Met +

Thr217Gln Gly206Pro + Trp208Ser + Thr212Gly + Tyr213Cys +

Gly223Glu Ala204Asn + Gly206Pro + Trp208Val + Pro214Asn +

Tyr218Glu Ile209Gly + Tyr210Ala + Tyr218Pro + Gly223Ser +

Thr224Ser Pro205Gln + Tyr213His + Tyr218Met 4- Ser220Glu +

Leu221Gly Ala204Asn + Tyr210Cys + Thr212Gln + Tyr218Leu +

Ser222Asp Gly206Ser + Ile209Leu 4- Thr212Gly + Pro214Gln +

Thr215Glu Ser207Asp + Pro214Ser + Thr215Asn + Thr217Asn +

Leu221Ser Pro205Gly + Trp208Thr + Thr217Gly + Ser220Glu +

Gly223Ser Gly206Ser + Trp208Glu + Tyr210Gln 4- Thr217Gln +

Leu221Pro Gly206Gln + Ser207Glu 4- Thr212Asn + Pro214Ser + Leu221Ala Ala2:.45er + Trp208Leu + Ile209Asn + Thr215Asp +

Thr224Asn Gly206Ser + Ile209Pro + Thr217Ser + Ser220Glu +

Gly223Gln Pro205Gln 4- Trp208Pro + Thr212Pro + Pro214Glu +

Ala219Ser Gly206Pro + Ser207Glu + Tyr210 et 4- Thr212Gly +

Tyr213Leu Ala204His + Tyr210Pro + Tyr213Ala + Thr217Glu +

Thr224Ser Trp20SCys + Ile209Met + Tyr210Ala + Tyr213Met +

Ala219Asn Trp208Thr + Tyr210Ala + Tyr218Met + Ala219Gl_: +

Ser220Asp Trp208Asn + Ile209Pro + Ala219Asp + Ser220Asp +

Gly223Ser Pro205Ser + Tyr213Thr 4- Pro214Asp + Thr215Asp +

Tyr218Val Ile209Ser + Tyr213Pro + Leu221Met + Ser222Asp +

Gly223Asp Ala204Thr + Thr217Gln + Tyr218Ile + Ser222Glu +

Gly223Asp Pro205Ser + Tyr210Ala + Thr215Asn + Ser222Glu +

Gly223Glu Tyr210Pro + Thr212Pro + Leu221Asn + Ser222Asp +

Gly223Asp Trp208Pro + Ile209Pro + Tyr213Ile 4- Ser222Asp +

Gly223Asp Gly206Gln + Tyr213Gly 4- Thr217Gly + Ser222Glu +

Gly223Glu Trp208Ser + Ile209Pro + Thr212Pro + Ser216Glu +

Thr217Asp Gly206Asn + Tyr218Met + Ala219His 4- Ser220Glu +

Leu221Asp Ile209Ala + Thr215Glu + Ser216Glu + Gly223Gln +

Thr224Asn Ile209Gly + Pro214Ser + Thr215Glu + Ser216Asp +

Tyr218Ile Ala204Ser + Trp208Val 4- Ile209Ser + Thr215Glu +

Ser2lβAsp Thr212Pro 4- Thr215Asp 4- Ser216Glu 4- Thr217Glu +

Thr224Ser Ala204His + Ser207Glu 4- Trp208His 4- Tyr210His +

Ser222Asp Ser207Glu + Tyr213Asn 4- Leu221Thr 4- Ser222Asp +

Thr224Gly Gly206Ser + Ser207Glu 4- Trp208Pro + Ala219Thr +

Ser222Asp Pro205Gly 4- Ser207Glu 4- Thr212Gln + Tyr213Gln +

Ser222Asp Pro205Ser + Ser207Glu + Tyr213His 4- Thr217Pro +

Ser222Glu Gly206Pro ^■+^■ Ser207Asp + Tyr213Ile -J- Tyr218Asn +

Ser222Glu Gly206Pro f Trp208Glu + Tyr210Cys + Pro214Gln +

Ser220Asp Tyr210Glu -<- Tyr213Gly + Tyr218Leu + Ala219Asp +

Thr224Ser Gly206Pro ÷ Ser207Asp + Leu221Asp + Ser222Glu +

Thr224Gln Tyr213Ser - Pro214Ser + Thr215Glu + Thr217Asp +

Tyr218Ala Ala204Asn + Tyr210Gln + Thr212Asn + Pro214Glu +

Ser216Asp Pro205Gln + Gly206Pro 4- Pro214Glu + Ser216Asp +

Thr224Ser Ser207Asp + Thr212Pro + Thr215Gly + Thr217Gly +

Gly223Asp Ser207Glu + Tyr210Val + Pro214Asn + Leu221Thr +

Gly223Glu Ser207Asp + Ile209Thr + Tyr210Met + Thr212Pro +

Gly223Asp Ser207Glu + Tyr210Ser + Tyr218Leu + Ser220Asp +

Ser222Glu Ser207Asp + Thr217Gln + Ser220Asp + Ser222Asp +

Thr224Asn Trp208Thr + Thr217Glu 4- Tyr218Ser + Ala219Glu +

Leu221Cys Pro205Gly + Thr212Ser + Ala219Gln + Ser220Glu +

Ser222Glu Gly206Pro + Trp208His + Tyr210Gly + Ser220Glu +

Ser222Asp Ala204Gln + Gly206Ser + Trp208Gln + Ser220Glu +

Ser222Glu Ala204Ser + Tyr213Thr + Ser220Asp 4- Leu221Met +

Ser222Asp Thr212Gly -J- Thr215Asp + Ser216Asp 4- Thr217Gly +

Tyr218Asp Ala204Asn + Tyr210Glu 4- Tyr213Ala + Ser220Glu +

Ser222Glu Ala204His + Gly206Pro + Ser216Glu -J- Tyr218Asp +

Leu221Ser Gly206Pro + Ile209Ser + Tyr210His + Ser222Glu +

Thr224Asp Trp208Met 4- Tyr218Leu + Ala219Pro + Ser222Glu +

Thr224Glu Trp208Val 4- Tyr210Leu + Thr212Asn + Tyr218Asp --

Ser220Glu Ala204Ser 4- Tyr213Gln + Tyr218Asp + Ala219Pro +

Ser220Glu Thr212Asn + Tyr213Gln 4- Thr217Ser + Tyr218Glu +

Ser220Glu Pro214Ser + Leu221Glu + Ser222Asp + Gly223Ser +

Thr224Asp Pro205Gly * Tyr210Cys + Ala219Asp + Ser220Glu -t- Ser222Asp Gly206Gln + Tyr210Gly - Pro214Glu + Thr217Pro +

Tyr218Glu Trp208Asp 4- Pro214Ser 4- Thr217Gln + Ser220Asp +

Gly223Asp ≤er207Glu + Tyr210Ala - Thr215Asn + Ser220Asp +

Leu221Ala Ser207Asp + Trp208Cys + Ile209Ala + Thr215Asn +

Ser220Glu Ser207Asp + Tyr210Met + Tyr218Asn + Ser220Glu +

Leu221Cys Ser207Glu + Tyr210Pro 4- Thr215Gln + Tyr218Cys +

Ser220Glu Ser207Glu + Thr212Ser + Pro214Gln + Ser220Asp +

Leu221Pro Pro205Asn + Ser207Asp + Thr215Pro + Ser220Asp +

Leu221Thr Trp208Glu + Ile209Asn + Thr217Gly + Ala219Asp +

Ser222Glu Pro205Asn + Ser207Glu 4- Tyr213Leu 4- Ser220Asp +

Gly223Glu Ala204Ser + Pro205Gly + Ser207Asp + Thr212Ser +

Thr224Asp Ala204Pro + Gly206Gln 4- Ser207Glu + Tyr218Asn +

Thr224Glu Gly20όSer + Ser207Glu 4- Tyr210Met + Tyr213Cys +

Thr224Asp Pro205Asn + Ser207Glu + Ile209Val 4- Tyr213Pro +

Thr224Glu Ser207Asp + Ile209Val + Tyr213Ala + Gly223Gln +

Thr224Glu Pro205Asn + Ser207Glu + Ala219Glu + Ser220Glu +

Gly223Gln Gly206Asn + Ser207Glu + Trp208Asp 4- Ala219Asp +

Thr224Pro Ala204Gly + Gly206Ser + Ser207Asp + Tyr210Asp +

Leu221Gly Ala204Pro + Ser207Glu + Ile209Thr + Tyr210Glu +

Tyr218Leu Ile209Thr + Tyr213Gly 4- Ala219Asp + Ser222Glu +

Gly223Asp Pro205Gln + Tyr213Gln + Tyr218Glu 4- Leu221Glu +

Ser222Asp Gly206Gln + Ser207Glu 4* Thr212Asn + Ser220Glu +

Thr224Glu Ser207Glu + Thr217Gly + Tyr218Asn + Ser220Asp +

Thr224Glu Ala204Thr + Tyr210Cys + Ser220Glu 4- Leu221Ser +

Gly223Glu Ile209Pro 4- Ser216Glu 4- Ala219Glu + Ser220Asp +

Gly223Asn Gly206Gln + Tyr210Met 4- Thr212Pro 4- Ala219Glu +

Ser222Asp Prc205Asn - Tyr210Glu + Thr217Asp -<- Tyr218Ala +

Thr224Pro Aia204Gly - Trp208Gln + Thr212Gln + 5er216Aso +

Ala219Glu Ala204Asn - Trp208Ala + Ser216Asp + Ala219Glu +

Thr224Gly Ala204Asn + Tyr210Asp + Thr215Glu + Thr217Glu +

Ala219Gly Trp208Leu ⁺ Ile209Gly + Thr215Asp + Thr217Glu +

Ser220Asp Thr212Pro + Tyr213Leu + Ser216Glu + Tyr218Glu +

Leu221Glu Thr217Asp + Ala219Glu + Leu221Asn + Ser222Glu +

Thr224Ser Pro205Ser ^■*- Trp208Glu + Tyr218Glu + Leu221Gly +

Ser222Glu Ser207Glu + Thr215Pro + Thr217Asp + Ser220Glu +

Gly223Pro Thr217Asp + Ala219Asn + Leu221Glu + Ser222Glu +

Gly223Asn Trp208Tyr + Ile209Asn + Thr217Glu + Leu221Glu 4-

Ser222Asp Ala204Gly + Tyr210Glu + Pro214Glu + Ser216Glu +

Thr217Asn Pro214Asp + Thr215Gln + Ser216Glu + Ser220Glu +

Leu221 et Ala204Gln + Tyr210Glu + Thr212Asn + Thr217Glu +

Ser222Asp Trp208Leu + Ile209Val + Thr215Asp + Thr217Glu +

Leu221Glu Gly206Pro + Ile209Val + Tyr213Pro + Thr215Asp +

Ala219Glu Pro205Asn + Ser207Glu + Tyr218Asp + Ser222Asp +

Thr224Gly Gly206Ser + Tyr210Asp + Pro214Asp + Thr215Pro +

Leu221Asp Tyr210Ser + Pro214Asp + Tyr218Cys + Ser220Glu +

Ser222Glu Ser207Asp + Ile209Ser + Thr212Ser + Pro214Asp +

Thr215Asp

TABLE27

Loop 6 - Sextuple Substitution Variants

Gly206Asn + Tyr210Gly + Thr212Gln + Tyr213Gly

Tyr218Ile 4- Thr224Gly Pro205Asn + Gly206Ser 4- Tyr213Gly 4- Ser216Asp

Thr217Ser 4- Thr224Asn Ala204Ser + Pro205Ser 4- Tyr213Ser + Tyr218His

Ala219Gln 4- Leu221Asn Ser207Glu + Thr212Gln + Thr215Pro 4- Tyr218Gln

Leu221Met 4- Gly223Gln Pro205Ser 4- Ser207Asp + Tyr210Val + Tyr213Leu +

Leu221Gln 4- Thr224Gly Ala204Ser + Trp208Cys + Ile209His + Thr215Glu +

Thr217Pro + Gly223Ser Ala204Gly + Ile209Asn + Tyr210Ile + Ser2lβAsp +

Ala219Asn + Leu221Met Ala204Gly + Pro205Gln * Pro214Glu + Thr215Ser +

Tyr213Asn + Thr224Ser Gly2OβAsn + Trp208Cys + Thr212Gly + Tyr213Gly +

Ser216Asp + Leu221His Trp208Pro + Ile209Gly + Thr215Ser + Ala219Thr +

Leu221Thr + Thr224Asp Pro205Gly + Gly206Gln + Ile209Gly + Thr212Asn +

Pro214Asn + Ser220Glu Pro205Gly + Trp208Leu ^■+^• Thr217Gln 4- Leu221Gln +

Gly223Gln + Thr224Glu Ala204Gly + Gly206Asn + Tyr210Cys + Tyr213Thr +

Gly223Asp + Thr224Asn Gly2OβAsn + Trp208His + Ile209Gly + Thr217Gly +

Leu221Val + Thr224Pro Ala204Thr + Thr212Gln + Tyr213Gln + Thr217Ser +

Ser222Asp + Thr224Pro Trp208Gly + Thr212Gln + Tyr213Leu + Tyr218Gly +

Ser222Glu 4- Gly223Ser Pro205Ser + Trp208Ser + Tyr213Asn + Tyr218Gln +

Ser222Asp + Thr224Gln Ala204His + Pro205Gly + Ile209Asn + Tyr213Gln +

Thr215Ser + Thr217Ser Ala204Thr + Gly206Asn + Trp208Leu + Thr215Ser +

Ser216Asp + Ala219His Gly206Ser + Thr212Gln + Tyr213Val + Tyr218Cys +

Ser220Glu + Thr224Gln Gly206Asn + Trp208Ile + lle209Ser + Tyr218Gln +

Gly223Glu + Thr224Gly Gly2OβAsn + Ser207Glu + Ile209Cys + Thr212Ser +

Thr217Pro + Ala219His Ala204Asn 4- Tyr210Cys + Tyr213Asn + Tyr218Leu +

Leu221His + Ser222Glu Gly206Pro + Tyr213Asn + Pro214Asn + Thr215Gln +

Tyr218Ser + Leu221Thr Ala204Pro 4- Pro205Asn + Gly206Gln + Ile209His +

Thr212Gly + Gly223Glu Pro205Gln + Tyr213Gly + Thr215Gly + Tyr218Ala +

Gly223Glu + Thr224Pro Ala204His + Ser207Glu + Trp208Gly + Ile209His +

Tyr213Cys + Thr215Ser Gly206Ser + Trp208His + Tyr213Thr 4- Ala219Gln +

Ser220Glu + Gly223Ser Tyr213His 4- Thr215Gln + Thr217Asn + Tyr218Ala +

Ala219Glu + Leu221Ser Ala204Asn + Pro205Ser + Gly206Asn + Ile209Leu +

Thr215Asp + Thr224Pro Pro205Ser + Ile209Gly + Tyr213Leu + Thr217Asp + Tyr218Asn + Ala219His Ala204Asn + Pro205Gln 4- Ser207Asp - Tyr210Val +

Thr212Gly + Leu221Thr Ala204Asn + Trp208Thr + Ile209Gln 4- Tyr213Pro +

Leu221Pro - Ser222Asp Ala204Ser - Gly206Ser + Tyr213Gly + Pro214Gln +

Tyr218Val + Ser222Asp Ala204Pro + Tyr210Cys + Tyr213Ser + Pro214Gln +

Thr215Pro + Thr217Gly Thr212Ser + Pro214Asp + Thr217Gly 4- Ala219Thr +

Leu221Pro + Thr224Gln Pro205Gln + Gly20βGln + Ser207Glu + Tyr210Gly +

Thr212Pro + Tyr218Gly Pro205Gln + Gly206Pro + Trp208Asn + Ile209Met +

Tyr213Ser + Ala219Asp Ala204Asn + Ser207Glu + Trp208Ala + Tyr213Asn +

Leu221Thr + Thr224Gly Ala204Pro + Trp208His + Ile209Asn + Tyr213Leu +

Thr217Ser + Ser222Glu Gly206Pro + Trp208Ser + Tyr210Val + Thr212Gly +

Tyr213Cys + Ala219Asp Pro205Gly + Gly206Gln + Ile209Cys 4- Tyr210His +

Thr215Gln + Ala219Thr Trp208Gly 4* Ile209Ser + Thr212Gln + Tyr218Met +

Leu221Ser + Gly223Ser Pro205Asn + Ile209Asn 4- Thr212Asn + Thr217Glu +

Gly223Pro + Thr224Ser Pro205Gly + Trp208Thr + Thr212Ser + Ser216Glu +

Thr217Gly + Gly223Ser Gly206Ser + Trp208Glu + Tyr210Gln + Thr215Gly +

Thr217Gln + Leu221Pro Gly206Ser + Ser207Glu 4- Trp208Leu + Thr212Asn +

Leu221Ala + Thr224Asn Ala204Asn + Pro205Gln + Gly206Gln + Trp208Ile +

Ser216Asp 4- Tyr218Cys Ala204Thr + Tyr210Cys 4- Tyr213Ser + Thr215Ser +

Ser216Asp + Ala219His Trp208Leu + He209Asn + Thr212Gly + Thr215Ser +

Tyr218Pro + Leu221Gly Pro205Gly 4- Gly20βGln 4- Tyr218Gly + Ala219Asn +

Leu221Gln 4- Thr224Ser Pro205Gln 4- Trp208Val 4- Tyr210Pro + Thr215Gly +

Ala219Ser 4- Thr224Glu Pro205Gly + Trp208Met 4- Ile209Ser 4- Thr212Asn +

Tyr218Ala + Ala219Thr Thr212Gly 4- Tyr213Asn 4- Pro214Ser + Ser216Glu +

Thr217Pro 4- Leu221Thr Ala204Gly 4- Trp208Pro 4- Ile209Pro 4- Tyr213Gln +

Tyr218Ser 4- Ser220Glu Ala204Gly + Gly206Asn + Ile209Met 4- Thr212Pro +

Ala219Glu + Leu221Met Pro205Asn + Gly206Pro 4- Trp208Leu 4- Tyr210Pro +

Thr217Gln 4- Tyr218G.lu Pro205Asn - Gly206Gln -r Tyr213Val + Ser220Asp +

Gly223Se +- Thr224Asn Ala20 Thr + Prc205Gly + Gly206Gln + Pro214Gln +

Leu221Val + Gly223Gln Pro205Ser + lle2θ^"9Gln + Thr212Pro + Ala219Gly +

Ser220Glu + Gly223Gln Ala204Gly + Gly206Gln + Ile209Leu + Thr212Asn +

Pro214Gln + Thr215Ser Gly206Asn + Trp208Ser + Thr212Ser + Pro214Asn +

Ser220Glu + Gly223Ser Ala204Kιs + Tyr213Leu + Pro214Asn + Thr217Pro +

Ser220Glu + Gly223Gln Gly206Pro + Ile209Cys + Pro214Gln + Thr217Ser +

Tyr218Cys + Ala219Gln Ala204Thr + Tyr210Ala + Thr215Gly + Thr217Gln +

Ala219Pro + Ser220Asp Ser207Asp + Ile209Thr + Thr212Gly 4- Thr217Pro +

Ala219Gly + Thr224Pro Ala204Gly + Ser207Glu + Thr212Asn 4* Pro214Ser 4-

Tyr218Ser + Ala219Ser Pro205Gln + Gly206Gln + Thr212Ser -J- Tyr213Ala +

Thr215Ser + Gly223Glu Pro205Ser + Tyr210Ala + Pro214Ser + Thr215Asn +

Ser222Asp + Gly223Glu Ile209Pro + Tyr210Pro + Thr212Pro + Leu221Asn +

Ser222Asp 4- Gly223Asp Pro205Ser + Thr212Ser + Tyr213Ile + Ala219Pro 4-

Ser222Glu 4- Gly223Asp Ala204Asn ^■+^■ Ile209Cys + Tyr213Cys 4- Tyr218Ala +

Leu221Asp + Ser222Glu Ile209Val + Thr212Asn + Tyr213Met + Thr217Asn +

Gly223Asp + Thr224Asp Pro205Gly + Ile209Asn + Thr212Asn + Ser216Glu +

Thr217Glu + Tyr218Gln Ala204His + Trp208Ser + Thr212Pro + Tyr213Pro +

Ser216Glu + Thr217Asp Pro205Ser + Ile209Ala + Thr212Gly + Ser2lβAsp +

Thr217Glu + Ala219Thr Pro205Ser + Trp208Met + Pro214Asn + Ala219Gln +

Ser220Glu + Leu221Asp Ala204Pro + Gly206Ser + Tyr213Ile 4- Ser220Glu +

Leu221Asp + Gly223Ser Gly206Pro + Tyr210Gly + Tyr213His + Thr217Glu +

Tyr218Asp 4- Leu221Asn Thr212Pro + Tyr213Ser + Thr215Asp 4- Ser216Asp +

Tyr218Pro + Gly223Gln Ser207Glu + Ile209Asn + Thr212Ser 4- Thr217Gly +

Ser222Glu 4- Thr224Gln Pro205Gly + Ser207Glu + Tyr210Ser 4- Pro214Gln +

Ser222Asp 4- Gly223Asn Gly206Pro + Ser207Asp ^•+ Trp208Ser + Pro214Ser +

Leu221Asn + Ser222Asp Gly20βGln + Ser207Asp + Ile209Cys + Tyr210Met + Thr217Asn Ser222Glu Gly206Pro + Ser207Glu + Ile209Pro + Tyr21CIle +

Leu221Asn 4- Ser222Glu Pro205Ser + Ser207Glu + Trp208His + Thr215Pro +

Leu221Asn + Ser222Glu Pro205Asn + Ser207Asp 4- Thr215Gln + Tyr218Ile +

Leu221Gly + Ser222Glu Ala204Pro + Ser207Asp + Tyr210Cys + Thr212Pro +

Thr215Pro + Ser222Glu Pro205Gly + Pro214Asp 4- Thr215Glu + Ser2lβAsp +

Thr217Pro 4- Gly223Gln Ile209Thr + Thr217Gly + Ala219Glu + Ser220Asp +

Leu221Glu + Gly223Ser Ala204Gln + Ser207Asp + Ile209Thr + Tyr218Asn 4-

Ser222Glu + Gly223Asp Pro205Ser + Ser207Glu + Pro214Gly + Thr215Asn +

Ser222Asp + Gly223Glu Pro205Asn + Gly206Pro + Trp208Asp + Thr212Gln +

Ser222Asp + Gly223Ser Gly206Gln + Pro214Asp + Thr215Asp 4- Thr217Asp +

Gly223Ser + Thr224Gly Ala204Hιs + Ile209Thr + Ser216Asp + Thr217Asp +

Tyr218Asp + Leu221Ser Thr212Asn + Pro214Gln + Tyr218Met + Ser222Glu +

Gly223Asp + Thr224Glu Ala204Gln + Gly206Pro -t- Tyr210Glu + Pro214Gly +

Ala219Asp 4- Leu221Gln Ala204Gly + Pro205Gln + Trp208Asp + Ile209Gln +

Pro214Gly + Leu221Glu Ala204Asn + Gly206Gln + Ser207Glu + Trp208His +

Leu221Asp + Ser222Asp Trp208Gly + Tyr213Ser + Pro214Ser + Thr215Glu +

Thr217Asp + Tyr218Ala Ala204Asn + Pro205Ser 4- Tyr210Pro + Pro214Asp 4- Gly206Gln + Ile209Leu + Thr212Gly + Pro214Asp -t-

Ser216Glu + Leu221His Pro205Gln + Trp208Glu + Tyr210Asp + Thr212Gln +

Tyr213Asn + Thr215Ser Ser207Asp + Thr212Pro + Thr215Gly + Thr217Gln +

Leu221Met + Gly223Asp Ala204Asn + Pro205Gln + Ser207Glu + Trp208Met +

Ala219Pro + Gly223Asp Ala204Thr + Ser207Asp + Thr212Gly + Ala219Asn +

Gly223Asp + Thr224Asn Ser207Asp 4- Pro214Asn 4- Thr215Gly + Tyr218Ala +

Leu221Ser + Gly223Asp Ser207Glu + Thr212Asn + Tyr213Gln + Tyr218Pro +

Ser220Glu + Leu221Asp Ala204Asn + Gly206Ser + Ser207Asp + Tyr210Thr +

Ser220Asp + Leu221Asp Gly206Ser + Thr212Asn + Thr215Pro + Tyr218Val +

Leu221Asp + Gly223Asp Trp208Met + Tyr210Thr + Thr212Asn + Ala219Gln +

Leu221Asp - Gly223Asp Gly206Ser + Ser207Glu -r Pro214Ser + Ala219Asn +

Ser220Glu - Ser222Glu Ala204Hιs - Ser2 7Glu - Ala219Ser + Ser220Glu ^J-

Leu221Hιs + Ser222Asp Pro205Ser + Ser207Glu + Tyr213His + Thr217Pro +

Ser220Glu + Ser222Glu Ser207Glu + Ile209Met + Tyr210Gly + Tyr213Leu +

Ser220Glu + Ser222Asp Ser207Asp + Tyr213Pro + Pro214Gln + Ser220Glu +

Ser222Glu + Gly223Pro Gly206Ser + Trp2θ^"8Gln + Tyr210Glu + Thr212Asn +

Thr215Gly + Leu221Glu Pro205Gly + Thr212Ser + Tyr218Leu + Ala219Gln +

Ser220Glu + Ser222Glu Trp208Phe + Thr212Asn + Pro214Ser + Ser220Glu +

Ser222Asp + Gly223Gln Ala204Gly + Gly206Pro + Tyr210Ser + Tyr218Cys +

Ser220Glu + Ser222Asp Pro205Ser + Trp208Leu + Tyr213Ala 4- Thr217Ser +

Ser220Asp + Ser222Asp Pro205Gly + Trp208His + Tyr210His + Ser220Glu +

Leu221Gln + Ser222Asp Pro205Gly f Thr212Gln + Ala219Thr + Ser220Glu +

Ser222Asp + Gly223Pro Ala204Ser -r Gly206Pro + Tyr210Gly + Ser220Glu 4-

Leu221Ser + Ser222Asp lle209Ser + Tyr210Ser + Thr212Asn + Tyr213Cys +

Ser220Asp + Ser222Glu Ala204Asn + Pro205Gly + Ser207Asp + Ile209Ser +

Tyr213Val + Leu221Glu Ala204Asn + Gly206Pro + Thr215Glu + Ser216Glu +

Thr217Asn + Tyr218Glu Gly206Pro + Ile209Ser + Tyr210His + Thr217Gly +

Ser222Glu + Thr224Asp Gly206Gln + Thr217Gly + Ser220Asp + Ser222Glu +

Gly223Glu + Thr224Ser Gly206Gln + Trp208Gln + Ile209Gln + Ser216Asp +

Tyr218Asp + Ala219Glu Gly206Asn + Thr212Pro + Pro214Ser + Thr217Glu +

Tyr218Glu + Ser220Glu Gly206Gln + Trp208Ser + Tyr210Asp + Pro214Gln +

Thr217Glu + Ala219Asp Gly206Ser + Ser207Glu + Trp208Pro + Tyr210Asp +

Ala219Thr + Ser222Asp Trp208Gly + Tyr210His + Thr212Ser + Pro214Glu +

Tyr218Asp + Leu221Ile Ala204Gln + Gly206Gln + Tyr210Gly - Pro214Glu +

Thr217Pro + Tyr218Glu Ala204His + Pro214Glu + Thr215Gln + Thr217Gln +

Tyr218Glu + Leu221Pro Ser207Asp + Ile209Cys + Thr215Pro + Tyr218His + Ala219Thr - 3er220Glu Prc2C5Asn - Ser2C7Glu + Trp208Tyr + Ile209Asn +

