JP2020143063A - Water-soluble trans-membrane proteins and methods for preparing and using the same - Google Patents

Abstract

To provide computer implemented methods for executing a procedure to select a water-soluble variant of a G Protein-Coupled Receptor (GPCR).SOLUTION: Provided is a method comprising: entering a sequence of the GPCR for analysis; obtaining a variant of the GPCR, in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helical segments ("TM regions") of the GPCR are substituted; subsequently obtaining an α-helical secondary structure result for the variant to verify maintenance of α-helical secondary structures in the variant; and obtaining a trans-membrane region result for the variant to verify water solubility of the variant, thereby selecting the water-soluble variant of the GPCR.SELECTED DRAWING: None

Description

(Related application)
Both of these applications claim the priority of US Patent Application No. 14 / 669,753 and International Application PCT / US No. 2015/022780 filed on March 26, 2015, both of which. 35U. S. C. Under 119 (e), US Provisional Application Nos. 62 / 117,550 filed on February 18, 2015, US Provisional Application Nos. 61 / 993,783 filed on May 15, 2014, and Claims the benefit