ThrΣ17Glr_ - Ser220Glu Ser207Asp + Pro214Ser + Thr215Asn + Thr217Asn +

Ser220Glu + Leu221Ser Pro205Gly + Ser207Asp + Tyr210Ser + Ser220Asp +

Gly223Pro 4- Thr224Ser Gly206Gln + Ser207Glu + Ile209Gln + Ser220Asp +

Leu221Ala + Thr224Ser Ala204Ser + Pro205Gly + Ser207Asp + Thr212Ser +

Thr215Gln + Ser220Glu Pro205Ser + Ser207Asp + Thr217Gln + Ala219Hιs +

Ser220Glu -i- Gly223Glu Ser207Glu - Thr215Gln + Tyr218Val + Ser220Asp +

Gly223Glu + Thr224Asn Pro205Gly 4- Ile209Pro + Tyr210Asp + Ala219Asp +

Ser222Asp + Gly223Ser Gly206Asn + Tyr210Gln + Pro214Asn + Thr215Glu +

Tyr218Glu + Leu221Ala Ile209Asn + Tyr210Asp + Thr212Gln + Ser222Glu +

Gly223Pro + Thr224Asn Ala204Gly -J- Gly206Gln + Ser220Asp + Leu221Asp +

Gly223Asn + Thr224Glu Gly206Pro + Thr212Pro + Thr217Pro ^■>^■ Tyr218Asn +

Leu221Glu + Thr224Glu Ile209His + Tyr210Leu + Tyr213Leu + Pro214Asp +

Thr217Asp + Ser220Glu Ile209Asn + Tyr210Ile + Thr212Asn + Thr215Gly +

Ser220Asp + Gly223Glu Gly206Gln + Tyr210Ala + Thr215Asn + Ser216Glu +

Thr217Asp + Ser220Asp Trp208Thr + Tyr213Pro + Tyr218His + Ala219Glu +

Leu221Cys + Ser222Asp Ala204Gly + Trp208Gln + Thr212Gln + Ser216Asp +

Ala219Glu + Gly223Ser Gly206Ser + Pro214Gly + Ser216Asp + Tyr218Pro +

Ala219Glu + Leu221Pro Pro205Asn + Tyr210Asn + Thr217Glu 4- Ala219Gln +

Ser220Glu + Gly223Gln Ala204Thr + Tyr213Ala -J- Pro214Gln + Thr215Gln +

Thr217Asp + Ξer220Glu Trp208Gln + Tyr210His 4- Thr217Asp + Tyr218Met +

Ser220Asp + Thr224Ser Ala204Asn + Gly206Pro + Tyr210Asp + Pro214Glu +

Ser220Glu 4- Thr224Asn Tyr213Ile + Ser21βGlu + Tyr218His + Ala219Glu +

Leu221Asp + Thr224Ser Pro205Ser + Trp208Glu - Tyr218Glu + Leu221Gly +

Ser222Glu + Thr224Gly Ser207Asp + Tyr213Met + Pro214C-ly + Thr217Gln +

Tyr218Asp + Ala219Glu Trp208Phe + Ala219Asp + Leu221Ala + Ser222Asp +

Gly223Asn + Thr224Glu Gly206Pro + Ile209Hιs + Pro214Glu + Ser220Asp +

Leu221Glu + Thr224Pro Trp208Tyr + Ile209Asn + Thr212Asn + Thr217Glu +

Leu221Glu + Ser222Asp Pro205Gly 4^* Ser216Glu + Thr217Glu + Ala219Gln +

Leu221Glu + Gly223Gln Pro214Asn + Thr217Pro 4- Ala219Asp + Ser220Asp +

Leu221Gln + Thr224Asp Ala204His + Gly206Asn + Ile209His + Pro214Glu + Ser207Asp + Tyr210Glu 4- Thr217Asp + Tyr218Ala +

Leu221Ile + Thr224Pro Thr212Gly + Tyr213Ala + Pro214Glu + Leu221Glu +

Ser222Asp + Thr224Ser Pro205Gln + Gly206Asn + Thr212Gln + Tyr218Glu +

Ser222Asp + Gly223Asp Pro205Gln + Thr212Gly + Tyr213Ala + Thr217Glu +

Tyr218Glu + Ser222Glu lie209Gln + Tyr213Ala + Pro214Glu + Ala219Asp +

Ser222Asp + Thr224Pro Ala204Gly + Tyr210Pro + Pro214Glu + Ala219Glu +

Leu221Asn 4- Ser222Asp Gly206Pro + Thr212Asn + Tyr213Thr + Pro214Asp +

Ser220Asp + Ser222Glu Ala204Thr + Tyr213Ser + Pro214Asp + Ala219Asn +

Ser220Asp + Ser222Glu Ala204His + Gly206Asn + Tyr210Pro + Pro214Glu +

Ser220Glu 4- Ser222Glu Ser207Asp + Trp208Met + Pro214Glu + Thr215Asp +

Thr217Ser + Gly223Pro Trp208Cys + Ile209Ser + Pro214Glu + Ser222Asp +

Gly223Asp + Thr224Gly Pro205Asn + Thr212Asn + Tyr213Leu + Thr215Asp +

Ser222Asp + Gly223Glu

TABLE28

Loop 6 - Heptuple Substitution Mutation Variants

Gly206Asn + Tyr210Gly + Thr212Gln + Thr215Gln

Tyr218Ile + Leu221Ile + Thr224Gly Ala204Ser + Pro205Ser + Ile209Met + Tyr213Ser

Tyr218His + Ala219Gln + Leu221Asn Ala204Gly + Trp208Gln + Thr212Gln + Ser216Asp

Tyr218Gln + Leu221Met + Gly223Ser Gly206Gln + Ile209Leu + Tyr210Ala 4- Thr212Gly

Tyr213Gly + Ser216Asp + Gly223Gln Ala204Gln + Pro205Asn 4- Gly206Asn + Trp208Ile

Tyr210Gln + Thr215Gln + Ala219Asp Ala204Asn + Pro205Gln + Ile209Ala + Tyr213Leu

Thr217Glu + Tyr218Met + Leu221Gln Pro205Gly + Ile209Gln + Thr212Ser + Ala219Gln

Ser220Glu + Leu221Cys + Thr224Ser Gly20βAsn + Trp208Cys + Thr212Gly + Tyr213Gly +

Ser21βAsp + Thr217Gly + Leu221His Ala204Gln + Pro205Ser + Tyr210His + Thr212Pro +

Tyr218Pro + Ala219Ser + Thr224Glu Alal Gly - Gly2OβAsn 4- Tyr210Cys + Tyr213Thr +

Thr215Gly + Gly223Asp + Thr224Asn Ala204Gln + Prc205Ser + Gly206Gln + Thr212Asn +

Tyr218Cys + Ala219His t^* Gly223Glu Ala204Ser + Gly206Asn + Ile209Gly + Pro214Gly +

Thr217Gly + Leu221Val + Thr224Pro Pro205Gly + Ile209Gln + Pro214Asn + Tyr218Ile +

Ala219Gly + Leu221Met + Ser222Asp Ala204Asn + Trp208Ala 4- Ile209Gln + Pro214Gly +

Thr217Glu + Tyr218Leu + Gly223Asn Gly206Asn + Tyr210Glu + Tyr213Gly + Thr217Gln +

Tyr218Leu + Ala219Thr + Thr224Ser Gly206Pro + Ile209His + Tyr213Asn + Pro214Asn +

Thr215Gln + Tyr218Ser - Leu221Thr Pro205Gly + Gly206Ser + Trp208Hιs + Tyr213Thr +

Ala219Gln + Ser220Glu + Gly223Ser Pro205Gln + Gly206Ser + Trp208Val + Tyr210Leu +

Thr212Asn + Tyr218Met + Ser220Glu Ala204His + Gly206Asn + Tyr210Ser + Pro214Ser +

Thr217Gln + Tyr218His + Ser222Asp Gly206Gln + Tyr213Asn + Thr215Gly + Ser216Glu +

Thr217Gly + Tyr218His + Gly223Asn Gly206Gln + Ser207Glu + Tyr210Gly + Thr212Pro +

Tyr213Met + Gly223Pro + Thr224Pro Ala204Asn + Trp208Thr + Ile209Gln + Tyr213Pro +

Thr217Ser + Leu221Pro + Ser222Asp Ala204Ser 4- Gly206Gln + Trp208Asn + Thr212Ser +

Ala219Gln + Ser220Glu + Leu221Asn Pro205Gly + Gly206Gln + Ile209Asn + Tyr213Val +

Thr215Gln + Thr217Ser + Ala219Thr Ala204Ser 4- Pro205Gln + Ile209Asn + Thr212Asn +

Thr217Glu 4- Gly223Pro + Thr224Ser Thr212Gly + Tyr213Ile + Pro214Gly + Tyr218Gly +

Ala219Pro + Leu221Asn + Thr224Glu Ala204Asn + Tyr210Asp + Thr212Pro + Tyr213Asn +

Thr217Asn + Tyr218Cys + Leu221His Ala204Gln + Ile209Cys + Tyr210Gln + Thr212Gly +

Tyr213Gln + Gly223Pro + Thr224Asp Pro205Gln + Gly20βGln 4- Trp208Thr + Tyr210Leu +

Thr215Gly + Thr217Gly + Tyr218Leu Tyr210Ala + Tyr213Val + Pro214Gln + Ala219Ser +

Leu221Cys + Ser222Glu + Thr224Asn Ala204Asn + Gly206Pro + Ser207Asp + Trp208His +

Ile209His + Pro214Gln + Leu221Gln Ala204Asn + Gly206Asn + Trp208Ile + Ile209Asn +

Ser216Glu + Ala219Pro + Gly223Asn Ala204Ser + Gly206Ser + Ile209Ala + Thr212Ser +

Pro214Gln + Tyr218Asp + Leu221His Gly206Asn + Ile209Pro + Tyr213Cys + Thr215Gln + Ser216Glu + Thr217Pro + Ala219His Ala204Gln + Trp208His + Tyr210Gly + Thr212Gln +

Tyr218Met + Ala219Asn + Ser220Glu Ala204Pro + Ser207Asp + Tyr213Val + Pro214Gly +

Thr215Gly + Tyr218Ala + Leu221Gln Pro205Asn 4- Gly206Pro + Trp208Leu + Tyr210Pro +

Pro214Asn + Thr217Gln + Tyr218Glu Pro205Asn + Gly206Gln 4- Tyr213Val + Ala219Gln +

Ser220Asp + Gly223Ser + Thr224Asn Gly206Pro + Ile209Cys + Tyr210Ser + Thr212Asn +

Tyr213Cys + Pro214Gln + Tyr218Cys Ala204Hιs + Trp208Asn 4- Thr212Pro + Pro214Gln +

Thr215Asn + Thr217Gly + Thr224Asp Pro205Ser + Gly206Ser 4- Tyr210Ser + Thr215Pro +

Ala219Asn + Leu221Met + Ser222Asp Ala204Ser + Gly206Gln + Trρ208Pro + Thr212Asn +

Thr215Glu + Tyr218Ile + Gly223Asn Ala204Ser + Pro205Asn + Trp208Tyr + Thr215Gly +

Tyr218Pro + Ser220Glu + Thr224Gly Ala204Asn + Tyr210His + Pro214Asn + Tyr218Pro +

Ala219Gln + Ser220Glu + Leu221Ile Ala204Gly + Gly206Asn + Ser207Asp + Thr212Asn +

Tyr213Leu + Thr215Pro + Gly223Pro Ala204Asn + Tyr210Met 4- Thr212Gln + Pro214Gly +

Ala219Gly + Ser222Asp + Gly223Gln Tyr210Pro + Thr212Gly + Tyr213Leu + Thr215Gly +

Thr217Gly + Ala219Glu + Ser220Asp Gly206Ser + Trp208Asn + Ile209Pro + Ala219Asp +

Ser220Asp + Gly223Ser + Thr224Gly Pro205Ser + Tyr210Cys 4- Tyr213Thr + Pro214Asp +

Thr215Asp + Tyr218Val + Leu221Ser Trp208His + Ile209Val + Thr212Pro + Tyr213Leu +

Thr217Ser + Ser222Glu + Gly223Glu Pro205Gly + Gly206Gln + Tyr210Asn + Tyr213Gly +

Thr217Gly 4- Ser222Glu + Gly223Glu Ala204Thr + Thr212Gln + Tyr213Gln -*- Thr217Ser +

Ser222Asp 4- Gly223Asp + Thr224Pro Pro205Gly + Ile209Cys + Tyr213Val + Pro214Gln +

Thr215Pro + Ser222Glu + Gly223Asp Trp208Ser + Ile209Ser + Ser216Glu + Thr217Glu +

Tyr218Gly 4- Ala219His + Gly223Pro Gly206Pro + Tyr210Gln + Thr212Pro + Tyr213Leu +

Ser216Glu 4- Thr217Asp + Leu221Pro Trp208Met + Ile209Gln + Tyr210Met + Tyr218Gln +

Ser220Glu + Leu221Glu + Thr224Ser Ile209Gln + Thr212Ser + Tyr213Val + Thr215Asp +

Ser216Glu + Tyr218Met + Ala219Pro Tyr213Pro + Pro214Gln 4- Thr215Asp + Ser216Glu +

Thr217Asp + Tyr218Met + Leu221Ala Ala204His 4- Pro205Gly + Ser207Glu + Trp208His +

Tyr210His + Tyr213Cys + Ser222Asp Gly206Pro + Ser207Asp + Trp208Ser + Pro214Ser +

Leu221Asn + Ser222Asp + Gly223Pro Ser207Glu - Trp208Tyr - Ile209Gly - Tyr213Thr +

Pro214Ser + Tyr218Met + Ser222Glu Pro205Ser + Ser207Asp + Trp208Ser + Thr212Gly +

Tyr213Asn + Tyr218Gln + Ser222Asp Ala204Thr + Gly206Asn + Trp208Thr + Thr212Gln +

Pro214Glu + Thr215Asp + Ser216Asp Ala204Thr + Gly206Asn + Ser207Glu + Tyr210Ile +

Leu221Thr + Ser222Glu + Gly223Glu Ala204His + Ser207Glu + Thr215Gln + Thr217Gln +

Tyr218Asn + Ser222Glu + Gly223Glu Ala204Gln + Trp208Asp + Tyr210Thr + Thr212Ser +

Thr217Asn t^* Ser222Asp + Gly223Asn Ile209Thr + Ser216Asp + Thr217Asp + Tyr218Asp +

Leu221Ser + Gly223Ser 4^* Thr224Gly Ala204Asn + Gly206Gln + Ser207Glu + Trp208His +

Thr212Ser + Leu221Asp + Ser222Asp Gly206Ser + Ser207Asp + Tyr210Val + Thr212Gln +

Tyr218Ile + Leu221Asp + Ser222Asp Ala204Ser + Gly206Pro + Pro214Gly + Thr215Asp +

Thr217Asp + Tyr218Pro + Gly223Pro Gly206Gln -t- Ile209His + Pro214Asp + Thr215Pro +

Ser216Asp + Leu221Gly + Thr224Ser Gly206Asn + Ser207Glu + Trp208Asp + Ile209Gly +

Tyr213Gly + Ser220Glu + Ser222Glu Pro205Ser + Ser207Asp + Ile209Asn + Tyr213Met +

Thr217Gln 4- Ala219His + Gly223Glu Pro205Gly + Gly206Gln + Ser207Glu + Ala219Pro +

Ser220Asp 4- Leu221Glu + Thr224Ser Trp208Met 4- Tyr210Thr + Thr212Asn -J- Ala219Gln +

Leu221Asp + Gly223Asp + Thr224Gln Pro205Ser + Ser207Glu + Tyr213His + Thr217Pro +

Ala219Ser + Ser220Glu + Ser222Glu Ser207Asp 4- Tyr210Met + Pro214Ser + Tyr218Asn +

Ser220Glu + Leu221Cys + Ser222Glu Ala204Pro + Ser207Glu + Pro214Gln + Ser220Asp +

Leu221His + Ser222Asp + Thr224Ser Ser207Glu + Ile209Met + Tyr210Gly + Tyr213Leu +

Thr215Gln + Ser220Glu + Ser222Asp Gly206Pro + Ser207Asp + Ile209Ser + Tyr210His +

Thr217Gly + Ser222Glu + Thr224Asp Pro205Asn + Tyr210Cys + Tyr213Gly + Thr217Asn +

Tyr218Met 4- Ser220Glu + Ser222Asp Ile209Gly + Thr212Pro + Thr215Asn + Thr217Gly +

Ala219His + Ser220Glu + Ser222Glu Ala204Gln + Gly206Gln 4- Thr212Pro + Ser220Glu -t-

Leu221Val + Ser222Asp + Thr224Gln Ala204Gln + Pro205Ser + Gly206Ser + Trp208Gln +

Thr215Pro + Ser220Glu + Ser222Glu Ser207Glu + Trp208Leu 4- Tyr210Ile + Thr212Gly +

Tyr218Cys + Leu221Asp + Gly223Asn Pro205Gly 4- Gly206Pro + Ser207Asp 4- Ile209Met +

Leu221Asp + Ser222Glu + Thr224Asp Pro205Gly + Thr215Asn + Thr217Pro + Tyr218Val + Leu221Thr + Ser222Glu + Thr224Asp Trp208Phe i- Tyr213Met + Thr215Asn + Tyr218Asp +

Ala219Pro + Ser220Glu + Thr224Gly Ala204Prc + Pro205Gly + Trp208Leu + Thr217Gln +

Leu221Asp + Ser222Glu + Thr224Asp :ie209Ala + Thr212Gln -i- Thr217Gln + Tyr218Thr +

3er220Glu + Leu221Asp + Gly223Glu Ala204Asn + Ile209Cys + Tyr213Cys + Tyr218Ala +

Ala219Glu + Leu221Asp + Ser222Glu Gly206Ser + Ser207Glu + Trp208Hιs + Tyr210Asp +

Tyr218Val + Ala219Thr + Ser222Asp Fro205Gly + Ser207Asp + Trp208Thr + Thr212Ser +

Thr217Gly + Ser220Glu + Gly223Ser Ser207Glu + Trp208Asn + Tyr210Pro + Pro214Asn +

Tyr218Ser + Ser220Asp -J- Gly223Ser Ala204Gln + Ser207Asp + Trp208Gly 4- Ile209Ala +

Thr212Asn + Ser220Glu + Gly223Pro Pro205Gln - Ser207Asp + Ile209Gln + Pro214Asn +

Thr215Gln + Ser220Asp + Thr224Gly Pro205Asn + Ser207Glu + Ile209Val + Thr212Gly +

Tyr213Pro + Ala219Asn + Thr224Glu Ala204Gly + Ser207Glu + Ile209Ala + Thr212Pro +

Tyr213Cys + Ala219Glu + Ser220Asp Pro205Ser 4- Trp208Glu + Thr212Asn -J- Tyr218Glu +

Ala219His + Leu221Glu + Ser222Glu Pro205Ser + Ile209Pro + Tyr210Asp + Thr212Ser +

Tyr213Ile + Ala219Pro + Ser222Glu Gly20βSer + Trp208Thr 4- Tyr210Ala + Pro214Asp +

Tyr218Met + Ala219Glu + Ser220Asp Ile209Leu + Pro214Glu + Thr215Pro + Ala219Glu +

Ser220Glu + Gly223Pro + Thr224Gly Trp208Tyr + Thr212Asn + Thr217Glu + Ala219Gly +

Ser220Asp + Leu221Glu + Ser222Asp Ala204Thr + Tyr210Pro + Tyr213Asn + Pro214Glu -

Tyr218Asp + Ser220Asp + Thr224Gly Gly206Pro + Thr212Pro + Pro214Gly + Thr215Pro +

Ser220Asp + Leu221Asp + Thr224Glu Thr212Asn + Pro214Gln + Tyr218Met + Ala219Asp +

Ser222Glu + Gly223Asp + Thr224Glu Trp208Gly + Tyr210Pro + Thr212Gly + Ser220Glu +

Leu221Met + Gly223Asp + Thr224Glu Gly206Gln + Tyr213Asn + Pro214Asn + Thr217Asp +

Ala219His 4- Ser220Glu + Leu221Asp Pro205Gln + Ile209Pro + Tyr210Asn + Tyr218Thr +

Ala219Glu 4- Ser220Asp + Gly223Asp Ala204Thr + Ser207Asp + Tyr210Asp + Tyr213Met +

Ala219Asn + Ser220Glu + Thr224Asp Pro205Ser + Trp208Pro + Ile209Pro 4- Tyr213Ile +

Ala219Asp 4- Ser222Asp + Gly223Asp Ala204Asn + Thr212Pro + Thr217Glu + Tyr218Asp +

Ser220Glu + Ser222Asp + Thr224Gln Gly206Pro + Ser207Asp + Trp208Val + Tyr210Thr +

Tyr213Ala + Ser220Asp + Thr224Asp 70

?ro205Gly 4- Ser207Glu 4- Tyr210Ile + Pro214Ser +

Thr2l7Gly + Ser220Asp - Thr224Glu Ala204Thr - Tyr210Cys - Thr212Pro ~ Tyr218Ile +

Ser220Glu + Leu221Ser * Gly223Glu Ala204Gln - Trp208Pro + Pro214Asn -^■- Ala219Thr +

3er220Glu + Gly223Asp + Thr224Gln Pro205Gln + Tyr213Gly + Thr215Gly - Tyr218Ala +

Ser220Asp + Gly223Glu + Thr224Pro Pro205Gly + Gly206Ser + Thr217Pro + Ala219His +

Ser220Asp + Leu221Cys + Gly223Asp Pro205Ser + Tyr210Thr + Thr212Asn + Tyr213Cys +

Pro214Ser + Ala219Asp + Ser222Asp Trp208Asn + Thr217Glu + Tyr218Cys + Ala219Glu +

Ser220Asp + Gly223Glu + Thr224Ser Prc205Ser + Gly206Pro + Trp208Pro 4- He209Ala +

Thr215Asp + Tyr218Asp + Ser220Asp Trp208Thr + Thr212Gly + Tyr213Ile + Thr215Asp +

Leu221Asp + Ser222Glu + Gly223Glu Ala204Gly + Pro205Gly + Tyr210Glu + Thr217Glu +

Tyr218Cys + Ala219Gly + Leu221Val Ser207Asp + Thr215Pro + Thr217Asp + Tyr218Ile +

Leu221Ala + Ser222Glu + Gly223Asp Gly206Pro + Trp208Glu + Tyr210Cys + Pro214Gln +

Thr217Asp + Ser220Asp + Gly223Gln Trp208Thr + Tyr213Pro + Pro214Ser + Ser216Asp +

Tyr218Met + Ala219Asp + Thr224Pro Ala204Asn + Trp208Ala + Tyr213Asn + Ser21βAsp +

Ala219Glu + Leu22lCys + Thr224Gly Gly20βSer + Tyr210Ser + Pro214Gly + Ser21βAsp +

Tyr218Cys + Ala219Glu + Leu221Pro Ala204Thr + Tyr210Gln + Thr212Gln + Tyr213Ala +

Pro214Gln + Thr217Asp + Ser220Glu Pro205Gln + Gly206Ser + Ser207Glu + Tyr210Ser +

Ser216Asp + Thr217Glu + Tyr218Glu Tyr210Met + Tyr213Met + Ser216Asp + Ala219Glu +

Ser220Glu + Leu221Cys + Ser222Glu Ala204Gln + Ile209His + Ser21βGlu + Thr217Ser +

Ala219Glu + Ser220Asp + Ser222Glu Ile209Ser + Tyr210Leu 4- Tyr213Leu + Pro214Asp +

Thr217Asp -J- Ser220Glu + Leu221Glu Ala204His + Pro205Gln + Pro214Asn + Tyr218Gln +

Ala219Asp + Leu221Glu + Thr224Asp Ala204Gly + Ser207Glu 4- Pro214Glu + Tyr218Pro +

Ser220Glu + Ser222Asp + Thr224Asn Ala204Pro + Pro205Gly + Trp208Glu + Ile209Ser +

Tyr210Asp + Ala219Thr -J- Thr224Glu Ala204Gly + Ser207Glu 4- Trp208Glu + Tyr210Asp +

Ser216Glu + Thr217Gln + Thr224Gly Trp208Ala + Tyr210Cys + Thr212Ser + Thr217Glu +

Ala219Gln 4- Ser220Asp + Ser222Glu Gly20βAsn + Ser207Glu + Trp208Glu + Thr212Ser +

Ser216Glu + Leu221Met + Gly223Glu Ala204Thr + Ser207Asp + Ile209Cys + Thr215Asp + Tyr218Ile - Ser220Asp + Leu221Asp Ξer23^~Asp + Thr212Pro + Pro214Gln 4- Ser216Glu -<-

Tyr213Val * Ser220Asp + Ser222Asp Ile209Pro + Thr212Gly ^■>^■ Tyr213Ser f Pro214Glu +

Thr215Glu -<- Ala219Gln + Ser220Glu Pro205Asn + Ser207Glu + Ile209Met + Pro214Ser +

Ser216Asp + Ala219Glu + Leu221Asp Ala204Thr + Ser207Glu + Tyr210Leu 4- Ser216Glu +

Ala219Asp + Leu221Asp + Gly223Asn Gly206Asn + Ser207Glu + Pro214Gly + Thr215Glu +

Thr217Glu + Tyr218Thr + Ala219Glu Gly206Gln + Ser207Glu 4- Tyr210Ala + Thr215Asn +

Thr217Asp + Ser220Asp + Leu221Ala Ser207Glu + Trp208Leu + Tyr213Val + Pro214Asp +

Ala219Glu 4* Ser222Glu + Thr224Pro Ala204Thr + Ser207Asp + Trp208Leu + Tyr210Asp +

Thr212Pro + Thr215Asp + Leu221Glu Pro205Gln + Ser207Asp + Ile209Ala + Thr217Asn +

Tyr218Glu + Ala219Asp + Gly223Asp Ser207Glu + Ile209Thr + Tyr213Gly 4- Ser216Asp +

Ala219Asp + Ser222Glu + Gly223Pro Gly206Ser + Ser207Asp + Ile209Ala + Thr212Gln +

Thr217Glu + Ala219Glu + Leu221Asn Pro205Asn + Tyr210Glu + Thr217Asp + Tyr218Ala +

Leu221Ile + Ser222Glu + Thr224Pro Ser216Asp + Thr217Asp + Tyr218Cys + Ala219His +

Leu221Glu + Ser222Glu + Gly223Asn Ala204Gln + Trp208Asp + Pro214Gln + Ser216Glu +

Thr217Gln + Ser220Asp + Gly223Asp Pro205Gln + Tyr210Leu + Tyr213Thr + Ser216Glu +

Tyr218Glu + Ser220Glu + Gly223Glu Pro214Gly + Thr217Asp 4* Tyr218Gly + Ala219Glu +

Leu221Asn + Ser222Glu + Thr224Glu Gly206Pro + Ser207Asp + Trp208Asp + Thr212Gln +

Thr217Asp + Gly223Ser + Thr224Glu Ala204Pro + Pro205Ser + Gly20βPro + He209Val +

Tyr213Pro + Thr215Asp + Ala219Glu Ala204Pro 4- Gly206Pro + Tyr210Val + Pro214Glu +

Ser220Glu + Ser222Glu + Thr224Pro Ala204Gly + Gly206Pro + Thr212Gln + Pro214Glu +

Ser220Glu + Leu221Val + Ser222Asp Ala204Asn + Gly206Gln + Ile209Ser + Tyr210Ala +

Pro214Glu + Ser220Glu + Ser222Asp Pro205Gln + Trp208Gln + Pro214Glu + Thr217Pro +

Leu221Met + Ser222Asp + Gly223Glu Ala204Thr + Pro205Asn + Pro214Asp + Thr215Pro +

Leu221Cys + Ser222Asp + Gly223Asp Ile209Pro + Tyr210Pro + Thr212Pro + Thr217Asp +

Leu221Asn + Ser222Asp + Gly223Asp Gly206Gln + Trp208Thr + Ile209Ser + Tyr213Met +

Thr215Glu + Ser222Asp + Gly223Glu Pro205Asn + Trp208Ser + Thr212Gly 4- Tyr213Asn +

Thr215Asp + Ser222Asp + Gly223Glu Tyr210Gly + Thr212Pro + Pro214Gly + Ser216Asp +

Ala219His + Leu221Glu + Ser222Asp Pro205Gly + Gly206Pro + Ile209His + Thr215Asp +

Ala219Gly + Leu221Glu + Ser222Glu Pro205Gln + Tyr210Gly + Thr212Asn + Ser216Asp +

Ala219Pro + Ser220Glu + Leu221Asp Gly206Ser + Trp208Glu + Tyr210Gln + Thr215Gly +

Ser216Glu + Thr217Glu + Leu221Pro Pro205Gln + Gly206Gln + Ser216Glu + Thr217Asp + Leu221Ala 4- Ser222Glu + Thr224Pro

Gly206Ser + Ser207Glu + Trp208Asp + Ile209Leu +

Thr215Asp + Ser216Glu + Tyr218Gln Tyr210His + Thr212Gln + Thr215Asp + Ser21βGlu +

Leu221Glu + Gly223Ser + Thr224Pro Ala204Ser + Pro205Ser 4- Gly206Asn + Thr215Asp +

Ser216Glu + Tyr218Val + Gly223Asp Ala204Gln + Gly206Asn + Ser207Glu + Trp208Pro +

Pro214Asp + Thr215Asp + Ser222Asp Ser207Asp 4- Tyr210Gln + Thr212Gln + Thr215Asp + Ser216Asp 4- Leu221Gly + Ser222Asp

Ile209Met + Tyr210Leu + Thr217Glu 4- Tyr218Thr +

Ser220Glu + Gly223Glu + Thr224Asn Pro205Ser 4- Ser207Glu + Trp208His 4- Thr215Pro +

Thr217Asp + Leu221Asn + Ser222Glu Ala204Thr + Ser207Glu + Thr212Gly + Tyr213Leu +

Pro214Asn + Ser216Glu + Ser222Glu Ala204Pro + Ser207Asp 4- Tyr210Leu + Thr212Gln +

Tyr213Thr + Thr215Glu + Ser222Glu

TABLE 29

Multi-loop Double Mutation Variants

Glylβ5Gln + Leu221Asp Glyl08Glu + Alal64Gly Leul04His + Thrl67Pro Alal64His + Ser220Glu Thrl95Gln + Ser222Asp Trp208His + Ala219Pro Gly 69Glu + AsnlOβGln Trpll2Cys + Thrll3Pro Gly 67Gln + Trp208Asp Alall4Glu + Leul34Ile Serl09Glu + Glyl36Ser GlyllOSer + Ser207Glu Vall03Ala + Thr212Ser Alall4Glu + Thrl67Pro Thrl95Glu + Ala204Thr Gly 67Ser + Thr217Glu Trp208Tyr + Ser220Glu Alal68His + Thr217Asp Alal68Glu + Tyr213Asn GlyllOPro + Ser220Asp Tyrl71Asn + Leu221Met Thr 73Asp + Leul34Ser Thrl37Asn ^■+^■ Thr217Pro Ser 70Asp 4- Alal68Thr Thrl67Glu + Tyr210Pro Thrl95Glu + Tyr210Gly Tyr210Gln + Thr217Glu Gly 67Glu + Valll5Cys Ser 70Glu 4- Ala219Thr Glyl36Pro + Leu221Ala Gly 67Ser + Thrl95Ser Phel93His + Ser222Asp Asn 68Glu 4- Tyr210Ser Glylβ5Asp 4- Prol69Ser Trp208Pro + Thr215Gly Thr 73Gly + Leul04Gly Thrll3Asn + Ser216Glu Gly 72Gln + Glyl39Asp Tyr210Ile 4- Leu221Gly Thr 73Pro + Tyrl71Asp Leul04Gln + Thrl37Gly Gly 69Pro 4- Tyr210Ala Thr217Gly + Gly223Glu Thrl95Asp 4- Tyr213Ser Gly 72Asn + Ala219Asn Vall38Pro + Tyr210Ala Tyr218Met + Gly223Asn Ser220Glu + Leu221Asn Trpll2Ala + Glyl39Asn Glyl35Glu + Leu221Gly Thrll3Asn + Thr217Ser Thrl95Asn 4- Thr217Asp Tyr210Val + Ser220Glu Vall38Gly + Ala219Asp Asnl^ββGlu + Leu221His Glyl^βδGln + Leu221His Thrll3Gln + Serl92Asp Asn ^ββAsp + Thr 73Ser Thrl67Gln + Thr217Gly Valll5Pro + Glyl35Ser Ala219Gln + Gly223Asp Trp208His + Thr217Asn Trp208Leu + Tyr210Val Glyl08Asn + Serl91Glu Serl07Glu + Tyr210Ala Asn 68Gln + Ser220Glu Trpll2Cys + Leu221Asn Glyl08Gln + Serl09Asp Vall03Met + Alal68His Alal64Pro + Serl92Glu Pro214Ser + Ser220Glu Thr215Asn + Thr217Gln Ser220Glu + Leu221Pro Glyl36Ser + Trp208Phe Thrl95Asp + Tyr210Gln Leul04Gln + Ala219Asp Gly 69Pro + Leu221Pro Gly 69Asn + Ala204Gly Glyl35Glu + Ala219Asn Glyl36Asn + Ser220Glu Glyl36Ser + Trp208Val Glyl39Asp + Asnl40Gln Trp208Cys + Tyr210Asn Glyl65Ser + Ser220Asp GlyllOSer + Trp208Thr Alall4Asp + Glyl62Asn Serl92Asp + Leu221Cys Vall03Ser + Glyl35Glu Asnl40Glu + Trp208Ala Thrll3Asp + Leu221Ser Serl09Asp + GlyllOSer Serl09Glu 4- Thrll3Gln Thr 73Gly + Glyl36Asp Alal64His + Leu221Asn Gly 67Pro + Vall03His Alall4Asn + Thr212Gln Asnl40Asp + Gly223Pro Glyl39Gln + Pro214Asn Aspl05Glu + Thrl95Gly Glyl35Pro + Ser207Glu Leul04Glu + Alall4Pro Asn ββGlu + Gly 69Pro Valll5Cys + Ser207Glu Asn 68Glu 4- Leu221Ala Asn ββSer + Gly 69Asp Serl09Glu + Phel93Pro Trp208Ile + Ser220Glu Pro214Ser + Thr224Glu Glyl65Pro + Ser220Glu Thrl37Gln + Tyrl71Glu Trp208Glu + Pro214Gly Ser216Asp + Leu221Met Glyl36Ser + Thr217Gln Serl41Glu + Tyr210Leu Trp208Thr + Gly223Ser Asn ββSer + Asnl70Gln Asn ββGln + Asnl70Glu Prol69Glu -J- Tyr210Thr Tyrl71Leu ^■ Leu221Asp Glyl39Glu 4- Leu221Pro Thr 73Pro 4- Leul34Gly Tyr210Glu + Leu221Ala Glyl39Ser + Tyr210Gly Asnl63Asp + Alal64Ser Asnl63Gln + Thr217Asp Asnl40Asp -t- Tyr218Ser Glyl08Ser + Alal64Gln Vall38Ser 4- Trp208Leu Thr217Ser + Thr224Asp Serl09Asp + Tyr218Ala Asn ββGlu + Phel93Val Asnl40Asp + Ala204Gly Vall38Ser + Ser220Asp Glyl08Asn + Tyr210Thr Glyl39Asn + Tyrl71Pro Thrll3Pro + Trp208Thr Serl41Glu + Leu221Gly AsnlOβGlu 4- Thrl67Gly Trpll2Ser 4- Ala219Ser Aspl05Glu 4- AsnlββGln Alall4Pro + Tyrl71Asn Thr217Gln + Ser220Asp Thrll3Gly + Tyr210Asp Tyrl71Cys + Tyr210Glu AsnlββAsp + Leu221Val Alall4His + Serl91Asp Gly 67Glu + Thr217Asn Asn ββGlu + Thr215Gln ThrlllAsn + Ser220Glu Trp208Gln + Leu221Ser Gly 69Gln 4- Alall4Thr Asnl63Asp 4- Leu221Ala Pro214Gly + Leu221Ile Thr217Gly + Ser220Asp Ala219Thr + Ser220Asp Tyr210Cys + Ser220Glu Vall38Asp + Gly223Pro Thrl95Asn + Leu221Gly Leul04Thr + Thr215Ser Gly 69Gln + Leul34Thr Glyl65Pro + Leu221Gly Thrlβ7Ser + Thr217Glu Thr212Pro + Thr217Glu AsnlββSer + Tyr218Asp GlylOδGlu + Tyrl71Gln Gly 67Asn + Tyr210Ile Valll5Cys + Pro214Gly Thrl37Asp + Tyr210Val Leul04Met + Trp208Gly Leul34Ala + Serl92Asp Trpll2Gln + Ala219Gln GlylOδGlu 4- Valll5His Thrll3Asn + Glyl39Pro Trp208Gln + Gly223Asn Ser207Glu + Trp208Gln Asn ββSer + Tyr210Asn Ser207Glu + Tyr210Pro Vall03Ala + Thr215Glu Vall03Asn + Tyr210Ala Asn ββSer + Trp208Phe Thr215Asn + Ala219Thr Glyl36Asn + Glyl39Asn Tyrl71Thr + Ser216Glu Leul04Glu + Tyr210Asn Serl09Glu + Thr217Asn Leul34Ile + AsnlββSer Tyrl71Gly + Thr217Gln Valll5Cys + Thr224Asp Prol69Ser -t- Thr215Asp Gly 72Gln + Glyl35Asn Tyr210Thr + Ser220Asp Asn 68Glu 4- Thrll3Ser AsnlOβGln + Thrll3Asp Serl09Glu + Trp208Asn Leul34Gln + Thrl37Glu Alall4Thr + Asnl70Glu Ala219Pro + Ser220Glu Tyrl71Val + Trp208Pro Glyl39Ser + Tyr213Ser Serl07Asp + Thrll3Asn Thrlβ7Gly + Leu221Met Tyrl71Gly + Ser207Glu Gly 67Ser + Thr217Ser Valll5Gly + Thr217Ser Asn ββSer 4- Trp208Ile Serl91Glu + Trp208Cys Thrl37Asp + Trp208Ser Leu221Gly + Ser222Glu Ser216Glu + Leu221Thr Glylβ2Pro + Ser207Glu Thr217Asp + Ala219Thr Leul04Cys + Ser207Glu Gly 69Gln + Ser220Glu Gly β9Gln + Vall38Asp Prol69Gly + Tyr210Leu Thr ^">3Asp + GlyllOSer Thr215Gly + Thr217Ser Glyl35Ser + Tyrl71Ser ThrlllAsn + Phel93Leu Trpll2Leu 4- Ser207Asp Leul04Glu + Trp208Ala Leu221Ile + Thr224Asp Glyl36Glu + Glyl65Asn Tyr210Glu + Thr217Pro Trpll2Phe + Pro214Asp GlyllOGln + Trpll2Met Asn 68Ser + Trp208Asp Gly 69Ser + Leul04Cys Thrl37Glu + Thr215Pro AsnlββGln + Thr217Glu Tyr210Thr + Thr212Ser Leul34His + Trp208Asp Serl41Glu + Tyr210Val Serl91Glu + Tyr210Leu Glyl39Pro + Ser207Asp Glyl62Ser + Tyrl71Met Thr 73Gln + Vall03Ala Tyrl71Val + Phel93Asp Leu221Thr 4- Ser222Glu Gly 69Asp + Alall4Gln Glyl08Glu + Trp208Met Gly 72Ser + Tyr210Gln Thrl37Asn + Trp208Asp Asnl40Asp + Phel93Thr Serl92Glu + Trp208Ala Asnl70Glu -t- Pro205Ser Tyr210Ala + Tyr213Ser Tyrl71Gln + Tyr216Ile Asn ββAsp + Leu221Cys Asnl63Gln + Gly223Asn Vall03Thr + Leul34Ser Thrl67Ser + Asnl70Asp Ser 70Glu + Glyl62Ser Tyrl71Gln + Serl92Glu Tyr210Val + Gly223Ser Thrl37Glu + Leu221Pro Gly 69Asn 4- Thr217Asp Pro205Ser 4- Tyr21δGlu Tyr210Met + Leu221Asn Thr215Asp + Thr217Pro Leul34Val + Glyl36Glu Thr 73Pro + Leu221Glu Tyr213Val + Ser220Glu Thr 73Ser + Serl07Glu Trp208Ser + Leu221Pro Thrll3Asp + Leu221His Alall4Thr + Thr217Asp AlalβδSer + Tyr218Glu Trp206Asp + Leu221Asn Thrl37Asp + Trp2Oδlle Glyl35Gln + Serl91Glu Vall03Glu 4- Trp208Gly Asnl 0Asp + Leu221His Serl92Asp 4- Tyr2lOCys GlylOδAsn 4- Tyr2lOHis Aspl05Glu 4- Trp208Asn Vall03Pro 4- Tyr2lOPro Thr 73Gln 4- Trpl12Tyr Leul34Cys 4- Asnl70Ser AsnlββGlu 4- Tyr2lOCys AsnlOβSer + Leu221Met Serl09Glu + Leu221Cys Serl07Asp + Leu221Ala Alall4Ser + Leu221Asn Glyl62Asn + Leu221Val

TABLE 33

Multi-loop Triple Mutation Variants

Thr217Glu Gly 69Ser Gly223Asp Thr217Gly Ser220Glu Ala219Gly Valll5Ala Tyr210Pro Thr217Ser Glyl65Gln Ser220Asp Trp20δHis Trp208Asp Thr212Ser Ser220Glu Leu221Met Thr217Pro Tyr210Pro Leu221Ala Ser222Asp Thr215Gly Tyrl71Glu Leu221Gly Tyr210Ala Gly223Glu Ala219Asn Thrl37Asp 4- Vall3δPro + Tyr210Ala Serl09Glu + Valll5Met 4- Trp208Cys Glyl39Asn + Ser220Glu 4- Leu221Asn Trpll2Ala 4- Glyl35Glu - Leu221Gly Thrll3Asn + Thrl95Asn 4- Thr217Ser Alal64His 4- Ser220Asp + Leu221His Asn ββAsp + Thr 73Ser + Thrll3Gln Glyl35Ser 4- Thrl67Gln + Thr217Gly Tyrl71Asp + Trp20δHis - Thr217Asn GlylOδAsn + Trp208Leu 4- Tyr210Val Trpll2Cys -J- Ser220Glu 4- Leu221Asn Vall03Met + AlalβδHis + Serl91Asp Pro214Ser -J- Thr217Gln 4- Ser220Glu Thr215Asn + Ser220Glu + Leu221Pro Glyl36Ser 4- Trp20δPhe + Tyr210Gln Gly 69Pro 4- Ala219Asp 4- Leu221Pro Gly 69Asn 4- Ala204Gly 4- Ser220Asp Glyl36Ser 4- Glyl39Asp 4- Trp208Val Asnl40Gln + Trp208Cys 4- Tyr210Asn GlyllOSer -t- Glyl65Ser + Trp208Thr Vall03Ser 4- Glyl35Glu + Leu221Cys Asnl40Glu 4- Trp208Ala 4- Leu221Ser Gly 67Pro + Vall03His 4- Alal64His Asnl40Asp 4- Thr212Gln 4- Gly223Pro Glyl39Gln -(- Thrl95Gly 4- Pro214Asn Gly 69Pro + Leul04Glu + Alall4Pro Phel93Pro + Trp206Glu 4- Thr212Pro Glyl36Gln + Trp206Cys 4- Leu221His Ser 70Asp + Thr 73Gly + Phel93Ser Tyr213Thr + Tyr218Gly + Gly223Asp Thrl95Glu + Trp208Ser + Leu221Cys Asnlβ3Asp + Leu221Ser + Gly223Pro Asn ββGln 4- Glyl39Asn ^■ Ser220Asp Thr 73Ser + Pro214Asp + Leu221Ile ThrlllPro 4- Asnl40Asp + Trp208Phe AsnlββGlu 4- Pro214Asn 4- Thr217Ser Glyl08Ser + GlyllOGlu + Trp208Ala Leul34Asp + Glyl65Asn + Tyr210Thr Phel93Thr 4- Thr217Gln 4- Ser220Asp Gly 67Pro 4- Trpll2Gln 4- Serl92Asp Tyr210Ile 4- Thr217Gln 4- Ser222Asp Vall03Ser 4- Leul04Ile 4- Alal64Asn Alal 4Asp + Thrl67Gly + Thr217Asn ThrlllAsp + Thrl95Gly + Tyr210Leu Tyr210Thr 4- Ser220Glu 4- Leu221Met Glyl36Gln + Trp208Phe + Leu221Asp Thrll3Ser + Trp208Val + Tyr210Thr Leul34Asn + Asnl40Ser + Thrl67Gln Glyl36Ser 4- Trp208Val + Leu221Ala Glyl65Pro + Pro214Ser + Ser220Glu Glyl36Ser + Tyr210Leu + Thr217Gln Serl41Glu + Trp208Thr + Gly223Ser Asn ββSer + Thrll3Pro + Asnl70Glu Thr 73Pro 4- Tyr210Thr 4- Ser21βGlu Tr.r 73Pro - Tyr210Glu 4- Leu221Ala Glyl39Ser 4- Alal64Ser 4- Tyr210Gly GlylOδSer + Vall3δSer 4- Alal64Gln Trp208Leu 4- Thr217Ser 4- Thr224Asp Asn ββGlu 4- Phel93Val 4- Tyr21δAla Glyl08Asn + Tyrl71Pro 4- Tyr210Thr Thrll3Pro 4- Glyl39Asn 4- Trp206Thr Aspl05Glu + AsnlββGln 4- Tyrl71Asn Alall4Pro 4- Thr217Gln 4- Ser220Asp Gly 67Glu + Alall4His 4- Thr217Asn Serl41Glu + Trp208Gln -t- Leu221Ser Gly 69Gln + Alall4Thr + Asnl70Asp Asnl63Asp 4- Ala219Thr + Leu221Ala Ser 70Asp + Thr217Gly + Ala219Thr Thrl95Asn + Leu221Gly + Gly223Pro Gly 69Gln + Leul04Thr + Leul34Thr Glyl39Asp -t- Glyl65Pro + Leu221Gly Gly 67Asn 4- Glyl08Glu 4- Tyr210Ile Valll5Cys 4- Tyr210Val 4- Pro214Gly Leul04Met 4- Thrl37Asp 4- Trp206Gly Trpll2Gln + Leul34Ala + Serl92Asp GlylOδGlu 4- Valll5His + Ala219Gln Thrll3Asn 4- Glyl39Pro + Thrl67Asp Thrl37Asp + Trp20δGln + Gly223Asn Ser207Glu 4- Trp208Gln + Tyr210Asn Asn ββSer + Ser207Glu + Tyr210Pro Vall03Asn 4- Tyr210Ala + Thr215Glu Asn ββSer 4- Trp208Phe 4- Ala219Thr Glyl36Asn 4- Glyl39Asn + Thr215Asn Serl09Glu 4- AsnlββSer + Thr217Asn Leul34Ile 4- Tyrl71Gly + Thr217Gln Gly 72Gln + GlyllOAsp 4- Glyl35Asn Asn βδGlu 4- AsnlOβGln + Thrll3Ser Alall4Thr + Leul34Gln + Thrl37Glu Serl07Asp -t- Glyl39Ser + Tyr213Ser Thrll3Asn 4- Thrl67Gly + Leu221Met Gly 67Ser 4- Ser207Glu + Thr217Ser Asn ββSer + Trp20δlle + Thr217Ser Trp208Ser 4- Leu221Gly + Ser222Glu Glyl62Pro 4- Ser216Glu ^■ Leu221Thr Leul04Cys 4- AsnlOβGln + Ser207Glu Gly 69Gln 4- Prolβ9Gly + Tyr210Leu Thr 73Asp + GlyllOSer + Thr217Ser Gly 69Ser 4- Gly 72Asn + Thr215Gly Glyl35Ser + Tyrl71Ser + Serl91Glu ThrlllAsn + Phel93Leu + Thr217Glu Trp208Ala 4- Leu221Ile + Thr224Asp Trpll2Phe + Pro214Asp ^■ Thr217Pro Asn 68Ser 4- GlyllOGln + Trpll2Met Gly 69Ser 4- Leul04Cys + Trp206Asp Thrl37Glu 4- AsnlββGln + Thr215Pro GlylOδGlu 4- Tyr210Thr + Thr212Ser GlylOδAsp Tyr210Cys GlyllOAsn Ser220Asp Gly β9Asn Ser220Glu Glyl62Glu Thr217Asn Trpll2Tyr Ser220Glu Trp206Gly Pro214Asp Leul34Asn Leu221Asp Thrll3Ser Leu221Glu AsnlββSer Ser220Glu Trpll2Pro Pro214Asp Leul04Glu Alal64Ser Gly 69Pro Leu221His GlylOδGln Ser220Glu GlyllOAsn Ser220Glu Alal64Thr Tyr210Asp Trpll2Pro Pro214Glu Valll5Glu Tyr210Gln Gly 69Ser Tyr210Pro Alall4Ser Thr224Pro Thr 73Pro Thr212Gly Gly 72Gln Leu221His Tyr210Cys Tyr218Asp Gly 69Asn AsnlββSer Thr 73Gly Ser220Glu Thr 73Gly Thr224Ser Vall03Met Leu221Met ThrlllAsn Phel93Met Thr 73Asp Thr217Gln Asn ββGlu Leu221Gln Thrll3Asp Thrl67Pro Asn ββGln Tyr210Pro Asn βδSer Asnl70Gln Asn βδSer Tyr210Asn Thrll3Gln Leu221His Thrl67Pro Tyr210Leu Glyl62Glu Thr217Pro Leul04Met Thr212Gly Thrl37Gln Pro205Gln Valll5Thr Ser220Asp Ser 70Asp Tyr210Val Pro214Gly Thr224Asp Leul34Val Trp20δThr Serl09Asp Tyr210Ser Asn ββSer Trp208Gln Thr 73Pro Gly206Gln Leul34Ser Ser222Asp ValllδCys Asnl70Gln Alal64Asn Thr217Glu Asn ββGlu Thr215Pro Glyl35Ser Thr217Gln Asnl40Gln Trp20δMet Glyl65Gln Leu221Asn Thr 73Pro Leu221Cys Thrll3Ser 4- Leu221Ile + Gly223Asp Asn ββAsp 4- Glyl36Gln + Glylβ2Gln Serl9lGlu 4- Thr217Asn 4- Thr224Pro Leul04Gln + Glyl62Glu 4- Tyr210Gln Trpll2Gln + Asnl63Asp + Thr217Gly Leul34Pro 4- Thr217Asn 4- Leu221Glu Glyl65Pro + Tyr210Met + Thr217Glu Alal64Gly 4- Serl91Glu 4- Trp20δCys Valll5Cys + Glyl39Asn 4- Pro205Asn Glyl35Pro + Trp20δAla 4- Tyr210Pro Prol69Asn + Thrl95Glu 4- Tyr210His Serl07Asp + Trp20δMet 4- Tyr210Pro Thr 73Gly + Alalβ4Gly 4- Tyr210Glu Leul04Val + Pro205Gln 4- Leu221Asp ThrlllGln + Serl41Asp 4- Leu221Asn Trpll2Cys + Thrl67Gln + Thr217Pro Vall36Thr + Asnl70Glu 4- Leu221Pro Ser 70Asp + Leul34Ser + Thr224Asn Thrl37Asn -r Ser207Asp -t- Tyr216Thr Glyl35Glu + Alal64Asn + AΪa203Ser Leul34Gly + Trp208Glu + Thr212Ser Asnl63Asp + Trp208Asn + Tyr210Thr Valll5Thr + Serl92Glu + Thr217Ser Gly 72Pro + Serl09Asp + Tyr210Gln Serl09Glu + Asnl63Gln + Phel93Pro Glyl36Pro + Prol69Glu + Leu221Ser GlyllOAsn + Ser220Glu + Leu221Met Trpll2Gly + Tyr210Asp 4- Thr217Pro Trp208Ser + Tyr210Ser 4- Leu221Pro Trpll2Met + Serl92Glu 4- Leu221Ala Thrl37Gly + Thrl67Ser + Thr217Glu Serl09Asp + Glyl62Pro + Tyr210Ala Alall4Thr + Glyl62Asn + Serl91Glu Thrl95Asn + Tyr218Pro 4- Leu221Glu Thr 73Glu + Alal64Gln -t- Pro214Ser Asn βδGlu + Trp208Tyr + Thr217Asn Glyl65Pro Thr212Ser + Ser220Glu Gly 67Asn + Glyl39Gln + AlalβδAsp Serl41Glu + Pro214Gln + Leu221Gln Gly206Ser + Trp208Tyr -t- Thr215Gln Leul34Pro + Glyl62Asn + Phel93Ile Trpll2Ala + Thr217Asn + Ser220Asp ThrlllGly + Glyl39Glu + Thr212Asn Serl07Glu + Tyr213Ala 4- Thr217Pro Glyl65Ser + Thr217Glu + Gly223Pro Tyr210His + Thr217Asp + Leu221Ala Trp20δCys + Thr217Asp -J- Tyr216Asn Gly 69Gln + Vall03Ala + Ser220Glu AsnlOβSer 4- Tyr210Gly + Gly223Ser Trp206Cys + Tyr210Asn + Thr217Asp Asn ββGln + Glyl39Gln 4- Thr217Gly Thr 73Glu + Leul04Ile + Tyr210Ile Thrll3Ser + Prol69Asn + Thr217Ser Tyr210Gly 4- Pro214Gly 4- Leu221Glu Leul34Ile + Ala219Glu 4- Ser220Asp Asnl40Asp +^■ Serl41Glu 4- Tyr218Ser Tyr213Vaϊ + Ser220Glu - Leu221Glu Tyr210Val 4- Ser220Glu + Leu221Asp Alal64His + Ser220Glu + Leu221Glu Tyrl71His + Ser220Asp 4- Leu221Glu Tyr210Ile + Ser220Glu + Leu221Asp Trpll2Leu + Ser220Asp + Leu221Glu Glyl36Pro + Serl91Asp 4- Serl92Glu AlalβδSer + Thr217Asp 4- Tyr21δGlu AsnlββSer 4- Thr217Glu 4- Tyr21δAsp Glyl62Asp 4- Asnl63Glu + Leu221Met Trp20δSer -t- Tyr210Glu 4- Ser220Asp Alall4Thr + Glylβ5Asp + Phel93Glu Ser207Glu + Thr217Asn + Ser222Glu Asnl40Ser + Trp208Glu + Ser220Asp Trp20δAsp + Leu221Asn + Ser222Glu Glyl39Gln + Trp208Glu + Leu221Asρ Asn βδGlu -t- Aspl05Glu 4- AsnlOβAsp Thrl37Gln + Glyl39Asp + Tyrl71Glu Trp208Asp + Tyr210Glu + Leu221Ala Gly β9Pro + AsnlββGlu + AlalβδGlu Tyrl71Cys + Tyr210Glu + Leu221Asp Thrl37Glu + Prol69Asp + Asnl70Ser Glylβ2Glu + Tyrl71Asp + Thr217Ser Leul34Ile + Serl91Glu + Gly223Glu Valll5Pro + Ser207Glu + Ser220Asp Glyl39Pro + Ser207Asp + Ser220Glu Ser 70Glu + Vall03Pro 4- AsnlOβAsp Gly 69Asp 4- Thr 73Asp + Tyr21δlle Asn ββAsp 4- Trp208Gly + Pro214Glu Asn ββAsp 4- Ser 70Glu + Glyl36Ser Asnl63Asp 4- Serl91Asp + Ser207Glu Asn ββSer + Gly 69Asp + Serl09Glu Gly β7Glu + Trp20δlle + Thr215Asp Ser 70Glu + Tyr210Gln + Thr217Glu Ser 70Glu + Glyl35Pro 4- Thr217Glu Vall36Glu + Serl41Asp 4- Tyr210Gln Vall36Glu 4- Serl41Glu 4- Pro214Asn Serl07Asp - GlyllOAsp + Alall4Glu Serl09Glu ^■+^■ Alall4Glu + Glyl36Ser GlyllOGlu + Glyl36Glu + Tyr210Gln Glyl35Ser + Tyr210Asp + Thr217Glu Ser 70Glu + Prol69Asn + Leu221Asp Valll5Gly + Glyl62Asp + Thrl67Asp GlyllOPro + Thr217Asp + Ser220Asp Alal64His + Thr217Asp + Ser220Asp Tyr210Cys + Thr217Asp + Ser220Asp Vall03Ser + Thr217Asp + Ser220Asp Thr217Glu + Tyr21δVal + Ser220Glu Gly 69Ser + Thr217Glu + Ser220Glu Tyr210Val + Thr217Asp + Ser220Glu Gly 67G1U + Serl07Glu 4- ThrlllAsn

AsnlββAsp 4- Serl 91Asp 4- Leu221Val

Leul34Asp 4- Phel93Glu + Gly223Asp

Asn ββAsp -t- Ser2lβAsp 4- Thr217Glu

Vail03Glu 4- AsnlOβGlu 4- Thrll3Glu

Ser 70Asp 4- Glyl39Ser + Ser220Asp

Leul04Asp + Aspl05Glu 4- Serl91Glu

Alal14Glu 4- Ser2lβAsp 4- Thr217Glu

Thrl95Glu 4- Ser2IβAsp 4* Thr217Glu

Vail15Glu + Ser220Asp + Leu221Glu

Serl09Asp + Ser220Asp 4- Leu221Glu

Thr215Glu + Ser2lβAsp + Ser220Asp

Serl92Asp + Phel93Glu + Thr217Asp

Glylβ5Asp + Tyr2lOGlu 4- Ser220Asp

Ser 70Glu + Aspl05Glu + Tyr218Asp

Gly 69Glu + Alal68Glu 4- Thrl95Glu

TABLE34

Multi-loop Quadruple Mutation Variants

Leul04Gly 4- Tyr210Pro + Thr217Glu Gly 69Ser + Vall03Gln + Gly223Asp Alal64Glu + Trp208Gln 4- Thr217Gly Trp20δGln * Ala219Gly 4- Ser220Glu Serl09Glu + ThrlllGln 4- Valll5Ala Alal64Gly + Glyl65Gln 4- Thrlβ7Pro AsnlOβGln + Trpll2Cys + Thrll3Pro Leul34Ser ^■+^■ Thrl37Asn + Thr217Pro Glyl36Pro + Thrl95Ser 4- Leu221Ala Leul04Gly ^■+^■ Trp208Pro + Thr215Gly Glyl36Pro + Phel93Met + Thr217Pro Tyrl71Ile + Phel93Ile + Thr224Gly Alal64Thr + Ser207Asp + Trp20δLeu Trpll2Pro 4- Trp206Thr + Tyr210Glu Leul34Val + Tyr210His + Thr217Pro Trp20δPhe + Ala219Asp + Leu221Met Tyrl71Leu 4- Trp208Val 4- Tyr210Thr Serl41Glu + Trp208Thr + Tyr210Leu Thr 73Pro + Thrll3Pro + Asnl70Glu Glyl39Ser + Tyr210Glu + Leu221Ala Vall36Ξer + Trp208Leu 4- Thr217Ser GlyllOGln + Trpll2Met + Serl92Asp Vall03Ala + Glyl62Ser 4- Alal64Glu Asnl 0Asp 4- Phel93Thr + Trp208Ala Asnl63Gln ^■+^■ Ser220Glu + Gly223Asn Tyrl71Gln + Tyr210Val 4- Gly223Ser Leul34Val + Glyl3βGlu 4- Thr217Pro Glyl62Gln + Trp208Ser + Leu221Pro Glyl62Asn + Serl92Asp + Tyr210Cys Serl09Glu + Trp208Asn + Tyr210Pro Trpll2Tyr 4- Leul34Cys + Asnl70Ser Glyl62Asn + AsnlββSer + Leu221Asn Gly 67Ser + Leul34Ser + Glyl62Asn + Alal64Gln

Leul34Ile + Glyl35Pro + Alal68Thr + Trp206Met

Trpll2Ser + AlalβδHis + Thr217Ser + Leu221Ala

GlyllOSer + ThrlllGln + Thr215Ser + Ser222Asp

ThrlllPro + Thrl37Asn + Prol69Gln + Ala219Glu

Thr 73Glu + AsnlOβSer + Trp208His + Leu221Thr

Asn ββAsp + Gly 67Pro + Gly 69Ser + Thr224Ser

Leul04Cys + Valll5His + Serl91Asp + Tyr21δVal

Leul34Glu + Thrl95Asn + Trp208Ser + Leu221Ser

Vall03Ser + Glyl39Asn + Thr217Asn + Leu221Cys

AsnlOβGln + Alal64Pro + AsnlββAsp 4- Phel93Pro

Aspl05Glu + Glyl35Pro 4- Vall3δSer + Thr217Pro

Gly 67Asp + Thrll3Asn + Alall4Asn -t- Tyr210Cys

AsnlββAsp 4- Thrl95Gly -J- Tyr210Gly + Thr224Gln

Trpll2Pro + AsnlββSer 4- Phel93His + Tyr21δlle

Gly 69Pro + Leul04Glu 4- Valll5Ser + Alal64Ser

Trpll2Pro + Valll5Glu 4- Glyl39Asn + Tyr210Gln

Gly 69Ser + Thr 73Ser + Tyr210Pro + Thr224Pro

Gly 72Gln + Thr 73Pro + GlyllOAsp + Leu221His

ThrlllAsn + Glyl36Pro + Tyr210Thr + Tyr21δAsp

GlylOδAsp + Glyl36Asn + AsnlββSer 4- Leu221Thr

Vall03Met + Pro205Ser + Leu221Met + Thr224Ser

Gly 69Asn + Thr 73Asp + Trp20δGln + Tyr210Leu

Thr 73Gly + AsnlββSer 4- Thrl67Pro -t- Leu221Gln

Asn ββGln + Thrll3Asp 4- Tyrl71Gly 4- Tyr210Pro

Glyl62Glu + Thrl67Pro + Trp20δVal 4- Tyr210Leu

Ser207Asp + Trp20δAla 4- Thr212Gly + Thr217Pro

Leul04Met + Thrl37Gln + Asnl63Asp + Pro205Gln

Serl09Asp + Leul34Val + Glylβ5Gln + Trp20δThr

Alall4Asn + Valll5Asn 4- Trp206Gln -f- Tyr210Ser

Asn ββGlu + Alal64Asn + Trp20δCys 4- Thr215Pro

Glyl35Ser + AlalβδPro 4- Ser207Asp + Thr217Gln

Thrl67Asp + Trp20δMet 4- Thr217Gln + Leu221Asn

Thr 73Pro + AsnlOβGlu 4- Thrll3Ser 4- Leu221Cys

Glyl62Gln + Serl91Glu + Thr217Asn 4- Thr224Pro

Gly 69Gln + Leul04Gln + Glyl62Glu + Tyr210Gln

Leul04Gln + Tyrl71Pro 4- Thr217Gly + Leu221Glu

GlyllOGlu + Phel93Asn + Trp208Cys + Gly223Pro

Alal64Gly + Serl91Glu 4- Pro205Asn + Trp206Cys

Glyl35Pro + Ser207Glu 4- Trp20δAla + Tyr210Cys

Thr 73Glu + Leul04Val + Pro205Gln + Leu221Cys

Trpll2Cys + Vall38Thr + Thrl67Gln + Thr217Pro

Serl09Asp + Alal64His + Gly223Pro + Thr224Asn

Asn 68Gln + Tyr210Pro 4- Thr212Ser ^■+^■ Ser220Asp

Leul34Gly + Trp208Glu -J- Tyr210Met + Thr212Ser

Asnl63Asp 4- Asnl70Gln 4- Trp208Asn 4- Tyr210Thr

Trpll2Gly + Trp208Ser 4- Tyr210Asp 4- Thr217Pro

Trpll2Met + Serl92Glu 4- Trp208Ser + Tyr210Val

Trpll2Leu + Ser220Asp + Leu221Ala + Thr224Asn

Leul04Met + ThrlllAsp 4- Alal64Gln + Pro214Ser

Glyl39Gln + Glyl65Pro 4- Thr212Ser + Ser220Glu

Thrll3Asp + Leul34His + Trp208Ile 4- Leu221Ala

Aspl05Glu 4- Pro214Gln 4- Thr215Gln + Leu221Gln Leul34Pro + Gly206Ser + Trp20δTyr Trpll2Ala + Thr217Asn + Ser220Asp Serl07Giu + Tyr213Ala + Thr217Pro Gly 67Gln + Thrll3Asn + Ser220Glu Asn ββGln + Tyr210Asn + Thr217Asp Thr 73Glu + Glyl39Gln + Thr217Gly Thrll3Ser + Tyr210Ile + Thr217Ser Leul34Pro + Tyr210Met 4- Ser220Asp Thr 73Glu 4- Tyrl71His + Leu221Ala Vall38Met + Thr217Asn + Ala219His Glyl36Gln 4- Ser220Asp + Thr224Gln Gly 69Asn + Tyr210Glu + Thr217Gly Thr 73Ser 4- Thr212Pro + Ser222Glu Thrl37Pro + Tyrl71Gly + Ser220Glu Tyr210Pro 4- Ser220Asp + Leu221Pro Vall03Asp + Phel93His + Thr217Gly Asn 68Asp 4- Vall38Ala + Trp208His Aspl05Glu -t- Glylβ5Ser + Leu221Gln Asn 68Gln 4- Thr217Ser + Ser220Asp Tyrl71Asn 4- Tyr210Asn + Tyr218Asn Asn ββSer -J- Tyr210Cys 4- Gly223Glu Alall4Gln + Tyr210Gly + Thr224Pro Asn ββSer + Alal64Asp + AlalβδGln Leul04Gly 4- Serl92Asp + Leu221Asn GlylOδAsp + Trp20δThr + Gly223Asn Asn ββGlu 4- Alal64Pro + Gly223Pro GlyllOPro + Ser216Glu + Leu221His Valll5Met + Trp20δHis + Tyr210Asp Leul04Ser 4- GlyllOPro + Thrl95Gly Leul04Asn + Trp20δSer + Leu221Val AsnlOβGln + Tyrl71Thr + Leu221Pro Pro205Asn 4- Thr217Gly + Tyr218Leu Leul04Pro + Thrl37Asp + Thr217Pro GlyllOSer + Asnl70Glu + Thr217Asn Thrll3Gly + Trp208His + Tyr213Ile AsnlββGln + Thr217Ser + Tyr218Met Glyl62Asp + Trp208Tyr + Thr217Gln Gly 69Ser + Trp20δMet -t- Tyr210Leu Vall03Asp + Ile209Val + Tyr210Gln ThrlllAsn -t- Trp20δTyr + Thr212Gly Serl07Glu ^• Glyl62Ser ^■+^• Trp208Cys Vall03Thr + AlalβδGlu 4- Thr217Ser Glyl39Pro + Leu221Pro + Thr224Pro Leul34Ser 4- Tyr210Ala + Thr217Gln Vall03Asn + Tyr218Met + Thr224Asn Asn ββAsp + Asnl63Ser 4- Leu221Val Gly 69Glu + Thrl37Pro 4- Trp208Ile Gly 69Pro + Tyr210Ser + Leu221Ile Asn ββGln + Trpll2Gly 4- Trp20δPro Trpll2Ala + Glyl35Ser + Asnl63Asp Gly 67Asn + Tyr210Thr + Thr217Pro AsnlββAsp + Ile209His + Tyr210Val Thr 73Ser + Tyrl71Glu + Thr217Asn ValllδGlu + Ala219Gly

Leul04Pro + Ser222Asp

Serl41Asp + Tyr210Leu

Gly 67Ser + Tyr210Gln

Tyrl71Met + Thr217Gln

Asn 68Gln + Leu221Ala

Leul04Asn + Pro214Glu

Glyl65Ser + Leu221Thr

Gly 67Ser 4- Ser220Asp

Serl09Asp + Thr217Pro

ThrlllGly + Leu221Ile

Trpll2Asn + Thr224Gln

ValllδAla -t- Leu221Cys

Glyl35Ser 4- Leu221Pro

Asn ββGln + Tyr21δThr

Thr 73Gly 4- Leu221Asn

Leul34Ser + Ser220Glu

Glyl35Pro 4- Leu221Gln

Asn 68Gln + Thr215Gln

Vall36Gln 4- Leu221Cys

Phel93Gln + Thr217Gln

Gly β7Asn + Pro205Ser

Asn ββSer + Thr217Asp

Pro214Gly + Leu221His

Asn ββGln + Tyr21δMet

ThrlllGlu + Leu221Thr

Serl41Asp + Ala204Ser

GlylOδPro + Tyr213Ser

Trp20δHis + Ser220Asp

Gly 69G1U + Thr224Ser

Serl09Asp + Thr217Ser

Thr 73Asp + Leu221Pro

Glyl62Pro + Ser220Glu

Asnl70Gln + Tyr210Gly

Thrll3Ser + Pro205Ser

Trpll2Pro + Leu221Asn

Glyl62Glu + Pro214Asn

Alall4Pro + Phel93Ile

Alal64Gln + Tyr210Leu

Vall03Pro + Glyl65Gln

Glyl62Ser + Leu221Gln

Serl41Asp + Leu221Met

Asn ββSer + Tyr210Thr

Leul34Asp + Trp206Val

Serl91Asp 4- Thr217Ser

Gly 67Asn + Thr215Pro

Alall4Thr + Ala203Pro

Serl91Glu + Tyr210Ala

Asnl40Asp + Tyr21δSer

Thr 73Ser + Leu221Glu

Phel93Met + Leu221Glu

Thrll3Ser + Leu221Glu

Thr 73Pro + Phel93Glu Alall4Asn 4- Trp2OδPro + Tyr210Glu - Ser220Glu Trp20δCys 4- Tyr2lOGlu + Ala219Asn 4- Ser220Asp Alall4Pro 4- Glyl39Pro + Tyr210Asp 4- Ser220Glu Tyr210Glu 4- Thr217Gln + Tyr21δGln 4- Ser220Glu Asnl40Ser 4- Trp2OδGlu 4- Ser220Asp 4- Leu221Asp Vall03Pro 4- Trp2OδGlu 4- Tyr210Ser + Ser220Glu Thr 73Gln 4- Serl07Asp 4- Serl09Glu 4- Asnl63Gln Gly 67Gln + Serl07Asp -t- Serl09Glu 4- Leu221Cys AsplOSGlu + AsnlOβSer 4- Serl09Glu -J- Thr217Pro Leul04Glu + Aspl05Glu 4- Serl07Asp + Leu221Ser Gly206Pro -t- Pro214Asp 4- Thr217Glu + Leu221Asn Ser207Asp 4- Trp2OδGlu 4- Ser220Asp 4- Leu221Ser Glyl35Asp 4- Glyl62Glu 4- Tyrl71Asp 4- Thr217Ser Gly 69Glu 4- Leul04Asp 4- Aspl05Glu 4- Tyr210Ala AlalβδAsn + Ser2 20Asp 4- Leu221Pro + Ser222Asp Phel93Gln 4- Ser207Glu + Thr217Ser + Leu221Glu GlyllOGln 4- Glyl65Gln + Ser222Asp + Thr224Asp AsnlOβGlu 4- Serl09Asp + Phel93Ile + Trp208Leu GlyllOAsn 4- Thrl37Glu 4- Prol69Asp + Asnl70Ser Glyl36Glu + Glylβ2Glu + Trp208Ala -J- Thr217Asn Leul34Ile + Serl91Glu 4- Leu221His + Gly223Glu Glyl39Pro + Tyrl71Met 4- Ser207Asp + Ser220Glu Vall03Pro + ThrlUGly 4- Ser207Asp + Ser220Asp GlylOδAsn -J- Thrl95Gly 4- Ser207Asp + Ser220Asp Ser 70Glu 4- Vail03Pro -t- AsnlOβAsp + AlalβδPro Thrll3Gly 4- Glyl62Ser ^■ Asnl63Asp 4- Asnl70Glu Leul04Ile 4- Serl07Glu -J- GlyllOAsp + Asnl70Gln Thr 73Gly + Glyl 35Asp + Prol69Asp + Trp208Thr Tyr210Met 4- Thr215Asp -J- Tyr218Glu + Leu221Asn Leul04Thr 4- Glyl36Glu -J- Prol69Asp + Leu221Val Asn ββAsp + Thr 73Glu -J- Tyr218Ile + Leu221Cys Aspl05Glu 4- ThrlHAsp 4- Alall4Pro + Vall3δMet Asn βδGlu -t- Thr 73Glu + Trp206Tyr + Thr217Asn Ser207Glu 4- Tyr213Ile 4- Ala219Asp + Ser222Asp Asnlβ3Asp 4- Alal64Ser 4- Serl91Asp + Ser207Glu Thr 73Glu 4- AlalβδSer 4- Tyr210Glu + Tyr218Glu Gly 67Glu 4- Trp2Oδlle 4- Pro214Asn + Thr215Asp Aspl05Glu -J- ThrlUGlu 4- Thrll3Glu + Asnl63Gln Ser 70Glu 4- Tyr2lOHis 4- Thr217Asp + Leu221Ala Gly β7Glu 4- Glyl08Asp + Thrl37Gln + Thrl67Pro Serl07Asp + GlyllOAsp 4- Alall4Glu + Tyr218Asn Ser 70Asp + Tyr2lOGlu + Thr217Asn + Ser220Glu Thrll3Gly 4- Trp2OδGlu 4- Ser220Asp + Thr224Asp Vall36Met 4- Ala219Glu 4- Ser222Asp + Thr224Gln Ser 70Glu 4- Trpl12Tyr 4- Tyr216Cys + Leu221Glu Gly 67Ser 4- Ser 70Glu 4- Prol69Asn + Leu221Asp Valll5Cys + Glyl39Asn -t- Thr217Asp + Ser220Asp Gly 69Ser + Vail15Thr 4- Thr217Glu + Ser220Glu Alal68His + Trp208Tyr 4- Thr217Asp + Ser220Glu ThrlllGln 4- Glyl35Pro + Thr217Asp + Ser220Glu Alal64Thr + Serl91Glu + Trp206Glu + Thr215Asn Asn βδGlu + Vail03Glu + Serl09Glu + AsnlββGln Ser 70Asp 4- Glyl39Ser + Thr217Glu + Ser220Asp Leul34Asp + Phel93Glu + Ala203Ser + Gly223Asp Asn ββAsp 4- Asn βδSer + Ser216Asp + Thr217Glu Alal64Glu 4- Trp208Cys + Leu221Glu + Ser222Asp Ser 70Glu 4- Ser207Glu + Ala219Ser + Ser220Glu Gly 67Asp + Ser 70Asp + Thr 73Gly + Tyr210Glu Trp20δlle + Thr217Glu + Tyr21δAsp + Ser222Asp Leul04Ile + ThrlllGlu + Glyl35Asp + Asnlβ3Gln Alall4Glu + Leul34Ile + Ala219Glu + Ser220Asp AsnlββGlu + Thrl67Gly + Ser222Asp 4- Gly223Asp Glyl39Glu + Tyr210Ala + Ser222Asp 4- Gly223Glu Vall03Gly 4- GlyllOGlu + ThrlllGlu 4- Ser222Glu Alall4Glu + Asnl70Ser + Ser216Asp 4- Thr217Glu GlylOδAsp 4- Serl09Asp + Tyr210Gln 4- Thr217Glu Asn 68Ser 4^* Serl09Asp + GlyllOGlu 4- Ser220Glu Asn 68Glu 4- Asnl63Ser + Ser207Asp + Trp20δGlu Ser207Asp -t- Trp20δGlu + Thr217Glu + Leu221Cys Gly β9Pro 4- Aspl05Glu + AsnlOβAsp + AsnlββAsp Serl09Asp 4- Tyr210Gln + Ser220Glu 4- Leu221Asp Tyrl71Gly + Serl92Glu + Ser220Asp 4- Leu221Glu Asnl70Glu + Serl91Glu + Serl92Glu 4- Pro205Ser Asn ββAsp + Tyr210Gln + Thr217Glu + Tyr21δGlu AsnlOβAsp 4- Serl07Glu 4- Thrl37Asp 4- Tyr210Gln Serl92Asp 4- Phel93Glu 4- Pro205Ser 4- Thr217Asp Ser 70Asp 4- Glyl62Asp -J- Asnl63Glu - Leu221Ser Gly 69Asn 4- AsnlOβGlu + Tyr210Asp + Ser220Glu Vall03Ser -t- Asnl40Asp -t- Tyr210Glu + Ser220Asp Gly 69Glu 4- Phel93His + Ser207Glu + Ser222Asp Glyl62Glu 4- Asnl63Gln + Trp20δAsp + Ser220Glu Valll5Cys 4- Asnl70Asp 4- Trp20δAsp + Ser220Asp GlyllOGlu + Trp20δAsp 4- Leu221Asn + Ser222Glu Prol69Asp 4- Tyrl71Glu + Pro214Asn + Gly223Glu Asnl63Glu + Ser207Glu 4- Trp208Met + Tyr210Thr Gly 67Glu 4- Ser 70Glu + Valll5Cys -t- Glyl62Glu Glyl62Glu 4- Alal64Glu 4- Ser220Glu + Leu221Gly Asn ββGlu 4- Vall03Asp 4- Glyl08Asn 4- Leu221Met Aspl05Glu 4- Serl09Glu + Tyr21δAsp + Leu221Ala Gly 67Asp 4- Alal64Asp + AlalβδGlu + Tyr213Asn Gly 69Pro -t- Serl41Asp 4- Pro214Asp + Thr217Asp Thr 73Glu + Trpll2Ile 4- Trp206Gly + Ser220Asp Gly 72Ser + Serl07Asp 4- ThrlllAsp 4- AsnlββGln Glyl65Glu + Serl92Asp 4- Thr217Asp 4- Leu221Ile Glyl35Glu 4- Asnl40Glu + Ala204His 4- Pro214Ser Aspl05Glu 4- GlyllOAsp 4- Asnl70Asp + Ala219Gly Asn ββAsp 4- Asn βδAsp + Leul34Gly ^■ Asnl63Glu Leul04Thr -t- AlalβδAsp 4- Asnl70Asp 4- Ser220Glu Thr 73Asp 4- Serl09Glu + Pro214Glu + Thr217Asn Thr 73Asp 4- Valll5Cys 4- Pro214Asp + Thr224Glu Serl09Asp 4- Thrll3Asp 4- Leul34Asn + Phel93Pro Gly 69Ser + GlylOδAsp + GlyllOAsp + Ser220Glu Glyl39Asp -J- Serl41Asp 4- Alal64Asp + Leu221Val Asn ββAsp + Trp208Met + Ser220Asp + Ser222Glu Trpll2Asp + Tyr210Thr + Ser220Glu + Ser222Glu Serl09Asp 4- Ser207Glu + Tyr210Gly + Leu221Glu Gly 69Asp 4- Aspl05Glu + Thrl37Asp + Trp208Asn Serl09Asp + ThrlUGlu + Serl91Glu + Tyr2lOLeu Glyl39Gln - Prolβ9Asp + Serl91Asp + Ser207Glu Gly 67Ser + Asnl40Asp 4- Ser2 22Asp + Thr224Glu Vall38Asp + Asnl70Glu 4- Trp2OδHis + Thr215Glu Asn 68Asp 4- ThrlUGly 4- Vail3δAsp 4- Asnl70Asp Leul04Glu + Serl07Asp + Glyl35Gln + Tyrl71Asp Leul04Pro 4- Trpl12Asp 4- Asnl40Glu 4- Ser222Glu Glyl39Glu + Asnl70Asp + Gly2OβPro + Ser220Glu Alal64Asp + Asnl70Asp 4- Ser2lβAsp Thr217Pro Thrll3Glu + Alal14His 4- Ser207Glu Ser220Glu GlyllOGlu + Vail15Gln 4- Ser207Asp Thr224Glu AsnlOβGlu + Asnl63Glu -f- Asnl70Glu Leu221His Gly 69Asp + Thr 73Glu + Thrl13Asn Serl41Glu Gly 69Asp + Thr 73Asp + ThrlUGly + Glyl39Glu Serl07Asp + Asnl63Glu + Serl92Asp + Trp2OδMet Asn βδAsp + GlylOδAsp 4- Ser207Asp + Ile209Ala Glyl35Asp + Glyl39Pro + Prol69Asp + Trp2OδGlu Asn ββGlu + Thrl67Gly + Trp2OδGlu 4- Pro214Asp Asn ββAsp + Serl09Asp + Trp2OδGly + Pro214Glu Gly 69Glu 4- Asnl40Ser 4- Serl92Glu + Pro214Asp Leul04Gly 4- Glyl39Glu 4- Serl91Asp + Thr224Asp AsnlOβGln + Thrl13Asp + Asnl63Asp + AsnlββGlu Leul34Glu + Ser207Asp + Trp208Pro + Tyr2lOGlu Serl91Glu + Thrl95Asn 4- Tyr218Pro + Leu221Glu Asnl70Asp + Phel93Ser + Ser220Glu + Thr224Asp Aspl05Glu 4- Alal14Glu + Serl92Glu + Thr224Asn Gly 67Asp + Glyl08Asp + Ser207Asp + Tyr2lOCys Asn βδAsp 4- GlyllOGlu + Thrl13Gly + Leu221Ala Leul04Ile 4- Alal14Glu 4- Ser220Glu 4- Gly223Glu Serl07Asp 4- Ser220Glu 4- Leu221Pro 4- Gly223Asp Vall03Asp 4- Vail38Glu + Serl41Glu + Gly2OβGln Vall3δGlu 4- Serl41Asp + Tyr2lOGln + Ser220Asp Asn βδAsp + Vail15Gly + Thr217Asp + Ser220Glu Asn ββAsp + Leul04Gly + Alal68Thr + Ser2lβGlu Vall03Glu + AsnlOβGlu + Thrl13Pro Ser220Asp Aspl05Glu + Thrl13Asp + Glyl 39Asn Serl41Glu Ser 70Glu 4- Aspl05Glu 4- Glyl39Asp Tyr2lOAla Asn ββGlu 4- Tyr2lOAsp + Thr217Glu Leu221Ala GlylOδGlu + Glyl35Ser Tyr2lOAsp Thr217Glu Gly 69Asp + GlylOδGln Trpl12Asp + Serl41Glu Serl07Glu 4- GlyllOPro Thr217Asp + Ser220Asp Glyl35Glu 4- Thr217G1U Tyr218Val + Ser220Glu Asnl63Asp + Tyrl71Thr Thr217Asp + Ser220Asp Alall4Thr + Thrl37Glu + Thr217Asp + Ser220Asp AsnlββGlu + Thr212Pro + Thr217Asp 4- Ser220Asp Gly 67Asp + Serl07Asp + Tyr210Asn + Ser222Glu Asnl40Ser + Trp2OδGly + Thr217Glu + Leu221Asp AsnlββGln + Thr215Pro + Thr217Glu + Leu221Asp Valll5Gln + AlalβδGln + Phel93Asp 4- Leu221Asp Trp208Ile 4- Pro214Ser 4- Ser220Glu + Thr224Glu GlylOδGlu 4- Thrl37Glu 4- Asnl63Asp + Trp2OδSer Glyl39Pro + Alal64Glu + Serl91Asp + Pro214Glu Thr 73Asp + ThrlllPro + AsnlββAsp + Serl91Asp

Ser 70Glu + Alal68His + Thrl95Glu + Ser216Asp

Ξerl09Glu + Thrl37Asp + Glyl39Ser + Alal64Gln

Asnl70Gin - Tyr210Asp + Pro214Glu 4- Gly223Ser

Ser 70Glu 4- Glyl39Asn + Alal64Glu + Ser220Asp

Asn ββAsp - Thrll3Glu + Serl41Asp + Trp208Gly

Asn ββAsp 4- GlylOδGlu + Thr215Gly + Leu221Ser

Asn βδSer + Thr 73Glu + AsnlββGlu + Leu221Asp

Thr 73Asp 4- Serl92Asp + Ser220Asp + Leu221Met

Serl07Asρ 4- ThrlllAsp + Serl92Glu + Thr217Ser

TABLE 35 Multi-loop Quintuple Mutation Variants

Asn ββGln 4- Gly 67Ser 4- Leul04Gly 4- Tyr210Pro +

Thr217Glu Gly β9Ser + Vall03Gln + Trpll2Cys + Thr217Gly +

Gly223Asp Valll5Pro 4- Leul34Ile + Alal64Glu + Phel93Asn +

Trp20δGln Leul04Gly + Thrll3Ser + Alal68Glu + Asnl70Ser +

Ala219Gly Gly 69Ser + Serl09Asp + ThrlllGln + Valll5Ala +

Tyr210Pro Vall03Thr + Glyl08Glu + Valll5Pro + Tyrl71Ser +

Trp206Asn Leul34Ala + Vall3δCys + Asnl63Gln 4- Tyrl71Ser +

Ser220Asp Asn ββSer + Valll5Gly 4- Serl91Glu 4- Trp206Ile +

Thr217Ser Gly 72Asn 4- Trpll2Phe + Valll5Ala 4- AsnlββGln +

Trp20δGly Leul34His 4- Glyl65Gln + Trp208Pro 4- Tyr210Val +

Thr217Glu Leul04Pro 4- Tyrl71Leu 4- Trp208Leu + Ala219Glu +

Leu221Ser AsnlββGlu 4- Thrlβ7Pro + Tyr210Pro + Tyr213Ile +

Pro214Asn Glyl39Gln 4- Asnl70Glu 4- Trp206Cys 4- Thr215Ser +

Leu221Asn Alall4Glu 4- Glyl35Ser + AlalβδThr 4- Ala203Thr +

Trp208 et Alal64His 4- AlalβδHis 4- Phel93Ala + Thr217Ser +

Ser220Asp Gly 67Pro 4- Gly 69Ser + Serl91Asp 4- Tyr21δVal +

Thr224Ser Glyl39Asn + Alal64Pro 4- Phel93Pro 4- Thr217Asn +

Leu221Cys Leul04Met 4- Ser207Asp 4- Trp20δAla + Thr212Gly +

Thr217Pro Leul34Val 4- Trp206Thr + Pro214Gly 4- Leu221Ile +

Thr224Asp Serl09Asp + Alall4Asn + Valll5Asn + Glyl65Gln +

Tyr210Ser Leul04Ser - GlyllOPro + Glyl36Glu + AlalβδThr +

Ala219Gln GlyllOPro * Leul34Pro + AsnlββAsp + Asnl70Ser +

Trp20δAsn Thrll3Gln - Prol69Gly + Asnl70Gln + Tyr218Gln +

Leu221Ser Thrl37Ser + Thrl95Gln + Ser207Glu + Trp208Gln +

Thr217Pro Gly 69Gln + Vall03Gly 4- Trpll2Met + Trp208Asn +

Pro214Gly Glyl36Pro 4- Glyl62Ser + Trp208Ile + Tyr210Gly +

Pro214Gln Thrl37Asp 4- Glyl65Gln + Thrl95Ser + Trp20δPro +

Thr212Ser Asn ββSer + Prol69Ser + Tyr210Asn + Tyr218Asn +

Gly223Glu Ser 70Glu + AsnlOβGln + Vall38Ala + AlalβδGln +

Leu221Val Gly 69Ser + Thrll3Asp + Alall4Gln 4- Trp20δThr +

Ala219Ser Thrll3Gln -J- AlalβδAsp 4- Prol69Ser + Asnl70Ser +

Ala219Thr Glyl39Ser + Asnl70Gln + Tyrl71Leu 4- Trp208Cys +

Thr215Ser Gly 67Ser + Alall4Asn + Glyl39Ser + Tyr210Thr +

Tyr218Ser Trpll2Glu - Alal64Pro + Tyrl71Pro + Thrl95Asn +

Thr217Ser Gly 67Glu 4- Valll5Asn + Glyl62Gln + GlylβSGln +

Phel93Val Glyl35Ser * Tyrl71Cys + Trp20δHis + Tyr210Met +

Leu221Val Gly 69Asn 4- AsnlOβSer + AsnlββGlu + Tyr210Cys -t-

Leu221Gly Glyl35Ser 4- Tyrl71Asn + Trp206His + Ser216Glu +

Leu221Gly Gly 69Asn + Serl09Asp + Glyl65Asn + Phel93Asn +

Trp20δHis Prol69Ser + Ser207Glu + Pro214Asn + Thr215Asn +

Thr217Gly Ser 70Asp 4- Asnl63Ser + Tyr210Val 4- Tyr213Asn +

Leu221Ala GlylOδGln 4- GlyllOSer + Glyl35Gln + Thrl37Asn +

Trp20δGlu Asn βδSer + Alal64Gln + Asnl70Glu 4- Thr217Ser +

Gly223Ser Gly 69Ser f Trpll2Cys + Alal64Gly + Leu221Ser +

Gly223Glu Gly 69Glu + Gly 72Gln + Thrl37Pro + Trp20δlle +

Tyr218Pro Vall03Gln - Vall38His + Glyl62Ser + AsnlββAsp + Trp208His ValllSGln + Leul34Cys + AlalβδSer + Prol69Asp +

Tyr210Ser Gly 67Gln + Trpll2Leu + Phel93His + Thrl95Glu 4-

Thr217Ser Gly 67Gln + Leul04Asn + Alal64Glu + Ala204Ser +

Pro205Ser Glyl35Ser + Vall38Ser + Asnl40Glu + Tyr210Leu +

Thr217Gly Gly 67Asn + Thr 73Ser 4- Tyrl71Leu + Phel93Tyr +

Tyr210Pro Glyl35Pro + Trp208His 4- Tyr210Leu -J- Ala219Ser +

Leu221Gln Asn 66Gln + AsnlOβGlu 4- Thrl37Gln + Prol69Gln +

Thr215Gln Thrl37Asp + Vall3δGln + Trp20δlle + Tyr218Leu +

Leu221Cys Gly 67Asn + Glyl65Asn -t- Phel93Tyr + Pro205Ser +

Trp208Cys Asn ^ββSer + Thr 73Ser -J- Serl07Asp 4- Alal68Ser +

Tyr210Cys Thrl37Gly + Tyr210Pro 4- Thr215Pro 4- Ser216Asp +

Thr217Ser Thr 73Pro + Trpll2Asp 4- Glyl39Pro 4- Thr215Pro +

Leu221His Leul04Cys + ThrlllPro + Trp208Pro 4- Leu221Cys +

Ser222Asp Thrll3Gln + Ala204His 4- Trp208Phe + Thr217Asp +

Leu221Cys ThrlllGly + Glyl39Gln 4- Tyrl71Leu 4- Tyr210Met +

Thr217Glu GlyllOAsn 4- ValllSGln + Prol69Gln 4- Thrl95Gly +

Ser220Asp Tyrl71Asp 4- Gly206Ser -J- Thr217Gln + Ala219Gly +

L __ιeeuu2__2__1ιTihnrr Serl09Glu + ThrlllSer + Valll5Cys + Vall36Cys +

T Tyyrrll7711SSeerr

Leul04His Asnl63A^■isspp ++ TTrrpp2206Thr + Tyr210Cy

T Thhrτ-2?1177A&s<-:nn Glyl36Asn + Glyl39Pro + Serl41Glu + Prol69Gln + Q9UHiisc Gly 69Ser 4- Glyl36Ser 4- Thrl37Asn 4- Thrl95Pro +

Ala219Glu AsnlOβAsp 4- Glyl62Ser 4- Asnl63Gln 4- Prol69Gly +

Leu221Pro AlalβδGly + Phel93Met 4- Pro205Asn + Tyr210Asn +

Leu221Asn Gly β9Pro 4- Glyl39Asp + Alal64Gly 4- Tyr213Ser +

Thr217Ser Gly 72Ser + Leul04Gln + AsnlββGlu 4- Prol69Asn +

Thr215Gly Leul34Cys 4- Trp208Asn + Tyr210Ala 4- Tyr21δGly 4-

Thr224Gly Leul04Gln + Thrl95Asn + Trp20δVal + Tyr210Pro +

Leu221Cys Gly ^β7Pro + Thr 73Asn + Trp208Pro + Ala219Asn +

Ser220Asp Thr 73Gln + Glyl08Pro + Glyl39Pro + Serl92Glu +

Gly223Gln Asn 68Glu + Thr 73Gln + Prol69Gln + Tyr210Pro +

Leu221Met Leul04His + Thrll3Gln + Asnl40Gln + Thrl67Asn +

Phel93Asp Gly 67Pro + Asn βδSer + Glyl39Asp + Thr215Gly +

Thr217Pro Glyl36Ser + Vall36Met 4- Thrl67Gln + Tyr21δAsn +

Ser220Asp Aspl05Glu + GlyllOSer + Trpll2His + Tyr210Gln +

Ala219Thr Asn βδAsp 4- AsnlOβGln 4- Alal64Ser + Thrl95Gln +

Trp20δCys Leul04Val + Leul34Ala + Thrl37Asn 4- Asnl70Asp +

Leu221Ile Gly 67Ser + Leul34Met 4- Trp20δAla + Tyr210Ser +

Thr217Pro Alall4Gly + Glyl35Asp + Glyl39Ser + Asnl70Ser +

Thr217Ser ThrlllAsn + Phel93Ser + Pro205Gln + Trp20δThr +

Leu221Glu Serl09Asp 4- Vall38Asn + Phel93Ala 4- Trp208Cys +

Thr212Gln Ser 70Asp 4- AsnlββSer + He209Pro 4- Tyr210Met +

Leu221Ser AsnlOβSer + Leul34Cys 4* Glyl36Ser + AlalβδThr +

Tyr210Glu Gly 67Gln + Leul34Gln + Glyl35Asp 4- Phel93His +

Thr224Gly Asn βδSer -J- Leul04Ala + Glyl62Asp + Tyrl71Met +

Thr217Gly Asnl63Glu 4- Thr212Gln + Thr215Asn 4- Thr217Asn +

Leu221Met Gly 69Gln + AsnlOβGln + Ser207Glu 4- Trp20δThr +

Thr217Gly Glyl39Glu 4- AlalβδHis + Thrl95Gly 4- Leu221His +

Thr224Asn Gly 72Ser + Alall4Asn + Leul34His 4- Prol69Glu +

Trp20δTyr Vall3δSer + Alal64Asn + Tyr210Cys + Tyr21δLeu +

Ser220Asp Leul04Gln 4- Tyrl71Glu 4- Trp20δGly + Thr217Gln +

Ala219His AsnlOβSer + Tyrl71Gly 4- Phel93Gln 4- Thr215Gln +

Ala219Gln Gly 72Ser + GlylOδAsn 4- Trpll2Ile 4- AsnlββGlu +

Leu221Cys Asn βδSer 4- Leul04Thr + Thr215Ser + Ala219Thr + Thr224Asp Vall38Ser + Asnl63Asp + Phel93Cys + Pro205Asn +

Thr217Ser Gly 67Ser + Tyr210Pro + Tyr218Thr + Ser220Asp +

Leu221Ser Glyl35Asn + Glyl36Gln + Glyl62Ser 4- Tyr210Asn +

Pro214Asn Thrl95Ser + Trp208Pro + Tyr210Gly + Ala219Ser +

Leu221Ile Asn bSGlu + ThrlllPro + Asnl63Ser + Phel93Tyr +

Leu221Val Gly ^β7Ser + Thr 73Pro + Asnl70Glu + Tyr210Leu +

Leu221Val Glyl35Asn + Glyl39Ser + Phel93Asp 4- Thr217Gly +

Leu221Gly Glyl36Gln + Glyl39Gln + Glyl65Ser + Tyr210Asp +

Thr217Pro Serl41Glu + Glyl62Pro + Thrlβ7Gly 4- Tyr210Ile +

Thr217Ser Leul04Pro + GlyllOPro + Glyl36Pro 4- Pro205Asn +

Thr217Glu Valll5Ala 4- Alal64Thr + Serl92Asp 4- Trp208Leu -t-

Tyr210Met Thr 73Asn + Vall03Asn + Thr215Asn + Thr217Gln +

Ser222Asp Vall03Pro + Trp208Tyr + Ile209Cys + Tyr210Thr +

Thr217Gln Trpll2Phe + Alall4Ser + Prol69Gln -J- Ser220Glu +

Leu221His GlylOδAsn + Leul34Met 4- Tyr210Ser + Tyr213Ile +

Pro214Glu Alal^βδPro + Trp20δHis + Tyr210Ala + Ser220Glu +

Leu221Gln Glyl39Pro 4- Asnl40Glu 4- Serl41Asp + AsnlββSer +

Trp208Thr Vall03Asn + ThrlllGly + Tyr210Gln + Ser216Asp +

Thr217Glu Vall38His + Glyl39Asn + Ser207Asp 4- Trp208Glu +

Leu221Gln AlalβδAsn + Prol69Gly + Thr217Pro 4- Ser220Asp +

Leu221Asp Tyr210Leu 4- Ala219Gly 4- Ser220Asp + Leu221Asp +

Gly223Ser Vall03Ala 4- Prol69Asn + Thr217Gln 4- Ser220Asp +

Leu221Asp Thr 73Asn 4- Asnl70Asp 4- Tyrl71Glu 4- Tyr210Ile 4-

Leu221Ser Glyl36Ser 4- Serl91Asp + Serl92Glu + Tyr216Cys +

Leu221Asn Thr 73Pro + Valll5Ala + Serl92Asp + Phel93Glu +

Trp20δGln Thrl37Gln + Glyl62Asp + Asnl63Asp + Ala203His +

Pro205Gln Asnl63Gln -r Tyr210Asp + Thr215Pro 4- Ser220Glu +

Gly223Pro AsplOSGlu - AsnlO^βGlu + Serl07Asp + Trp208Phe +

Tyr210Ser Trpll2Gly + Phel93Gln + Ser207Asp + Trp208His +

Ser222Asp Glyl08Asn + Ser207Glu + Tyr213Thr + Thr215Pro +

Ser222Asp Leul34Met -t- Ser207Asp + Trp208Val + Tyr210His +

Ser222Asp Gly 69Glu 4- AsnlOβAsp 4- Alall4His 4- AlalβδSer +

Thr217Gln Leul04Pro 4- Trp206Glu + Thr212Asn + Thr215Ser +

Ser222Asp ThrlllPro + Thrl37Asn + Prol69Gln + Tyr210Glu +

Ala219Glu Thr 73Gly + Valll5Gly + Trp208Glu + Tvr210Asp +

Ser220Glu Glyl35Gln + Glyl36Glu + Asnl70Ser + Tyrl71Glu +

Trp208Gln Thr 73Ser + Serl07Asp 4- Serl09Asp + Glyl36Pro 4-

Tyr210Gly Leul34Pro + Prol69Gln + Pro214Glu + Ser216Glu +

Thr217Gly Aspl05Glu 4- Serl09Glu + Prol69Gly + Tyrl71Gln +

Thr212Ser Glyl35Pro 4- Vall38Ala + Ser207Asp + Tyr210Thr +

Gly223Glu Glyl35Pro 4- Ser207Glu + Trp208Ala + Tyr210Pro +

Gly223Glu Prol69Gln 4- Phel93Val 4- Tyr210Glu 4- Thr215Asn +

Leu221Asp Alall4Gln + Glyl36Ser + Ala219Asn + Ser220Asp +

Ser222Asp Leul34Ser 4- Thrl67Gly + Ser220Asp + Leu221Pro +

Ser222Asp GlylOδGln + Trpll2Leu + Tyr218Glu + Ser220Asp +

Leu221Glu GlylOδPro + Alall4Asn + Ser207Asp + Tyr210Glu +

Ser220Glu Thrl37Glu 4- Glyl62Glu 4- Prol69Asn + Asnl70Ser +

Tyrl71Asp Asn βδGln + Glyl62Glu + Alal64Thr + Glyl65Asp --

Tyrl71Ala Thrl37Pro 4- Glyl39Asp 4- Asnl70Glu 4- Trp208Met +

Tyr210Thr Asn βδGln + Vall38Ser + Glyl62Pro + Alal64Glu 4-

Asnl70Asp Asn 68Gln + Ser207Glu 4- Trp20δThr + Thr217Ser 4-

Ser220Asp Vall3δAla 4- Phel93Thr + Ser207Asp + Tyr210Met +

Ser220Asp Phel93Ala 4- Trp20δGlu 4- He209Ser 4- Leu221Cys 4- Gly223Glu ThrlllGly * Phel93Gln + Ser207Asp + Ser220Asp +

Gly223Glu Ser 70Asp - Aspl05Glu + GlylOδGlu + ThrlllSer +

Tyr210Val Glyl36Glu + Glyl62Asp 4- Glyl65Ser + Alal68Pro +

Thrl95Glu Leul04Gly 4- Glyl65Glu 4- Prol69Glu + Thr217Asn +

Leu221Ala Thrl95Asn 4- Tyr210Asp + Thr217Glu + Ser220Glu +

Gly223Pro Trp208Phe + Tyr210Asp + Tyr213Cys 4- Thr217Glu +

Ser220Glu Ser 70Asp + Glyl08Glu + Tyrl71Gly + Thr217Gln +

Leu221Pro Thrl95Gly + Ser207Glu 4- Tyr210Glu + Tyr213Cys +

Ala219Ser Asn βδGlu + Aspl05Glu + GlyllOAsp + ThrlllPro +

Asnl40Ser Ser 70Asp + Vall03Gln + Pro214Gln + Ser220Asp +

Leu221Glu Asn ^ββAsp + Thr 73Glu 4- AsnlOβSer + Trp20δHis +

Leu221Thr Alal64Asp -J- Thrl67Asp + Tyrl71Pro * Ala219Gln +

Leu221Thr Alall4His + Ala219Gly + Ser220Glu -J- Ser222Glu +

Thr224Glu AsnlOβAsp 4- Serl09Asp + ThrlllAsp 4- Thrl37Ser +

Pro205Asn Leul04Cys 4- Asnl63Glu 4- Serl92Asp 4- Ala219Pro +

Ser222Glu Glyl62Asp + Alal64Gln + Ser207Asp 4- Trp20δTyr +

Gly223Asp Ser 70Asp 4- Serl07Glu 4- Serl09Glu + Valll5Cys +

Glyl35Ser Alall4Gly -J- Ala219Asp + Ser220Glu + Leu221Asn +

Gly223Glu Asn ββAsp 4- Aspl05Glu + Valll5Gln 4- Thrl37Ser +

Trp20δHis Gly 69Glu + Thr 73Glu 4- Vall03His + Serl07Asp +

Leu221Ala GlylOδPro 4- Trpll2Gln + Glyl62Asn -J- Ser220Glu +

Gly223Asp Asn βδAsp 4- Asnl70Gln + Tyrl71Pro 4- Thr215Asp +

Ser216Asp Ser 70Asp + Tyrl71Ser + Thrl95Gly 4- Tyr210Asp +

Ser220Glu Thrll3Gly 4- Glyl62Ser + Trp20δGlu + Ser220Asp +

Thr224Asp Ser 70Glu 4- Asnl40Ser 4- Ala203Gln 4- Ala219Glu +

Gly223Pro Trpll2Pro 4- Asnl70Ser + Ser216Asp 4- Thr217Asp +

Ser220Asp Thr 73Asn + Vall38Asp + Prol69Glu + Asnl70Ser +

Thr217Gly Ser 70Asp + Aspl05Glu + Glyl62Pro + Trp208Val +

Thr217Ser Gly 67Asn + Asnl40Ser + Ala219Glu + Leu221 et +

Ser222Glu Gly 72Ser + GlyllOPro + Trpll2Ala + Tyr210Asp +

Thr217Glu GlylOδPro + Trpll2Leu + Leul34Cys + Glyl35Asp +

Alal64Asp Asn ββGln + Trp208Ala + Thr217Glu + Ser220Glu +

Leu221Thr Thr 73Gly + Leul34Ser + Thr217Glu + Ser220Glu +

Leu221Asn Gly 67Ser + Alal68Gly + Pro214Gln + Thr217Glu +

Ser220Asp Valll5Cys + Glyl39Asn 4- Tyr210Cys + Thr217Asp +

Ser220Asp Leul34Glu + Thrl37Glu + Thrl95Asn + Trp206Ser +

Leu221Ser Alal64Gln + Tyr210Val + Thr215Asp + Thr217Glu +

Ser220Asp Gly 67Asp + Ser 70Asp 4- Thr217Gly + Tyr21δAsp +

Leu221Ile Ser 70Asp + ThrlllAsn + Trp20δCys -J- Thr217Glu +

Ser220Glu Leul34Glu + Glyl62Ser 4- AsnlββGln + Ser207Asp 4-

Gly223Asp Glyl62Pro 4- Thrl67Asn + Prol69Glu + Serl91Glu +

Phel93Asp Ser 70Glu + Trpll2Tyr + Ser207Glu + Tyr21δCys +

Leu221Glu Asn ββGlu 4- Leul04Glu + Serl07Asp 4- Trp20δSer +

Thr215Pro Leul04Ala + Glyl35Asp 4- Thrl95Asp 4- Tyr213Ile +

Thr217Gly Thrl67Gln + Serl92Glu 4- Ser207Glu 4- Trp20δCys +

Ser220Asp Glyl65Pro + Serl91Asp 4- Trp20δGly 4- Ser220Asp +

Leu221Glu Glyl35Glu + Asnl63Asp 4- Tyr213Met 4- Leu221His +

Ser222Asp ThrlllAsp 4- Glyl36Glu 4- Glyl62Glu 4- Alal64Asn +

Pro214Ser Thrll3Gln + Tyr213Thr 4- Ser21βGlu 4- Thr217Glu +

Leu221Glu Ser 70Glu -J- GlylOδGlu 4- Alal64Gly 4- Glyl65Gln +

Leu221Asp Ser 70Glu + Leul04Gln 4- Ser220Asp 4- Leu221His +

Gly223Pro Glyl65Asp + Prol69Ser + Ser207Glu 4- Trp20δGly +

Ser222Asp AlalβδPro 4- Prol69Asn + Thr215Asp 4- Thr217Asp + Leu22lGlu GlyllOPro 4- Thrll3Ser + Glyl65Glu + Trp20βAsp +

Ser222Asp GlyllOGlu 4- Thrll3Asp + Glyl39Glu + Trp20δTyr +

Tyr210Ala Asn ^βδGlu + AsnlOβGlu + Tyrl71His + Tyr210Gln +

Ser216Glu Alal^β4Glu + Alal^βδHis + Ala219Glu + Ser220Asp +

Leu221Cys Asn βδAsp 4- Tyr210Gln 4- Ala219Glu 4- Ser220Glu +

Leu221Ser Thr 73Asp 4- Asnlβ3Ser 4- Ala219His + Ser222Glu +

Gly223Glu Gly 67Asp 4- Asn ^βδGlu 4- AlalβδGln + Prol69Glu +

Phel93Asn GlyllOAsp 4- Glyl39Gln + Asnl40Glu + Serl41Glu +

Tyr218Asn Serl09Glu + Ser216Glu + Thr217Asp 4- Tyr218Asn +

Leu221His Glyl35Pro + Glyl36Ser + Phel93Asp 4- Ser216Asp +

Thr217Glu Glyl65Gln + Serl92Glu 4- Ala203Ser + Ser216Asp +

Thr217Asp Gly 67Gln 4- Leul34His 4- Glyl36Asp 4- Ser207Asp +

Trp206Glu Vall03Ser 4- Ser207Asp + Trp20δGlu + Thr217Glu -t-

Leu221Cys Gly 69Pro + AsplOSGlu 4- AsnlOβAsp 4- AsnlββAsp +

Ile209Thr Trpll2Tyr 4- Asnl70Glu 4- Trp206Gln + Ser220Glu +

Leu221Glu Thrl37Gly 4- Glyl39Gln + Glyl62Asp 4- Ser220Glu +

Leu221Asp Thrll3Asp 4- Alal64Gly + Serl91Glu 4- Serl92Glu +

Thrl95Gly Serl07Glu + Thrll3Glu + Alall4Asp + Tyr210Pro +

Leu221Ala Asn ββAsp 4- Gly 67Asp + Leul34Pro 4- Tyr210Asn +

Ser222Asp Asn βδGln + Leul04Ala 4- Prol69Asp 4- Asnl70Glu 4-

Leu221Asp AsnlOβGlu 4- Serl07Asp + Glyl39Asp 4- Ala203Asn +

Thr217Ser Thr 73Gly 4- Thrl37Glu 4- Tyr210Asp 4- Thr217Gln +

Ser220Glu Trpll2Ile + Glyl36Glu + Phel93Thr + Tyr210Glu +

Ser220Glu Trpll2Ala 4- Asnl63Ser + Serl92Glu + Tyr210Glu +

Ser220Glu Ser 70Glu 4- Thr 73Ser 4- Vall03Asp 4- Pro214Glu +

Tyr218Gly AsnlOβGlu 4- Serl09Glu 4- Thrl95Gln 4- Trp208Pro +

Pro214Glu Asn 68Gln 4- Vall03Asp 4- Thrl37Gln + Ser207Asp +

Ser222Asp Thrl37Asp + Ser207Glu + Thr217Gly + Leu221Pro +

Ser222Asp Gly 69Asp + Thr 73Glu + ThrlllPro + Glyl39Asn +

Ser220Asp Asn ^ββGlu + Ser 70Asp + Pro214Gly + Ser220Glu +

Leu221Ser Ser 70Glu + Leul04Cys + Tyr210Ser + Thr215Glu ■+■

Ser220Glu Thrl67Pro 4- Ser216Glu 4- Ala219Glu + Leu221Met +

Ser222Asp Vall3δAsp 4- AsnlββGlu 4- Thrlβ7Asp -J- AlalβδThr -*

Leu221Ala Leul34Pro + Serl41Glu + Trp20δGlu 4- Thr217Ser +

Ser220Glu Glyl35Glu + Tyrl71Asn + Pro205Gln 4- Trp20δGlu +

Ser220Asp Trpll2Asn + Thrl67Glu 4- AlalβδGly 4- Trp20δAsp +

Ser220Glu Asnl40Asp 4- Prol69Gln + Trp208Glu + Thr215Gln +

Ser220Glu AsnlOβGln + Glyl36Asp + Trp208Glu 4- Thr217Ser +

Ser220Glu AsnlOβAsp + ThrlllAsn + Thrl67Gly + Tyr210Asp +

Ala21 Asp Asn ββGln 4- Prol69Asp 4- Tyrl71Glu + Pro214Asn +

Gly223Glu Trpll2Gly + Asnl63Asp + Thrl67Glu + Serl91Glu +

Gly223Ser Trpll2Asp 4- Thrll3Ser + Alall4Glu + Thr217Glu +

Gly223Gln Asn ββAsp + Thr 73Gly + Vall03Asp + ThrlllPro +

Thrl95Asn Asn βδAsp + Trpll2Met 4- Trp20δAsn 4- Pro214Asp +

Ser220Asp Aspl05Glu 4- Serl09Glu 4- Asnl63Glu 4- AsnlββSer +

Tyr21δGly GlyllOGln + Trpll2Asp 4- Leul34Pro 4- Pro214Asp +

Thr217Asp Gly 67Pro 4- Thrll3Glu 4- Glyl35Asp 4- Glyl62Asp +

Thr215Gln Ser 70Glu + Thrl95Ser + Pro214Ser + Ser220Glu +

Gly223Glu GlyllOPro 4- Vall36Glu + Trp20δGlu + Tyr210Glu +

Thr217Gly GlylOδAsn + Asnl63Glu 4- Ala219Asp + Leu221Val +

Thr224Asp Thr 73Asp 4- GlylOδAsn + Serl09Glu + Pro214Glu +

Thr217Asn Thrl37Asp + Glyl39Glu 4- Asnl70Ser 4- Trp20δCys +

Tyr210Asp Trpll2Phe + Glyl39Glu 4- Tyr213Thr + Ala219Glu + Leu221Glu Leul04Pro + Prol69Glu + Tyr210Glu + Leu221Asp +

Thr224Gly AsplOSGlu 4- Glyl65Glu + Thrlβ7Gln + AlalβδGlu +

Tyr210Pro Thrl37Asp + Trp206Asn + Tyr216Ser + Ser220Asp +

Ser222Asp Gly 69Glu + Thrll3Gln + Thr217Ser + Ser220Glu 4*

Ser222Glu Leul04His 4- Thrll3Asp + Ser207Asp + Thr217Ser +

Leu221Glu Alall4Thr - Glyl35Asp + Ser207Asp + Thr217Ser +

Leu221Asp Gly 69Ser + Leul04Asp + AsnlOβGlu + Serl91Glu +

Thr217Gly Vall03Ala 4- Tyrl71Val + Ser207Asp + Ser216Asp +

Tyr21δAsp Gly 69Ser 4- Leul34Ile 4- Glyl62Asp + Ser207Glu +

Ser220Glu Gly 72Pro 4- Thrll3Glu + AsnlββAsp + Thrl67Asp +

Thr217Gln AsnlββGlu + Thrl67Asp + Ser207Glu + Thr217Gly +

Leu221Val ThrlllAsn + Trpll2Glu + Vall3δGly + Asnlβ3Asp +

Tyrl71Glu Gly ^β7Pro 4- AsnlOβGlu 4- Leul34Pro 4- Thrl37Asp +

Prolβ9Glu Thrl95Glu 4- Ser207Asp 4- Ser220Asp 4- Leu221Val +

Thr224Gly Asn βδAsp 4- Gly 69Gln + Glyl62Asp + Prol69Ser +

Gly223Asp Thr 73Ser + Glyl65Asn + Ser207Asp + Tyr210Asp +

Ser21βGlu Vall03Glu 4- Alal64Asp 4- Asnl70Glu 4- Trp206Ala +

Tyr210Ile Alall4Thr + Alal64Asp + Asnl70Glu + Tyr210Ala +

Thr215Asp Asn βδGlu -t- Glyl39Pro 4- Alal64Pro 4- Serl91Asp 4-

Pro214Glu Asn 68Glu + Alal64Glu + Glylβ5Ser + Tyr210Gly +

Pro214Asp Gly 67Ser 4- Thrl67Gly + Ser207Glu + Ser216Asp +

Ser220Asp Aspl05Glu 4- GlyllOAsn + Ser207Asp + Ala219Gly +

Ser220Glu Vall03Gly 4- Asnl40Asp 4- Ser207Asp + Tyr213Asn +

Ser220Asp Serl09Glu 4- Glyl65Asn + Ser207Glu 4- Tyr210Cys +

Ser220Asp GlyllOAsn 4- Asnl40Glu 4- Ser207Glu 4- Thr217Ser +

Ser220Glu GlyllOAsp + Glyl36Pro + Gly206Gln + Ser207Glu +

Ser220Glu Thrll3Gln + Leul34Ile + Serl92Glu + Thrl95Asp +

Ser220Asp Glyl3^βGlu + Trp208Glu + Thr217Gly + Leu221Asn +

Gly223Asp Serl07Asp + GlyllOAsp + Leul34Ser + Asnl40Glu +

Tyr210Thr Thrl67Glu + Trp208Asp + Thr217Asn + Ala219Asp +

Gly223Asn Gly 69Glu + Alall4Asp + Thrl95Ser + Trp208Ser +

Pro214Glu Vall03Asp + Thrll3Glu + Phel93Tyr + Trp208Asp +

Ala219Pro AsnlO^βAsp + Glyl36Ser + Vall36Met + Serl91Asp +

Ser222Asp Glyl35Asp + Glyl39Glu - Tyr210Ala + Thr217Ser +

Ser222Asp Thrll3Asp + Asnl40Ser + Ala203Gln + Ser207Glu ^■+^■

Tyr210Glu GlylCδGlu + Thrll3Gln + Ser207Glu + Tyr210Asp +

Pro214Asn Glyl35Glu + Asnlβ3Asp + Tyr210Met + Tyr218Pro +

Ser220Asp Asnl63Asp + Alal64Thr + Serl91Glu + Thr217Asp +

Thr224Gln Asn 68Glu + Leul04Asp + Valll5Thr + Alal64Asp +

Pro214Gln Thr 73Gly + Serl07Glu + GlylOδPro 4- Leu221Glu +

Thr224Asp Gly 67Ser + Glyl62Asp + Thrl67Asp + Ser207Glu +

Thr217Ser Gly 67Gln + Serl09Glu + Vall3δSer + Pro214Glu +

Ala219Glu Serl07Glu 4- Glyl35Glu 4- Trp206His 4- Thr217Ser +

Thr224GΪu Asn ββGlu + Aspl05Glu + Vall3δHis 4- Serl92Glu +

Tyr210Ile Asn ββGlu + Aspl05Glu + Ala219Gln + Ser220Glu +

Leu221Asn Gly 69Ser + Ser 70Glu + GlyllOGlu + Thr217Asp +

Leu221Ala Tyrl71Glu + Serl92Asp + Ser207Glu + Trp20δPro +

Tyr210Met Gly 67Gln + Aspl05Glu + Alal64Asp 4- Prol69Asn +

Gly223Asp Thr 73Asp 4- Serl41Asp + Trp206Leu 4- Ser216Glu +

Thr217Pro Asn ββAsp 4- Trp206Gln + Ile209Thr + Tyr210Val +

Ser216Glu Asn ββSer + Vall03Glu + AsnlOβAsp 4- Serl91Glu +

Tyr210Ala Asn 66Gln + Leul34Glu 4- Serl92Glu 4- Leu221Gly +

Ser222Glu Ser 70Asp + Aspl05Glu 4- GlylOΘSer + Glyl39Glu 4- Thr217Gly Gly 67Ser + AsnlOβAsp + Glylβ5Gln + Ala219Asp +

Ser222Glu AsπlO^βGin - Leul34Met + Asnl40Asp + Ala219Asp +

Ser222Asp Gly 69G1 - Vall03Gln + Leul34Asp + Thrl67Gln +

Trp208Gly Thrl37Glu + Glyl65Glu + Tyr210Ser + Thr217Ser +

Leu221Pro Serl07Asp + Glyl62Asp 4- Thrl67Asp + Tyrl71Cys 4-

Trp20δlle AsnlO^βGlu + Trpll2Asp + Leul34Gly + Serl41Glu +

Phel93Ser Alall4Thr + Asnl40Asp + Phel93Thr + Thr217Glu +

Ser220Glu Thrll3Ser + ValllSAsp + Leul34Val + Thr217Asp +

Ser220Glu GlyllOAsp + Asnl70Gln 4- Thr217Glu 4- Tyr21δAla +

Ser220Asp Thrl67Asp + Phel93Thr + Thr217Asp + Ala219Gln +

Ser220Asp GlylOδGlu + Pro214Asn + Thr217Glu + Ser220Asp +

Leu221Val AsnlO^βGln + Glyl35Glu + Vall38His + Thr217Glu +

Ser220Glu GlyllOGlu + Tyr210Met + Thr217Glu + Ser220Asp +

Leu221His Gly 67Giu + Serl07Glu 4- GlyllOSer + Thrll3Glu +

Thr215Ser Gly 67Glu + Serl07Glu + ThrlllPro + AlalβδHis +

Ser220Glu ThrlllAsp + Glyl36Asp 4- Trp208Glu 4- Tyr210Met +

Thr217Gln Aspl05Glu + Glyl62Ser 4- AsnlββAsp 4- Serl91Asp +

Trp208Phe Aspl05Glu + AsnlββGlu + Serl91Glu + Trp208Ala +

Leu221His Gly 67Glu + Vall03Glu + Alal64Asn + Ser207Asp +

Tyr21δAla Gly 72Asn + Leul04Asp 4- AsnlOβGln + Thrll3Asp +

Thr217Asp Ser 70Asp 4- Glyl35Glu 4- AlalβδGlu 4- Tyr210Ile +

Ala219Gly AsnlOβGln + Serl09Glu + Thrll3Asp 4- Thrl37Glu +

Trp206Asn Alall4Ser + Vall3δThr 4- Prol69Asn 4- Phel93Asp +

Trp206Glu Asn βδAsp + Vall3δAsp + Alal64Gly 4- AlalβδGlu +

Thr217Gly Gly 69Asn + Vall38Asp 4- Alal68Glu 4- Ser207Asp +

Thr217Pro GlyllOSer + ThrlllGln 4- Alal64Glu 4- Thr215Ser +

Ser222Asp Leul04Ser + Glyl08Glu + Valll5Glu + Vall38Hιs +

Serl41Glu Ser 70Glu + Alall4Ser + Thrl37Ser + Ser207Asp + Gly 69Asp + GlyllOAsp + Thrl37Glu + Prolβ9Ser +

Tyrl71Hιs Ser 70Asp + Thrl37Glu + Alalβ4Hιs 4- Trp206Ile +

Ser220Glu Ser 70Glu + Thrl37Glu + Thr217Asn + Ser220Glu +

Gly223Asn GlyllOGln + Trpll2Cys + Valll5Glu 4- Asnl40Asp +

Ser216Glu Asn βδAsp + Serl09Glu + AsnlββGln + Trp20δPro +

Leu221Asp Thrll3Pro + Thrl67Glu + Tyrl71Glu + Thr217Gln +

Ser220Glu Trpll2Ile + Leul34Hιs + Glyl39Asp + Glyl62Glu +

Trp20δVal GlyllOAsp + Glyl62Gln + Trp20δlle + Tyr210Glu +

Gly223Asp Gly 67Asp + ThrlllAsp 4- Trp208Val 4- Tyr210Ser +

Ser216Glu Gly 69Asn + Thr 73Glu + AsnlOβAsp + Serl41Glu +

Glyl62Asn Gly 69Glu + Glyl35Asn + Prol69Asn + Trp208Ala +

Thr217Glu Ala203Gly + Trp206Ala + Pro214Glu 4- Ala219Gln +

Leu221Asp Thr 73Gly r GlyllOGlu + Pro214Asp 4- Ser220Glu +

Leu221Asn Vall03Ala + Thrl67Gly + Serl92Glu + Pro214Glu +

Ser220Asp Aspl05Glu 4- Tyrl71Gln + Pro214Asp + Thr217Ser +

Ser220Asp Tyrl71Val 4- Serl91Asp 4- Trp206Pro 4- Pro214Glu +

Ser220Glu Gly 67Asp -J- Glyl62Ser + Gly206Ser + Ser207Glu +

Thr217Glu Gly 67Glu -t- Trpll2Phe + Ser207Asp + Thr217Asp +

Ala219Gln Asn βδAsp 4- Asnl63Glu 4- Phel93Met 4- Gly206Ser +

Leu221Glu Asn ββAsp 4- Leul04Ala 4- Serl09Glu 4- AlalβδThr +

Ser21βGlu Vall36Met + Glyl39Asp 4- Glylβ2Ser + AlalβδGlu +

Ser220Asϋ

TABLE36

Multi-loop Sextuple Mutation Variants ββGln -J- Gly 67Ser 4- Gly 69Ser 4- Leul04Gly + Tyr210Pro 4- Thr217Glu Ser 70Asp + GlyllOPro 4- Glyl36Gln 4- Phel93Ser +

Trp208Cys + Leu221His Thrll3Pro + Glyl39Asn + Serl41Glu + Tyrl71Pro +

Trp206Thr 4- Leu221Gly Gly β9Gln + Leul04Thr + Serl07Asp + Leul34Thr +

Thr215Ser + Leu221Gly Leul04Met + Leul34Ala + Thrl37Asp + Trp20δGly +

Tyr210Val + Pro214Gly Gly 69Ser + Thr ^"-3Asp + GlyllOSer + Tyr210Leu +

Thr215Gly 4- Thr217Ser Asn ββSer + Gly 72Gln + Thr 73Pro 4- GlyllOAsp +

Trp208Val 4- Leu221His ThrlllAsn + Alal64His + Thrl67Glu 4- Phel93Met 4-

Tyr210Asn 4- Thr217Gln Asn ββGln + Thr 73Gly + Thrll3Asp 4- AsnlββSer --

Thrl67Pro 4- Tyr210Pro Leul04Met + Pro205Gln + Ser207Asp -t- Trp206Ala 4-

Thr212Gly 4- Thr217Pro Leul34Val + Glylβ5Gln + Trp20δThr 4- Pro214Gly +

Leu221Ile 4- Thr224Asp Asn ββSer + Serl09Asp + Alall4Asn 4- Valll5Asn +

Trp208Gln + Tyr210Ser AsnlOβGln + Alal64Pro + Glyl65Ser 4- Tyrl71Ala +

Ser220Glu 4- Thr224Ser GlyllOSer + Glyl36Pro + Ile209His + Thr217Gln +

Leu221Ile 4- Thr224Asp Asn ββGln 4- Valll5Pro + Trp20δAsp 4- Thr217Ser +

Leu221Ile 4- Thr224Pro Gly 67Glu 4- Thr 73Asn + Asnl70Ser 4- Tyr210Ser +

Thr212Gln + Gly223Pro Vall03Gly + Alal64Thr + Tyr210His 4- Ala219His +

Ser220Glu + Leu221Gly Thr 73Pro + Asnl40Gln + Thrlβ7Asp 4- Trp20δ et +

Thr217Gln 4- Leu221Asn Serl09Asp 4- Alal64His + Tyr210Pro + Thr212Ser +

Gly223Pro 4- Thr224Asn GlyllOGln + Tyr210Pro + Tyr213Thr -J- Tyr216Met +

Ser220Asp 4- Leu221His Vall3δPro + AlalβδGly + Prol69Gln + Tyr210Ser +

Thr217Gln -t- Ser220Asp Alall4Thr + Thrl37Asn + Glyl39Ser 4- Trp208Pro +

Pro214Gln + Ξer220Glu Vall03Met + Thrl37Ser + Ala203Ser + Tyr210Thr +

Leu221Met + Gly223Ser Leul04Glu ^•+^• ThrlllSer + Valll5His 4- Glyl65Asn +

Tyr210Met + Ala219His GlyllOPro + Leul34Pro 4- AsnlββAsp 4- Asnl70Ser +

Trp208Asn 4- Thr217Asn Thrl67Glu + Prol69Gly + Asnl70Gln 4- Trp208Gly +

Thr215Ser 4- Leu221Ser ThrlllSer + Thrl37Ser + Thrl95Gln + Ser207Glu +

Tyr210Val -t- Thr217Pro AsnlOβAsp + Glyl36Pro + Glyl62Ser 4- Trp208Ile -t- Tyr210Gly + Pro214Gln Ser 70Asp + Glyl65Gln + Phel93Asn + Thrl95Ser +

Trp208Pro 4- Thr212Ser Gly 72Ser + Alall4Gln + AsnlββGln + Phel93Glu +

Tyr210Gly 4- Thr224Pro Asn ββSer + Glyl36Ser + Glyl62Pro + Asnl63Ser +

Alal64Asp + Alal68Gln Gly ^β7Pro + AsnlOβGln + Serl91Asp + Trp208His +

Tyr218His + Leu221Asn Trpll2Leu + AlalβδPro 4- Trp208His + Tyr210Ser +

Thr217Gly + Ser220Asp Asn ^ββSer + ThrlllGly + ValllδGly 4- AlalβδSer +

Asnl70Gln + Pro214Gly GlyllOSer + Trpll2Val + Glyl62Asn + Thrlβ7Pro +

Prolβ9Gln + Ser216Asp Gly β9Asn + AsnlOβSer + Glyl39Glu 4- Asnl70Ser +

Tyr210Cys 4- Leu221Gly Gly 69Asn + Serl41Asp + Phel93Asn + Tyr210Ile -t-

Ala219Asn + Leu221Ser Serl09Asp -J- Prol69Ser 4- Trp20δHis + Pro214Asn +

Thr215Asn + Thr217Gly Asn βδAsp + Glyl62Gln 4- Alal64His + Trp206Pro +

Tyr210Leu 4- Leu221Val Vall03Thr + Glyl39Pro + Alal64Thr + Thr217Ser +

Leu221Pro + Thr224Pro Gly 69Glu + Gly 72Gln + Thrl37Pro + Trp206Ile +

Tyr216Pro 4- Gly223Gln Gly 67Pro + Leul04Cys -J- Vall3δMet 4- Glyl39Ser +

Ala203Gln + Ala219Asn Trpll2Ser + Phel93Ala + Thr217Gly 4- Tyr21δGlu +

Leu221Met + Thr224Asn Asnl63Gln + Trp206Leu + Tyr210Ser + Thr217Glu +

Ala219Gly + Gly223Pro Vall03His 4- GlyllOGln + Glyl39Ser + Serl92Asp +

Phel93Asn 4- Thr217Gln GlylOδGln + Prol69Asn 4- Thrl95Ser + Trp20δAla +

Thr215Pro + Thr217Glu Serl09Asp + ThrlllAsn + Trpll2Gln + Thrl67Gln +

Thrl95Gly + Tyr210Asn Gly 72Gln 4- Trpll2Leu + Glyl36Asp 4- Tyrl71His +

Tyr210Thr + Leu221Ser Gly 69Glu 4- Alal64Pro + Trp206Val + Tyr210Thr +

Tyr21δGln 4- Thr224Ser Alall4Pro 4- Leul34Glu 4- Asnl40Ser 4- Alal64Gln +

Phel93Ile 4- Trp20δAsn Asn ββSer 4- Vall3δMet 4- Glyl39Asn -J- Asnl63Gln +

Glyl65Gln 4- Tyr210Thr AsnlOβGln 4- Serl91Asp 4- Phel93Leu 4- Trp20δVal +

Pro214Gln + Thr217Ser Alall4Thr + AsnlββSer + Tyrl71Gly + Serl91Glu +

Ala203Pro + Tyr210Ala Asn βδGln + Tyrl71Asp + Phel93Tyr + Gly20βSer +

Tyr210Val -J- Leu221Thr Leul04Ala + Alall4Ser - Asnl63Asp + Thrl95Gly +

Gly206Gln + Leu221Ala GlyllOAsn + Valll5Asn + Trp20δCys -t- Tyr210His +

Ala219Thr + Gly223Asp Asn ^ββAsp 1- Gly 69Asn 4- ValllSAla + Alal64Ser +

Trp208Gln + Thr217Gln Valll5Met + Thrl37Ser + Glyl62Asn + AsnlββGlu +

Tyrl71His + Pro214Ser AsnlO^βGln + GlyllOGln + Trp20δHis 4- Thr217Pro +

Ala219Asp + Ser220Asp Gly 67Asn + Gly 72Asn + Alall4Gly + Asnl40Gln + Gly 69Gln + Thr 73Ser + Prol69Gln + Trp20δlle 4-

Ser220Glu + Leu221Asp Asnl70Glu + Tyrl71Asp + Phel93Ser + Trp20δHis +

Leu221Thr + Thr224Gly Gly 67Ser + Gly ^β9Glu + Ser 70Glu + Vall03Gly +

Tyr210Gln + Ala219Asn Trpll2Gly + AsnlββSer + Trp20δMet + Tyr210Glu +

Ser220Glu + Thr224Asn Leul34Pro 4- Thrl95Gly + Trp206Glu -J- Tyr210Asp 4-

Thr217Ser + Ser220Glu Leul04Gln + Trpll2Gln + Tyrl71Pro + Trp20δGlu -f-

Thr217Gly + Leu221Glu Aspl05Glu + GlylOδAsp 4- Glyl3βGln + Thrlβ7Gln +

Tyrl71Gln + Leu221Val Serl07Glu + Serl09Glu 4- Vall38Met + AlalβδHis +

Prol69Gln + Phel93Ser Gly 67Glu + Ser 70Glu + Trpll2His 4- Tyrl71Ile +

Trp20δHis 4- Tyr210Ala Vall03Pro 4- Trp206Glu 4- Tyr210Ser + Ala219Glu +

Ser220Glu + Ser222Asp Serl91Asp 4- Ser207Glu 4- Tyr210Ala + Thr215Gly +

Thr217Gln + Gly223Ser AsnlOβGlu + Serl09Asp + AlalβδThr + Tyrl71Met +

Phel93Ile + Trp208Leu Asn βδSer + Thrl37Gln + Serl91Asp + Ser207Asp +

Trp206Asp 4- Leu221Thr Vall03Gln + Vall3δHis 4- AsnlββAsp + Serl92Glu +

Trp208His 4- Leu221Gly Vall03Pro 4- Glyl39Pro 4- Asnl63Asp + Glyl65Gln +

AlalβδGlu 4- Tyr210Leu Asn 68Ser 4- Thrll3Gln + Leul34Ile + Serl91Glu +

Leu221His 4- Gly223Glu GlyllOGln + Ser207Glu 4- Trp208Gln + Thr2l7Pro +

Tyr218Gly 4- Ser220Asp Glylβ2Ser + Phel93Ala + Trp208Glu 4- He209Ser +

Leu221Cys 4- Gly223Glu Ser 70Asp + Aspl05Glu + GlylOδGlu + Asnl40Gln +

Trp20δLeu 4- Thr217Gly GlyllOAsn + Thrl37Glu 4- Prol69Asp -J- Asnl70Ser +

Thrl95Glu 4- Tyr210His sn βδGln 4- Glyl62Glu 4- Alal64Thr + Glyl65Asp 4- Tyrl71Ala - Serl92Asp Vall38Met + Glyl39Asn + Asnl70Gln + Tyr210Glu +

Thr21^"7Asp - Ser220Glu Glyl39Ser + Asnl63Asp - Alal64Ser + Serl91Asp 4-

Tyr210Gly + Leu221Ala Ser 70Asp + Thr 73Asp 4- Thrl67Ser + Trp208Ser +

Thr217Asp + Ala219Asp Asn ββGlu 4- Leul04Glu 4- Aspl05Glu + Serl07Asp ^■+^■

Leu221Ser + Gly223Pro Asn ββGlu + Thr 73Asp 4- GlyllOPro 4- AlalβδGly +

Tyr21δVal -r Leu221Ile Gly 67Asn + Aspl05Glu + AsnlOβGlu + ThrlllGlu +

GlylβSAsn - Thr217Gly Asn ββAsp + Tyr210Ser - Pro214Glu + Thr215Asp 4-

Thr217Gln 4- Tyr216Asp Thr 73Asn + Serl92Glu + Ala203Thr + Ser207Asp +

Trp20δGlu 4- Leu221Glu Vall36Gly + Prol69Glu 4- Serl92Glu 4- Thrl95Glu +

Trp20δPro 4- Thr212Gln Thr 73Asp 4- GlylOδAsn + Leul34Ser + Tyr210Ala +

Thr217Gln 4- Tyr216Glu Thr 73Asn + Alall4Thr + Vall36Ser + Glyl39Asp +

Serl41Glu + Asnl70Glu GlylOδGln + Glyl35Pro + Glyl62Asn + Ser220Glu +

Leu221Ala 4- Gly223Glu Vall03Gln + Trpll2Cys + Alal64Glu + Trp20δGln +

Thr217Gly + Gly223Asp ThrlllGln + Leul34His + Serl91Asp + Phel93Asρ +

Thrl95Gln + Trp206Asp Vall03Ser + Leul04Ile 4- GlyllOGlu + Glyl36Glu +

Alal64Asn + Tyr210Gln Leul34Ser + Thrl67Gly + Serl91Glu + Ser220Asp +

Leu221Pro -J- Ser222Asp Gly 69Gln + Trp20δAsp + Pro214Glu 4- Ser220Asp +

Ser222Glu 4- Gly223Asn Serl07Asp - Serl09Glu + Trpll2Pro -J- Glyl35Asp +

AlalβδThr + Thr212Gly Asnl40Ser -J- Thrl67Asp + Trp208Glu 4- Thr217Asn +

Ser220Asp 4- Leu221Asp Glyl35Asp + Glyl62Glu 4- Tyrl71Asp 4- Tyr210Ala +

Thr217Ser 4- Ser222Asp Valll5Asp + Tyr210Gln + Ser220Glu 4- Leu221Asp +

Ser222Asp 4- Gly223Ser Alall4His + AsnlββAsp + Tyrl71Cys + Tyr210Glu +

Ser220Asp 4- Leu221Asp Valll5Cys 4- Glyl39Asn + Alal64Gly + Pro205Asn +

Thr217Asp 4- Ser220Asp Thrl67Gln + Tyr210Ile + Thr215Gly + Thr217Asp +

Tyr218Asn 4- Ser220Asp Thrl37Ser 4- Trp208Leu 4- Tyr210Leu 4- Thr217Glu +

Ser220Asp 4- Leu221Gly ThrlllAsn + Glyl35Ser + Trp208Asp + Tyr210Glu +

Thr217Glu 4- Leu221Ala ill

Thrll3Asn + Alall4Gly + Thrl37Asρ 4- Thrl67Asp +

Trp208Gln 4- Gly223Asn Ser 70Asp -t- Thr 73Glu + Asnl63Asp 4- Thrl67Pro +

Pro214Glu + Thr217Gly Glyl35Glu + Tyrl71Asn + Pro205Gln + Ser207Glu +

Trp20δGlu + Ser220Asp Gly 69Asp + Aspl05Glu 4- GlylOδAsp + Ser207Asp +

Trp20δThr + Gly223Asn Thrll3Gly + Alall4Pro ^■+- Tyr210Asp 4- Pro214Asp 4-

Thr217Gln + Ser220Asp Asn βδGlu 4- Ser 70Asp + Aspl05Glu + Alall4Thr +

Glyl35Ser + Ser220Asp ThrlllSer + Thrl37Glu + Ser207Glu + Trp20δThr 4-

Ser220Glu + Leu221Asp Gly 69Asp + Leul04Asp + Aspl05Glu + Asnl40Glu 4-

AsnlββSer 4- Leu221His Thrl37Asp + Glyl39Glu + Asnl70Glu + Trp20δCys +

Tyr210Asp + Leu221Thr Gly 67Pro + Trpll2Gln + Serl92Asp + Tyr210Ile +

Ser220Asp + Ser222Asp Vall03Gln + Glyl08Asp + Glyl35Glu + Thrl37Glu +

AlalβδGln + Tyr210Asn Valll5Asp + Leul34Glu + AsnlββGln 4- Asnl70Asp +

Tyr210Val 4- Thr212Pro Asn ββAsp + Asn βδSer + Thrl67Gln + Ile209Leu +

Ser2lβAsp + Thr217Glu Vall03Glu + GlylOδAsp 4- GlyllOGlu 4- Glyl65Pro +

Thrl95Glu + Thr217Gln Asn ββAsp + Ser 70Glu + Thr 73Asp + Serl09Asp +

Glyl36Pro + Tyr210Ile Gly 69Asp 4- Ser 70Glu + Aspl05Glu + Asnl63Glu +

Tyr210Met + Thr217Gln Asn βδGln + Leul34Glu + Ser207Asp 4- Trp20δPro +

Tyr210Asp + Leu221Asp Asn ββSer + Valll5Gly + Glyl62Asp + Serl91Glu +

Trp208Ile + Thr217Ser Ser 70Glu + Tyr213Thr + Thr217Asn 4- Tyr21δGly +

Ser222Glu + Gly223Asp Trpll2Met + Glyl65Asp 4- Prol69Ser + Ser207Glu +

Trp208Gly + Ser222Asp Gly 69Ser + Serl09Asp + GlyllOGlu + Alall4Gly +

Glyl36Asp + Phel93Asp Alalβ4Gln + Glyl65Pro + Serl92Asp + Phel93Asp +

Ser207Glu + Thr217Asp Asn 68Glu + Thr 73Asn + Glyl62Gln 4- Trp208Gln +

Ala219Glu + Ser220Asp Ser 70Glu + Leul34Ser - Glylβ5Asp + AsnlββAsp +

Alal68Gly + Ile209Gly Leul04Asp + Aspl05Glu + Trp206Gly + Tyr210Glu +

Tyr21δAla + Gly223Pro Asn 68Asp + Gly 69Glu + ThrlllAsn + Trpll2His 4-

Glyl36Asn + Prol69Glu Glyl08Ser + Asnl40Asp + Serl41Glu + Alal64Gln + Ser207Glu + Tyr218Ser Leul04Cys + Glyl62Pro 4- Ser207Glu + Ser216Glu +

Thr2l7Asp + Ala219Thr Alal64Asn + Asnl^ββAsp + Gly206Gln + Tyr210Thr +

Ser2lβAsp + Thr217Asp GiylOPAsp + Serl09Asp + AsnlββSer + Thr217Gln +

Ser220^*Asp + Leu221Thr Serl09Asp + GlyllOAsp + Vall3δAla + Tyrl71His +

Ala204His + Ser222Glu Asn 68Gln + Thrl37Gly + Prol69Asp + Ser207Glu +

Trp208Glu 4- Tyr21δGln Trpll2Asp + Thrll3Glu + Vall36Gly + Serl91Glu +

Trp208Pro + Leu221Ser Leul04Gly + Aspl05Glu + AsnlOβGlu + Gly206Ser +

Trp20δHιs + Tyr21δGlu GlylOδAsp + Leul34Asn + Glyl36Gln + Trp20δPhe +

Ser22θ^"Asp + Leu221Asp GlylOSGln + Trpll2Tyr + Asnl70Glu + Trp206Gln +

Ser220Glu + Leu221Glu Gly 72Asn + AsnlββAsp + Phel93Ser + Trp20δTyr +

Ser220Glu + Leu221Glu Asn ββGlu + Gly 67Glu + ThrlllAsn + Thr215Gln +

Thr217Asn + Ser220Glu AsnlOβAsp + Serl07Asp + Glyl65Asp + Phel93Val +

Trp20δVal + Leu221Thr Leul04Val + Glyl65Gln + Serl92Asp + Phel93Glu +

Tyr210Glu + Leu221Cys Glyl36Gln - Prol69Ser + Serl92Asp + Phel93Asp +

Trp20δLeu + 3er220Glu Serl07Asp + ValllSThr + Thrl37Gln + Glyl62Asp +

Asnl63Asp + Ala203His Vall03Met + Serl07Glu + Trp206Val + Thr215Glu +

Ser216Glu + Gly223Ser Vall03Ser + Asnl40Asp + Serl41Glu + Tyr210Glu +

Ser220Asp 4- Gly223Asn Asnl63Asp + Asnl70Glu + Thrl95Asn + Ser207Asp +

Tyr210Met + Tyr21δPro Asn ββAsp + Glyl39Gln + Tyr210Glu + Thr217Ser +

Ser220Asp + Thr224Asn Gly 69Asp + AsnlOβAsp + GlyllOAsp + Glyl65Asn +

Prol69Ser + Thr217Asp Phel93Ile + Thr215Ser + Thr217Glu + Ser220Asp +

Leu221Pro + Gly223Glu Vall03Gly + Aspl05Glu + Thrl95Asn + Ser207Asp +

Ser222Asp + Gly223Asn Gly 69Asp + Asnl40Gln + Alal64His + AlalβδSer +

Ser207Asp + Ser222Asp Asn 68Asp + ThrlllAsn + Alall4Gly + Glyl35Gln +

Ser207Glu + Ser222Glu AsnlOβAsp + Glyl39Asn + Glyl62Glu + Prol69Ser +

Serl91Asp + Thr224Asp Thrll3Asp + Serl92Asp + Ser207Glu 4- Tyr213Thr +

Thr215Pro + Ser222Asp Gly 69Pro + Asnlβ3Asp + Thrl95Glu + Trp206Ser +

Tyr210Glu + Leu221Cys Gly 69Pro -r Ser 70Asp + GlyllOSer + Glyl35Glu +

Asnl70Glu + Leu221Ile Gly 69Glu ⁺ Alal64His 4- Thrl95Gln + Ser220Glu +

Leu221Glu + Thr224Glu Gly 69Gln + Glyl36Asp + Vall36Asp + Prol69Gly +

Asnl70Gln + Ser220Glu Glyl36Glu + Vall3δAsp + Thrl95Asn + Ser220Glu +

Leu221Gly + Gly223Pro Asn βδGln + Serl07Glu + Serl09Glu + Glyl39Gln +

Alal64Gln + Ser207Asp Serl07Glu + Serl09Glu + Prol69Gln + Tyr210Met +

Thr217Pro + Ser222Asp Gly 67Asp + Ser 70Glu + Vall03Ala + ThrlllGlu +

Tyr210Asn + Pro214Ser Asn β^βGlu 4- Gly 67Asp + Leul34His + Serl91Asp +

Ser207Glu + Trp206Thr Asn β^βGlu + Vall03Asp + GlylOδAsn + Asnl70Ser +

Thr217Asn + Leu221Met Thr 73Glu + Trpll2Pro 4- AsnlββSer + Phel93His +

Tyr218Ile + Ser220Glu Valll5His + Ser207Glu + Trp208Tyr 4- Tyr210Leu +

Pro214Glu + Leu221Glu Asn ββGln + Serl07Glu + Serl09Glu + Ser207Glu +

Gly223Asp + Thr224Gly Asn ββGlu + Asn 68Asp + Thr 73Gln + Thrl95Ser +

Ser207Asp + Tyr21δPro Ser 70Asp + Leul04Ile + Asnlβ3Asp + Thr217Gln +

Leu221Val + Thr224Glu Gly 67Asp + Vall03Glu + AsnlOβAsp + AlalβδAsn +

Thrl95Glu + Trp208Leu Thrl37Gly 4- Glyl62Glu + Phel93Asp + Trp20δAsp +

Thr217Asn + Tyr218Val Glyl35Pro + Glyl39Glu + Ser207Glu 4- Trp208Ala +

Tyr210Cys + Gly223Glu AsnlOβSer + Thrl37Asp + Glyl39Glu + Serl41Asp +

Ala204Gln + Trp206Glu Trpll2Ala + Asnl63Glu + Serl92Glu + Trp206Val +

Tyr210Glu 4- Ser220Glu GlyllOAsp + Trpll2Asp 4- Tyr210Thr + Ser220Glu +

Leu221Met + Ser222Glu Gly 67Asp 4- Gly 69Asp + Thr 73Pro 4* Serl09Asp +

Trp208Gln + Ser222Asp Asn ββSer + Vall03Glu + Aspl05Glu + Trpll2His +

Serl91Glu + Tyr210Ala Gly 67Glu + Gly 69Asn + Glyl62Glu + Ser207Glu +

Thr217Asn + Gly223Glu Ser 70Asp 4- Glyl65Asp + AlalβδAsp + Trp20δPro +

Thr217Gly + Leu221Ile Gly 67Glu + Vall03Asn + Glyl3βGln + Trp208Pro +

Ser216Glu + Leu221Met Gly 67Asn + Glyl39Glu + Serl41Glu + Tyr213Gly + Ser220Asp * Leu221Asn Gly β7Glu + Leul04Ser 4- Tyr210Ser + Ser220Asp +

Ser222Giu ^■i- Gly223Gln Gly β7Asp + Asn βδGlu -t- Glyl36Asp + AlalβθGln +

Prol69Glu 4- Phel93Asn Asn ββGlu + Gly 69Asp + Asnl40Gln 4- Serl92Asp +

Phel93Asp 4- Tyr210Leu Leul04Asp + AsnlOβGlu + Asnl70Glu 4- Tyr210Ile +

Thr215Ser + Thr224Pro Leul04Asp ^J- Serl07Glu + Glyl35Asp + Glyl39Asn +

Alal64Glu + Thr217Gly Trpll2Glu + Glyl39Glu + Alal64Glu + Thrl95Glu +

Thr212Ser + Tyr213Ser Asn 68Asp + ThrlllGly + Thrll3Ser + Vall38Asp +

Asnl70Asp + Phel93Ser Asnl63Gln + AlalβδAsp + Asnl70Asp 4- Ser207Glu +

Thr217Gly + Ser220Asp Gly 67Asp + AsnlOβGlu + Thrll3Ser + Glyl36Gln +

AsnlββGlu + Leu221Cys Asn ββGln + GlyllOAsp + Alall4His + Glyl36Glu 4-

Thrl37Asp + Ser222Glu Gly 67Asp + Asn βδSer + Gly 72Pro + Glyl39Ser +

Thrl67Glu + Thrl95Asp Asn ββAsp + Asn 66Gln + Serl91Glu + Ser207Asp +

Trp208Val + Ser220Asp Serl09Glu + Alall4Glu + Leul34Ile 4* Glyl36Ser +

Ala219Glu + Ser220Asp Valll5Glu + AlalβδHis + Prol69Asn + Tyr210Asp + Asn ββGlu + AsnlOβAsp + Asnl70Ser 4- Trp208Thr +

Ser2lβAsp + Tyr21δGly Glyl62Asn + Asnl70Asp + Phel93Glu + Tyr210Gln +

Ser220Glu + Thr224Asp GlylOδSer + Leul34Pro + Thrl37Asp + Prol69Glu +

Ser207Glu + Thr217Gly Thrl37Asp + Vall36Glu + Asnl63Glu 4- Alal64Asn +

Alal68Ser + Thr217Glu Glyl62Glu + Trp206Glu ^■+^■ Tyr210Val 4- Thr217Glu +

Ser220Glu + Gly223Pro Glyl35Asp + Vall38Thr + Ser207Asp 4- Thr217Pro +

Tyr21δlle + Ser220Glu Leul04Gly + Serl92Asp + Thrl95Asp + Thr217Pro +

Ser220Glu + Leu221Asn Asn ββSer + Glyl36Glu 4- Vall38Glu 4- Trp20δLeu +

Thr217Asp + Ser220Glu Gly 69Ser + Vall03Asp + Serl09Asp + Serl91Asp +

Gly206Gln + Tyr210Pro Alall4Gly + Glyl62Asp + Pro214Gly + Thr215Asp +

Thr217Glu + Ser220Glu Thrl37Gln + Glyl39Asp + Glyl65Pro 4- Tyrl71Glu +

Thr217Glu + Ser220Glu Gly 67Glu + Aspl05Glu + Asnl40Glu + Phel93Gly +

Trp206Leu + Leu221Cys Leul04Giu + Trpll2Leu + Phel93Leu + Ser207Asp +

Trp203Ala + Thr224Asp Ser 70Glu + Thrll3Gly + Glyl62Ser + Asnl63Asp +

Alalβ4Glu + Ser220Asp Gly 69Glu + Ser 70Asp + ThrlllGlu + Ser220Asp +

Leu221Pro + Gly223Ser Asn ^ββGln + Leul04Pro 4- Glyl36Glu + Asnl63Asp +

Tyrl71Ile + Leu221Glu Ser 70Asp + Leul04Glu + AlalβδAsn + Prol69Glu +

Thrl95Asp + Trp20δPhe Ser 70Glu + Glyl62Glu + Trp20δGlu + Thr217Gly +

Leu221Thr + Ser222Asp Gly 69Asn + Thrl37Glu + Glyl39Pro + AsnlββAsp +

Prol69Asp + Trp20δAsp Gly β9Glu + Leul04Glu + Valll5Ser + Asnl40Ser +

Serl92Glu + Pro214Asp Leul04His + Aspl05Glu + Vall3δHis + Asnl63Asp +

Serl92Glu + Thr217Asn Alall4Asn + Asnl63Glu + Asnl70Ser + Serl92Asp +

Ala204Asn + Thr217Asp Asn 68Gln + Thr 73Asp + Serl09Glu 4- Trp208Phe +

Pro214Glu + Ser220Asp Valll5Cys + Leul34Asp + Glyl35Pro + Alal64Thr +

Asnl70Asp + Trp20δAsp Asn ββGlu + Gly 69Glu + Trpll2Gln + Glyl35Asn +

Glyl39Asp + Asnl70Glu Thr 73Gly + Serl07Glu + Glyl35Asp + Prol69Asp +

Trp20δThr + Leu221Asn Gly 67Ser + Ser 70Glu + Glyl36Asp + Prol69Asn +

Leu221Asp + Gly223Asp Ser 70Asp + GlyllOGlu + Vall3δSer + Tyr210Val +

Leu221Asp + Gly223Asp Asn ββAsp 4- Leul04Glu + Serl09Glu 4- Tyr210Asn +

Thr217Asn + Ser220Asp Asn ββGlu + Gly 67Asp + Asnl63Glu + Ser207Glu +

Thr215Gly + Leu221Thr Gly 69Glu + Tyr210Glu + Thr215Asp 4- Thr217Gln +

Tyr218Gln + Ser220Glu Ser 70Asp + Thrl37Glu 4- Alal64His + Thrl67Gly +

Trp20δAsp 4- Ser220Glu Vall36Met + Glyl39Gln + Asnl40Glu + AlalβδPro +

Tyrl71Glu + Thr215Glu Vall03Gly 4- AsnlOβSer + Serl07Asp + ThrlllAsp +

Ser222Glu + Gly223Glu Gly 72Gln + AsnlOβGlu + GlyllOAsp + Thrll3Glu +

Glyl35Asn + Ala219Glu Serl09Glu + Thrll3Asp + Trp20δlle + Ser220Glu +

Leu221Asp 4- Thr224Ser Gly 67Asn + Ser 70Asp + GlylOδAsp + Asnl70Glu +

Thr215Pro + Leu221Thr Gly 67Asp + Serl07Asp + Thrlβ7Gly + Tyr210Asn +

Ser220Asp + Ser222Glu Gly 69Asp + Thr 73Glu + AsnlOβGln + ThrlllAsn + Vall38Glu + Thr217Asp ThrlllAsn -t- Serl91Asp + Thrl95Asp + Ala204Thr +

Ala219Ser + Ser220Glu Serl09Asp + Serl91Glu + Tyr210Val + Thr215Asn +

Leu221Gly + Ser222Glu Thr 73Glu - Leul04Asp + AsnlOβGln + Thrl95Ser +

Ser207Glu + Ser220Asp Asn ββAsp + Thr 73Asp + Leul04Glu + Glyl3βGln +

Glyl62Asp + Leu221Gly Gly 72Asn + Leul04Asp + Asnl63Asp + Tyrl71Ser +

Serl91Glu 4- Thr217Asn Valll5Asp + Glyl36Gln + Tyr210Glu + Tyr218Cys +

Leu221Asn + Ser222Asp Thr 73Asp + Asnl40Ser + Asnl63Glu 4- Thrl95Asp +

Ser220Asp + Leu221Pro Asn βδGlu + Leul04Asp + Prol69Asn + Trp208Val +

Thr217Asn + Gly223Glu GlyllOGlu + Thrl67Gln + Serl92Glu 4- Ser207Glu +

Trp20δCys + Ser220Asp Vall03Gly + Glyl36Glu + Asnl40Asp + Ser207Asp +

Tyr213Asn + Ser220Asp Thrll3Asp + Glyl36Asp + Thrl37Gln + Asnl63Glu 4-

AsnlββGlu + Trp20δPhe Asn ββGlu + Gly 69Glu ^■+^■ Serl09Asp + Serl92Asp +

Phel93Val + Tyr216Ala Ser 70Glu + Serl41Asp + Asnl63Ser + Trp20δMet +

Tyr210Pro + Thr215Asp Aspl05Glu + GlyllOAsn + ThrlllGlu + Leul34Ala +

Serl41Glu + Pheι93Tyr Asn ββGlu + Gly 69Ser + GlylOδAsp + ThrlllAsp +

Thrl95Gly + Tyr210Leu Glyl65Glu + Tyrl71Asn + Ser207Glu + Thr215Glu +

Thr217Gly 4- Thr224Glu Asn ββGln + Thr 73Ser + Glyl39Asn + Pro214Asp +

Ser220Asp + Leu221Ile AsnlββGlu -t- Serl92Asp + Trp20δThr + Tyr210Gly +

Thr215Asp + Ser222Glu Asn ββAsp + Serl91Asp + Thr215Asn + Ala219Asp +

Ser222Asp + Gly223Gln Aspl05Glu + GlylOδGlu + Thrl95Asn + Ile209Leu +

Pro214Asp 4* Ser220Asp ThrlllGlu + Vall3δAsp + Asnl63Ser + Tyr218Met +

Leu221Glu + Gly223Asp Gly 67Glu + Vall3δGlu + Serl41Glu + Trp208Ile +

Pro214Asn 4- Thr215Asp Trpll2Leu + Thrll3Gln + Glyl65Glu 4- Thrl67Asp +

Thr217Glu + Ser220Asp Gly 67Gln + Asn βδAsp + Gly 69Asp + Phel93Ala +

Ser216Glu + Ser222Glu Ser 70Asp + Valll5Glu - Glyl62Ser + AlalβδThr +

Tyr210Gly + Thr217Asp Ser 70Asp + Glyl36Asn + Glyl65Glu + Tyr210Asn +

Thr217Asp + Leu221His Thr 73Ser + Vall03Asn + Serl09Asp + Serl92Asp +

Ser207Asp 4- Trp20δlle AsnlOβAsp + Serl92Glu + Phel93Pro 4- Ser207Glu +

Thr2l7Gln + Tyr218Ile Serl41Asp -* Thrl67Asp + AlalβδAsp + Serl91Asp +

Ala219^*Asn - Leu221Thr Asn ββGln + Serl41Asp + Asnl70Ser + Thr217Gln +

Ser220Asp + Gly223Asp Thr 73Gly + Aspl05Glu + Alall4His + Ser220Glu +

Leu221Asn + Gly223Glu AsnlOβAsp 4- Asnl40Gln + Trp208Ser + Tyr210Cys +

Ser220Asp + Gly223Glu Gly 67Gln + Aspl05Glu + Alal64Asp 4* Prol69Asn +

Tyr218Leu + Gly223Asp Trpll2Val + AsnlββAsp + Phel93Gly + Pro214Glu +

Ser220Asp + Ser222Glu GlylOδAsp + ThrlllAsn + Serl92Glu + Thr217Asn +

Ala219Glu + Ser220Asp Vall03Ser + Valll5Asp + Glyl39Glu + AsnlββSer +

Ser207Glu + Ser220Glu Leul04Asp +^■ Aspl05Glu + Asnl40Glu + Thrl67Asp +

Pro214Gly + Ala219Gly Gly 67Asp + Asn βδAsp + ThrlllAsp + Alal64Gln +

Phel93Ser + Ser207Asp Serl09Glu + Trp208Asp + Ser216Glu + Thr217Asp +

Tyr21δAsn + Leu221His Serl41Asp + Alal68Glu + Tyr210Met + Ser216Glu +

Thr217Asp + Tyr218Ala Serl09Asp + GlyllOAsp + AsnlββAsp 4- Asnl70Ser 4-

Thr215Ser + Leu221Asp GlyllOGlu + Thrl67Glu + AlalβθAsp + Trp206Met +

Ser220Asp + Thr224Gln Aspl05Glu + AsnlOβAsp 4- Glylβ5Asp + Thrl95Ser +

Tyr21δlle + Ser220Asp AsnlOβGlu + Serl41Asp + Tyr216Ser + Ala219Thr +

Ser220Asp + Leu221Glu Thr 73Gln + ThrlllGlu + AsnlββAsp + Trp20δSer +

Ser220Asp + Leu221Asp Serl07Glu + AlalβδAsn + Prol69Asn + Serl91Asp 4-

Serl92Asp + Thr217Asp Alall4Glu + AsnlββSer + Thr212Pro + Thr217Glu +

Tyr216Asp + Thr224Asp AsnlOβGlu + Serl07Asp + Glyl39Asp + Ala203Asn +

Thr217Ser + Ser220Asp Thrll3Asn + Glyl39Ser + Serl41Glu + Thr215Glu +

Ser2lβAsp + Ser220Asp Gly 69Gln + Asnl40Glu 4- Glyl62Glu 4- Asnl63Asp +

Tyr210Gln + Ser220Asp Asn βδAsp + Alall4Pro + Glyl35Glu + Trp206Asp +

Leu221Ile + Gly223Asp Asn βδAsp 4* Alal64Glu + Trp20δCys + Tyr210Glu +

Ala219Thr + Ser220Asp ThrlllAsp + AsnlββAsp 4- Asnl70Gln + Tyr210Asp 4- fhr212Ser + Ser220Glu Serl41Glu + Glyl65Asp + Trp208Ile + Tyr210Asp +

Thr217Gln + Ser220Asp Asn ββGln + Vall03Asp + Glyl08Pro + Thrl37Glu +

Tyr210Glu + Ser220Glu GlylOδGlu + Thrl37Gln + Asnl63Glu 4- Tyr210Glu +

Ser220Asp + Leu221Cys Gly β9Gln + Ser 70Glu + Glyl35Glu + Asnl63Asp +

Thr217Gly + Ser220Asp AsnlOβGlu + Asnl40Glu + Asnl63Glu + Asnl70Glu +

Thrl95Asn + Leu221His AsnlOβGln + AsnlββAsp 4- AlalβδAsn 4- Asnl70Glu +

Ser207Glu + Ser220Asp Asn ββAsp + Ser 70Asp + GlyllOGln + Glyl36Asp +

Pro214Gln + Ser220Asp Asn βδSer + Prol69Asp + Serl92Glu + Ala219Asp +

Gly223Ser + Thr224Asp Thr 73Pro + Glyl35Ser + Phel93Glu + Gly206Gln +

Tyr210Asp + Thr217Glu Trpll2Glu + Valll5Gln + Asnl70Gln + Thr217Asp +

Tyr216Met + Ser220Asp GlyllOAsn + ValllSGlu + Thrl37Gln + Ile209Gly +

Thr217Asp + Ser220Glu GlylOδGlu + Thrll3Gln + Pro214Asn + Thr217Glu +

Tyr21δGln 4- Ser220Asp GlyllOSer + ThrlllGly + Thrll3Glu + Phel93Pro +

Thr217Glu ^• Ser220Asp AsnlββGlu 4- Trp208Ile + Tyr210Met + Thr217Glu +

Tyr218Val + Ser220Asp Ser 70Glu + Vall03Glu + Valll5Gln 4- Prol69Ser +

Tyr210Ala -t- Ser220Asp Asn ββAsp + Serl07Asp + Trp20δMet + Tyr210Pro +

Ser220Asp + Ser222Glu GlyllOSer + ThrlllAsp + Glyl35Gln + Glyl36Asp +

Thrl37Asn + Trp206Glu Thrl67Asp + AlalβδSer + Prol69Glu + Trp20δThr +

Thr215Asp 4- Leu221Asp ValllSGln + AlalβδGln + Prol69Gln + Phel93Asp + ^*Tyr210Ser + Leu221Asp Asn ββGlu + Glyl36Pro + Asnl40Gln + Serl91Asp +

Trp208Asp + Leu221His Glyl36Glu + Glyl39Glu + Serl92Asp + Ala203Asn +

Trp208Asp + Leu221His Gly 67Asn + Thrll3Gln + Thrl37Glu + Thrl67Asp +

Ala219Gln 4- Leu221Glu Gly 69Asn + Thr 73Asp + ThrlllPro + AsnlββAsp 4-

Serl91Asp + Leu221Val Vall38Pro + AsnlββGlu 4- Serl91Asp + Tyr218Asn +

Ser220Asp + Leu221Gln Aspl05Glu + Glyl39Asn + Glyl65Glu + Trp208Met +

Tyr218Thr + Gly223Asp GlyllOAsn + ThrlllPro + Alall4Asp + Asnl70Asp +

Trp208Asp + Tyr210Asp Gly 67Gln + Gly 69Glu + Serl07Asp + Thrlβ7Glu +

Alal68Asn + Ser207Asp Gly β9Glu + Glyl65Glu + Trp20δCys + Tyr210Cys +

Pro214Asp 4- Ser220Asp Ser 70Glu + Gly 72Gln + Vall03Thr + Serl09Glu +

Thrll3Glu + Ser220Asp Gly 69Pro + Valll5Glu 4- Tyr210Asn + Ser216Asp +

Ser220Glu + Ser222Glu Glyl36Glu + Trp203Ser + Ser216Asp + Ser220Asp +

Leu221Asn + Ser222Asp Leul04Glu + Valll5Pro + Glyl62Ser + AsnlββAsp +

Ser220Asp + Ser222Asp Gly 67Glu + Vall38Asp + Thr217Ser + Ser220Asp +

Leu221His + Ser222Asp Asn 68Asp + Asnl70Glu + Gly206Asn + Ser207Asp +

Trp20δlle + Leu221Asp AsnlO^βGln + Serl09Glu 4- Thrll3Asp + Leul34Gln +

Thrl37Glu + Trp20δAsn AsnlOβGlu + Glyl39Gln 4 Thrl67Glu + Prol69Gln +

Serl91Asp + Ser207Glu Serl07Glu 4* Valll5Asp + Glylβ5Asn + Trp20δThr +

Tyr21δAsp + Ser220Glu Gly 72Asn + AsnlOβGln + Glyl39Asp + Prol69Glu +

Trp20δPro + Ser220Glu Vall36Asp + Serl41Glu + Prol69Ser + Ser207Glu +

Ala219Glu + Leu221Thr AsnlOβGln + Alall4Asp + Glyl35Asp + Thrl95Asn +

Ser207Glu + Ser220Asp Ser 70Asp + AsnlββGlu 4- Asnl70Gln + Ser216Glu +

Thr217Gln + Thr224Gly Gly 69Asp + Asnl40Ser + Glyl62Pro + Trp20δGly +

Thr217Glu + Leu221Asp Asn βδAsp + Serl07Asp + Alall4Gln + Glyl62Asp 4-

Thr217Pro + Ser220Glu Ser 70Asp + ThrlllAsp + Ile209Met + Thr217Gly +

Ser220Glu + Leu221Asn Ser 70Asp + Leul04His + ThrlllGln + Tyrl71Ser +

Phel93Asp + Ser220Glu Thirl13Gly + Glyl39Pro 4- Serl41Glu + Tyrl71Glu +

Serl92Asp + He209Ser Trpll2Ala + Thrll3Glu + Vall3δGln + Serl41Asp +

Ser216Asp + Leu221Ser Gly 69Asn + AsnlOβAsp + Trpll2Gly + Glyl62Asp +

Phel93Glu + Ser220Glu Asn βδSer + Vall03Glu + AsnlOβAsp + AlalβδPro +

Prol69Asp 4- Pro214Glu Leul04Glu + Alall4Ser + Asnl40Ser + Glyl65Asp +

Ser207Asp + Ser220Glu Asn βδAsp + Serl09Glu + Glyl39Glu + Ala204Ser +

Leu221His + Gly223Asn ThrlllGly + Vall3δPro + Glyl39Asp + Ser207Glu +

Ser216Glu + Ser220Asp AsnlOβAsp 4- Asnl70Ser + Serl92Glu + Thrl95Asp 4- Trp208His + Ser220Glu AsnlOβAsp + Glyl62Asp + AsnlββGln + Thrl67Asp +

Pro214Asp + Aia219Thr ValllSGlu + Glyl36Glu + Ser207Glu + Tyr218Gly +

Ala219Pro + Leu221Pro Glyl3βSer - Glyl62Asp + Asnl63Gln + Alal64His + Asn 68Ser + Thr 73Glu 4- AsnlββGlu + Asnl70Gln +

Tyrl71Gly + Leu221Asp Thrll3Glu + Valll5Cys 4- Thrl37Glu + Serl91Glu +

Thr217Ser + Thr224Glu Gly 67Asp + Serl07Asp + Glyl39Asp + Glyl65Pro +

Thrl67^~Ser + Thr217Glu Thr 73Gly + Glyl08Asp + Thrl37Glu + Glylβ2Glu +

Tyr210Gln + Ser220Asp Leul04Asp + Glyl39Pro + Tyrl71Asp + Serl91Asp +

Ser222Glu + Thr224Ser Thrll3Asp + Prol69Glu + Tyr210Gln + Pro214Ser +

Ser220Glu + Gly223Asp GlylOδAsp + Glyl3βGlu + Serl41Asp 4- Thrl95Asp +

Thr215Ser + Thr217Gln Serl07Asp 4- Asnlβ3Glu -t- Prol69Gly + Ser207Asp +

Trp20δAsn + Gly223Asn GlyllOAsp + Asnlβ3Asp + Serl91Asp + Thrl95Gln +

Tyr210Glu 4- Thr217Pro Vall03Glu + GlylOδAsp + Ala203Gly + Trp20δ et +

Ala219Asp + Thr224Glu Thr 73Gln + ThrlllAsp + Leul34Asp + Glyl36Ser +

Asnl63Gln + Ser207Asp

π. Cleaning Compositions

In another embodiment of the present invention, an effective amount of one or more of the enzyme variants are included in compositions useful for cleaning a variety of surfaces in need of proteinaceous stain removal. Such cleaning compositions include detergent compositions for cleaning hard surfaces, unlimited in form (e.g., liquid and granular); detergent compositions for cleaning fabrics, unlimited in form (e.g., granular, liquid and bar formulations); dishwashing compositions (unlimited in form); oral cleaning compositions, unlimited in form (e.g., dentifrice, toothpaste and mouthwash formulations); denture cleaning compositions, unlimited in form (e.g., liquid, tablet); and contact lens cleaning compositions, unlimited in form (e.g., liquid, tablet).

The cleaning compositions also comprise, in addition to the Thermitase variants described hereinbefore, one or more cleaning composition materials compatible with the protease enzyme, the term "cleaning composition material", as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the Thermitase variant used in the composition, the specific selection of cleaning composition materials are readily made by considering the surface material to be cleaned, the desired form of the composition for the cleaning condition during use (e.g., through the wash detergent use). The term "compatible", as used herein, means the cleaning composition materials do not reduce the proteolytic activity of the Thermitase variant to such an extent that the protease is not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.

As used herein, "effective amount of enzyme variant" refers to the quantity of enzyme variant necessary to achieve the enzymatic activity necessary in the specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like. Preferably the cleaning compositions comprise from about 0.0001 % to about 10% of one or more enzyme variants of the present invention, more preferably from about 0.001% to about 1 %, more preferably still from about 0.01 % to about 0.1 %. Several examples of various cleaning compositions wherein the enzyme variants may be employed are discussed in further detail below. All parts, percentages and ratios used herein are by weight unless otherwise specified.

As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, oral cleaning compositions, denture cleaning compositions and contact lens cleaning compositions. A. Cleaning Compositions for Hard Surfaces. Dishes and Fabrics

The enzyme variants of the present invention can be used in a variety of detergent compositions where high sudsing and good insoluble substrate removal are desired. Thus the enzyme variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions and the like. Such compositions can be in the form of liquids, granules, bars and the like. Such compositions can be formulated as modern "concentrated" detergents which contain as much as 30%-60% by weight of surfactants.

The cleaning compositions herein can optionally, and preferably, contain various anionic, nonionic, zwitterionic, etc., surfactants. Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.

Nonlimiting examples of surfactants useful herein include the conventional C -| 1 -C 18 a-M benzene sulfonates and primary and random alkyl sulfates, the Cιn-C^*i 8 secondary (2,3) alkyl sulfates of the formulas CH₃(CH₂)x(CHOSO3)-M⁺)CH₃ and CH₃(CH₂)y(CHOSO₃-M⁺) CH₂CH₃ wherein x and (y+1 ) are integers of at least about 7, preferably at least about 9, and M is a water-solubilizing cation, especially sodium, the C-I Q- C^*i8 alkyl alkoxy sulfates (especially EO 1 -5 ethoxy sulfates), C-I Q-CI S alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C-_| o- Ci 8 alkyl polyglycosides, and their corresponding sulfated polyglycosides, Ci2-C-| 8 alpha-sulfonated fatty acid esters, C12-C18 alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C<|2-C-|8 betaines and sulfobetaines ("sultaines"), C-J O-C-I S amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. (Use of such surfactants in combination with the aforesaid amine oxide and/or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.) Other conventional useful surfactants are listed in standard texts. Particularly useful surfactants include the C10-C18 N-methyl glucamides disclosed in US Patent 5, 194,639, Connor et al., issued March 16, 1993, incorporated herein by reference.

A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C10-C16 alkolamides can be incorporated into the compositions, typically at about 1 % to about 10% levels. The C10-C14 onoethanol and diethanol amides illustrate a typical class of such suds boosters. Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous. If desired, soluble magnesium salts such as MgCl2, MgSO4, and the like, can be added at levels of, typically, from about 0.1 % to about 2%, to provide additionally sudsing.

The liquid detergent compositions herein can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydnc alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1 ,3-propanediol, ethylene glycol, glycerine, and 1 ,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.

The detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11.0. Finished products thus are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1 -10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies.

Likewise, the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001 % to about 1 % by weight. Various detersive and fabric care enzymes are well-known in the layndry detergent art.

Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1 % to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1 % to about 10% by weight.

Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various bnghteners, especially anionic bnghteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1 % to about 35% by weight Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials

Enzyme stabilizers may also be used in the cleaning compositions Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0 1 % to about 1 %) and calcium formate (preferably from about 0 1 % to about 1 %) 1 Hard surface cleaning compositions

As used herein "hard surface cleaning composition" refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like Hard surface cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0 001 % to about 10%, more preferably from about 01 % to about 5%, more preferably still from about 05% to about 1% by weight of active enzyme of the composition In addition to comprising one or more of the enzyme variants, such hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy/streaky residue on the glass surface

The surfactant component, when present, may comprise as little as 0 1 % of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%, more preferably from about 1 % to about 5% of surfactant.

Typically the compositions will contain from about 0 5% to about 50% of a detergency builder, preferably from about 1 % to about 10%

Preferably the pH should be in the range of about 8 to 12 Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary

Solvents may be included in the compositions Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trimethyl-1 ,3-pentanediol and 2-ethyl-1 ,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, preferably from about 3% to about 11 %.

Additionally, highly volatile solvents such as isopropanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface. When used, volatile solvents are typically present at levels of from about 2% to about 12% in the compositions.

The hard surface cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 7-12 Liquid Hard Surface Cleaning Compositions

Example No.

Component 8 9 10 11 12

*Disodium N-diethyleneglycol-N,N-iminodiacetate ^**Na4 ethylenediamine diacetic acid ***Diethyleneglycol monohexyl ether ****AII formulas adjusted to pH 7

In Examples 7-10, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gln66Asn, with substantially similar results.

In Examples 11-12, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gln66Asn and Gly206Asn, with substantially similar results.

Examples 13-18 Spray Compositions for Cleaning Hard Surfaces and Removing Household Mildew

Product pH is about 7.

In Examples 13-16, the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr111Asn, with substantially similar results.

In Examples 17-18, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr111Asn and Thr217Gly + Ser222Glu, with substantially similar results. 2. Dishwashing Compositions

In another embodiment of the present invention, dishwashing compositions comprise one or more enzyme variants of the present invention. As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to, granular and liquid forms. The dishwashing composition embodiment of the present invention is illustrated by the following examples. Examples 19-24 Dishwashing Composition

Product pH is adjusted to 7.

In Examples 19-22, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly139Asn, with substantially similar results.

In Examples 23-24, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly139Asn and Ser207Glu + Tyr210Ser + Gly223Asn, with substantially similar results. 3.. Fabric cleaning compositions

In another embodiment of the present invention, fabric cleaning compositions comprise one or more enzyme variants of the present invention. As used herein, "fabric cleaning composition" refers to all forms for detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms. Preferred fabric cleaning compositions are those in the liquid form. a. Granular fabric cleaning compositions

The granular fabric cleaning compositions of the present invention contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0 005% to about 5%, more preferably from about 0 01 % to about 1 % by weight of active enzyme of the composition In addition to one or more enzyme variants, the granular fabric cleaning compositions typically comprise at least one surfactant, one or more builders, and, in some cases, a bleaching agent

The granular fabric cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 25-28 Granular Fabric Cleaning Composition

In Examples 25-26, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala168Asn, with substantially similar results.

In Examples 27-28, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala168Asn and Tyr210Gly + Thr212Pro + Pro214Gly + Ser222Asp, with substantially similar results Examples 29-32 Granular Fabric Cleaning Composition

In Examples 29-30, the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr195Pro, with substantially similar results.

In Examples 31-32, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Thr195Pro and Trp208Phe + Tyr213Met + Thr215Pro + Ser220Glu + Thr224Gly, with substantially similar results.

Examples 33-36 Granular Fabric Cleaning Composition

In Examples 33-34, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly67Ser - Gly72Ser, with substantially similar results

In Examples 35-36, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly67Ser + Gly72Ser and Tyr171Thr with substantially similar results

Examples 37-40 Granular Fabric Cleaning Composition

In Examples 37-38, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser192Asp + Phe193Leu + Thr195Asn, with substantially similar results.

In Examples 39-40, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser192Asp + Phe193Leu + Thr195Asn and Tyr213Ser + Thr217Gly + Gly223Glu, with substantially similar results.

Examples 41-42 Granular Fabric Cleaning Composition

Examples 43-44 Granular Fabric Cleaning Composition

Example No.

Component 43 44

Sodium linear C-J2 alkyl benzene-sulfonate

Sodium sulfate

Zeolite A

Sodium nitrilotriacetate

Polyvinyl pyrrolidone

Tetraacetylethylene diamine

Boric acid

Perborate

Phenol sulphonate

Pro205Gly + Trp208Leu + Thr217Gln + Leu221 Gln + Gly223Gln + Thr224Glu

Fillers (e.g., silicates; carbonates; Up to 100 Up to 100 perfumes; water)

Example 45 Compact Granular Fabric Cleaning Composition

Component Weight %

Alkyl Sulphate 8.0

Alkyl Ethoxy Sulphate 2.0

Mixture of C25 and C45 alcohol 3 and 7 times ethoxylated 6.0

Polyhydroxy fatty acid amide 2.5

Zeolite 17.0

Layered silicate/citrate 16.0

Carbonate 7.0

Maleic acid acrylic acid copolymer 5.0

Soil release polymer 0.4

Carboxymethyl cellulose 0.4

Poly (4-vinylpyhdine) -N-oxide 0.1

Copolymer of vinylimidazole and vinylpyrrolidone 0.1

PEG2000 0.2

Asn66Ser + Ser70Glu + Gly72Asn 0.5

Lipase 0.2

Cellulase 0.2

Tetracetylethylene diamine 6.0 Percarbonate 22.0

Ethylene diamine disuccinic acid 0.3

Suds suppressor 3.5

Dιsodιum-4.4'-bιs (2-morpholιno -4-anιlιno-s-tπazιn-6- 0.25 ylamino) stilbene-2,2'-disulphonate

Disodium-4,4'-bιs (2-sulfostyril) biphenyl 0.05

Water, Perfume and Minors Up to 100

Example 46 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulphonate 7.6

C16-C18 alkyl sulfate 1.3

^14-15 alcohol 7 times ethoxylated 4.0

Coco-alkyl-dimethyl hydroxyethyl ammonium chloride 1.4

Dispersant 0.07

Silicone fluid 0.8

Trisodium citrate 5.0

Zeolite 4A 15.0

Maleic acid acrylic acid copolymer 4.0 Diethylene tπamine penta methylene phosphonic acid 0.4

Perborate 15.0 Tetraacetylethylene diamine 5.0

Smectite clay 10.0

Poly (oxy ethylene) (MW 300,000) 0.3 Ser109Glu + Thr113Gly 0.4

Lipase 0.2

A ylase 0.3

Cellulase 0.2

Sodium silicate 3.0

Sodium carbonate 10.0 Carboxymethyl cellulose 0.2

Brighteners 0.2

Water, perfume and minors Up to 100 Example 47 Granular Fabric Cleaning Composition

Component Weight %

Linear alkyl benzene sulfonate 6 92

Tallow alkyl sulfate 2 05

C 14"15 alcohol 7 times ethoxylated 4 4

C-12-15 ^a'^ky' ethoxy sulfate - 3 times ethoxylated 0 16

Zeolite 202

Citrate 5 5

Carbonate 15 4

Silicate 3 0

Maleic acid acrylic acid copolymer 4 0

Carboxymethyl cellulase 0 31

Soil release polymer 0 30

Glyl 35Gln + VaM 38Asp + Glyl 39Ser 0 2

Lipase 0 36

Cellulase 0 13

Perborate tetrahydrate 11 64

Perborate monohydrate 8 7

Tetraacetylethylene diamine 5 0

Diethylene tramine penta methyl phosphonic acid 0 38

Magnesium sulfate 0 40

Bnghtener 0 19

Perfume, si cone, suds suppressors 0 85

Minors Up to 100 b_ Liguid fabric cleaning compositions

Liquid fabric cleaning compositions of the present invention comprise an effective amount of one or more enzyme variants of the present invention, preferably from about 0 005% to about 5%, more preferably from about 0 01 % to about 1 %, by weight of active enzyme of the composition Such liquid fabric cleaning compositions typically additionally comprise an anionic surfactant, a fatty acid, a water-soluble detergency builder and water

The liquid fabric cleaning composition embodiment of the present invention is illustrated by the following examples Examples 48-52 Liquid Fabric Cleaning Compositions

In Examples 48-50 the Thermitase variants recited in Tables 2-36, among others, are substituted for Asn166Asp + Thr167Asn + Asn170Ser + Tyr171Thr, with substantially similar results.

In Examples 51-52, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Asn166Asp + Thr167Asn + Asn170Ser + Tyr171Thr and Ser191 Glu + Ser192Asp + Phe193Asp + Thr195Pro, with substantially similar results.

Examples 53-57 Liquid Fabric Cleaning Compositions

In Examples 53-55 the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro205Gln + Gly206Ser + Trp208Val + Tyr210Leu + Thr212Asn 4- Tyr218Met 4- Ser220Glu, with substantially similar results

In Examples 56-57, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro205Gln + Gly206Ser 4- Trp208Val + Tyr210Leu + Thr212Asn + Tyr218Met + Ser220Glu and Asn 68Asp + Val138Asp + Ala164Gly + Ala168Glu + Thr217Gly, with substantially similar results.

Examples 58-59 Liquid Fabric Cleaning Composition

In each of Examples 58 and 59 herein, the Thermitase variants recited in Tables 2-36, among others, are substituted for Leu134Cys + Thr137Glu + Val138Gly, with substantially similar results. Examples 60-62 Liquid Fabric Cleaning Composition

Bar fabric cleaning compositions of the present invention suitable for hand-washing soiled fabrics contain an effective amount of one or more enzyme variants of the present invention, preferably from about 0.001 % to about 10%, more preferably from about 0.01% to about 1 % by weight of the composition.

The bar fabric cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 63-66 Bar Fabric Cleaning Compositions

*Can be selected from convenient materials such as CaCO₃, talc, clay, silicates, and the like

In Examples 63-64 the Thermitase variants recited in Tables 2-36, among others, are substituted for Trp112Cys + Thr217Gly, with substantially similar results

In Examples 65-66, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Trp112Cys + Thr217Gly and VaM03Ala + Thr212Ser, with substantially similar results Examples 67-70 Bar Fabric Cleaning Compositions

*Can be selected from convenient materials such as CaCO₃, talc, clay, silicates, and the like.

In Example 67, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser109Glu, with substantially similar results.

In Example 68, the Thermitase variants recited in Tables 2-36, among others, are substituted for Pro169Glu, with substantially similar results.

In Examples 69-70, any combination of the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser109Glu and Pro169Glu, with substantially similar results. B. Additional Cleaning Compositions

In addition to the hard surface cleaning, dishwashing and fabric cleaning compositions discussed above, one or more enzyme variants of the present invention may be incorporated into a variety of other cleaning compositions where hydrolysis of an insoluble substrate is desired. Such additional cleaning compositions include but are not limited to, oral cleaning compositions, denture cleaning compositions, and contact lens cleaning compositions

1 Oral cleaning compositions

In another embodiment of the present invention, a pharmaceutically- acceptable amount of one or more enzyme variants of the present invention are included in compositions useful for removing proteinaceous stains from teeth or dentures As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like Preferably, the oral cleaning compositions comprise from about 0 0001 % to about 20% of one or more enzyme variants of the present invention, more preferably from about 0 001 % to about 10%, more preferably still from about 0 01 % to about 5%, by weight of the composition, and a pharmaceutically-acceptable carrier As used herein, "pharmaceutically-acceptable" means that drugs, medicaments or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and lower animals without undue toxiαty, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio

Typically, the pharmaceutically-acceptable oral cleaning carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99 99%, preferably from about 65% to about 99 99%, more preferably from about 65% to about 99%, by weight of the composition

The pharmaceutically-acceptable carrier components and optional components which may be included in the oral cleaning compositions of the present invention are well known to those skilled in the art A wide variety of composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in U.S Patent 5,096,700, Seibel, issued March 17, 1992, U S Patent 5,028,414, Sampathkumar, issued July 2, 1991 , and U S Patent 5,028,415, Benedict, Bush and Sunberg, issued July 2, 1991 , all of which are incorporated herein by reference

The oral cleaning composition embodiment of the present invention is illustrated by the following examples Examples 71 -74 Dentifrice Composition

*PEG-6 = Polyethylene glycol having a molecular weight of 600. **Precipitated silica identified as Zeodent 119 offered by J.M. Huber. ^***Carbopol offered by B.F. Goodrich Chemical Company. ****lota Carrageenan offered by Hercules Chemical Company.

In Examples 71-74 the Thermitase variants recited in Tables 2-36, among others, are substituted for Tyr218Ala, with substantially similar results.

Examples 75-78 Mouthwash Composition

In Examples 75-78, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ala164His + Ser220Glu, with substantially similar results.

Examples 79-82 Lozenge Composition

Example No.

Component 79 80 81 82

Leu221Val + Thr224Gln

Sorbitol

Mannitol

Starch

Sweetener

Flavor

Color

Corn Syrup

In Examples 79-82, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser Leu221Val + Thr224Gln, with substantially similar results. Examples 83-86 Chewing Gum Composition

Example No.

Component 83 84 85 86

Ser191Asp + Phe193lle + Thr195Gly 0.03 0.02 0.10 0.05

Sorbitol crystals

Paloja-T gum base^*

Sorbitol (70% aqueous solution)

Mannitol

Glycerine

Flavor

^•Supplied by L A. Dreyfus Company.

In Examples 83-86, the Thermitase variants recited in Tables 2-36, among others, are substituted for Ser191Asp + Phe193lle + Thr195Gly, with substantially similar results. 2. Denture cleaning compositions

In another embodiment of the present invention, denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more enzyme variants of the present invention. Such denture cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0.0001 % to about 50% of one or more of the enzyme variants, more preferably from about 0.001 % to about 35%, more preferably still from about 0.01 % to about 20%, by weight of the composition, and a denture cleansing carrier. Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see for example U.S. Patent 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more of the enzyme variants for removing proteinaceous stains from dentures.

The denture cleaning composition embodiment of the present invention is illustrated by the following examples. Examples 87-90 Two-layer Effervescent Denture Cleansing Tablet

^*Tetraacetylethylene diamine

In Examples 87-90, the Thermitase variants recited in Tables 2-36, among others, are substituted for Gly135Gln + Gly139Asn + Asn140Gln + Ser141Asp, with substantially similar results 3 Contact Lens Cleaning Compositions

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more enzyme variants of the present invention Such contact lens cleaning compositions comprise an effective amount of one or more of the enzyme variants, preferably from about 0 01 % to about 50% of one or more of the enzyme variants, more preferably from about 0 01 % to about 20%, more preferably still from about 1 % to about 5%, by weight of the composition and a contact lens cleaning carrier Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art (see for example U S Patent 4,863,627, Davies, Meaken and Rees, issued September 5, 1989, U S Patent Re 32,672, Huth, Lam and Kirai, reissued May 24, 1988, U S Patent 4,609 493, Schafer, issued September 2, 1986, U S Patent, 4,690,793, Ogunbiyi and Smith, issued September 1 , 1987, U S Patent 4,614,549, Ogunbiyi, Riedhammer and Smith, issued September 30, 1986, and U S Patent 4,285,738, Ogata, issued August 25, 1981 , each of which are incorporated herein by reference), and are generally appropriate for incorporation of one or more enzyme variants of the present invention for removing proteinaceous stains from contact lens

The contact lens cleaning composition embodiment of the present invention is illustrated by the following examples

Examples 91-94 Enzymatic Contact Lens Cleaning Solution

In Examples 91-94, the Thermitase variants recited in Tables 2-36, among others, are substituted for Leu221 Gln, with substantially similar results

While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications of the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention.

Missing upon filing

Missing upon filing

Claims

What is Claimed is:

1 A Thermitase variant having a modified amino acid sequence of

Thermitase wild-type amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region, wherein the modified amino acid sequence comprises a substitution at one or more positions in one or more of the loop regions, wherein

A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 66, 69, 70, 72 or 73, wherein

a. when a substitution occurs at position 66, the substituting amino acid is Asn;

b. when a substitution occurs at position 69, the substituting amino acid is Gln;

c. when a substitution occurs at position 70, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

d. when a substitution occurs at position 72, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and

e. when a substitution occurs at position 73, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

B when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 103, 104, 105 106, 108, 110, 112, 113 or 114, wherein

a. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

b. when a substitution occurs at position 104, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Gly, His, Ile, Met, Pro or Ser;

c. when a substitution occurs at position 105, the substituting amino acid is Glu;

d. when a substitution occurs at position 106, the substituting amino acid is Gln; e. when a substitution occurs at position 108, the substituting amino acid is Asn, Gln, Pro or Ser;

f. when a substitution occurs at position 110, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

g. when a substitution occurs at position 112, the substituting amino acid is Asn, Asp, Cys, Gln, Glu, His, Ile, Met, Phe, Pro, Thr or Tyr;

h. when a substitution occurs at position 113, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser; and

i. when a substitution occurs at position 114, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His,

Pro, Ser or Thr;

C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 134, 135, 136, 137, 138, 139 or 141 , wherein

a. when a substitution occurs at position 134, the substituting amino acid is Ala, Asn, Cys, Gln, Gly, His,

Met, Pro, Ser, Thr or Val,

b. when a substitution occurs at position 135, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser,

c. when a substitution occurs at position 136, the substituting amino acid is Pro,

d. when a substitution occurs at position 137, the substituting amino acid is Asn, Asp, Gln, Glu or Ser; e. when a substitution occurs at position 138, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu,

Gly, His, Met, Pro, Ser or Thr;

f. when a substitution occurs at position 139, the substituting amino acid is Asn, Gln, Pro or Ser; and g. when a substitution occurs at position 141 , the substituting amino acid is Asp or Glu,

D . when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 167, 169, or 171 , wherein a. when a substitution occurs at position 167, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

b. when a substitution occurs at position 169, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or

Ser; and

c. when a substitution occurs at position 171 , the substituting amino acid is His, Ile, Leu, Met or Pro, E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of position 193, wherein a. when a substitution occurs at position 193, the substituting amino acid is Asn, Cys, Gln, His, Ile, Met,

Thr or Tyr;

F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 204, 205 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 220, 221 , 223 or 224, wherein

a. when a substitution occurs at position 204, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His,

Pro, Ser or Thr;

b. when a substitution occurs at position 205, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or

Ser;

c. when a substitution occurs at position 206, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

d. when a substitution occurs at position 207, the substituting amino acid is Asp or Glu,

e. when a substitution occurs at position 208, the substituting amino acid is Asp or Glu,

f. when a substitution occurs at position 209, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu,

Gly, His, Leu, Met, Pro, Ser; Thr or Val,

g. when a substitution occurs at position 210, the substituting amino acid is Asp, His, Ile, Leu, Met or Pro, h . when a substitution occurs at position 211 , the substituting amino acid is Asp or Glu, i. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

j. when a substitution occurs at position 213, the substituting amino acid is Asp, Glu, His, Ile, Leu, Met,

Phe, Pro or Val,

k. when a substitution occurs at position 214, the substituting amino acid is Asn, Gln, Gly, or Ser;

I. when a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

m. when a substitution occurs at position 216, the substituting amino acid is Asp or Glu, n. when a substitution occurs at position 217, the substituting amino acid is Asp, Glu or Pro,

o. when a substitution occurs at position 220, the substituting amino acid is Asp or Glu, p. when a substitution occurs at position 221 , the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu,

Gly, His, Ile, Met, Pro, Ser; Thr or Val, q. when a substitution occurs at position 222, the substituting amino acid is Asp or Glu, r. when a substitution occurs at position 223, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser; and

s. when a substitution occurs at position 224, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or

Ser;

whereby the Thermitase variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Thermitase.

2 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the first loop region.

3 The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the second loop region .

4. The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the third loop region.

5. The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the fourth loop region.

6. The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the fifth loop region.

7. The Thermitase variant of Claim 1 , wherein one or more substitutions occur in the sixth loop region.

8. A cleaning composition selected from the group consisting of a hard surface cleaning composition, a dishwashing composition, an oral cleaning composition, a denture cleansing composition, a contact lens cleaning composition and a fabric cleaning composition, characterized in that the cleaning composition comprises the Thermitase variant of any of Claims 1-7 and a cleaning composition carrier; preferably the cleaning composition is a hard surface cleaning composition or the cleaning composition is a fabric cleaning composition; preferably the composition comprises at least about 5% surfactant and at least about 5% builder; by weight of the composition; preferably the composition further comprises cleaning composition materials selected from the group consisting of solvents, buffers, enzymes, soil release agents, clay soil removal agents, dispersing agents, brighteners, suds suppressors, fabric softeners, suds boosters, enzyme stabilizers, bleaching agents, dyes, perfumes, and mixtures thereof.

9. A DNA sequence encoding the Thermitase variant of any of Claims 1-7.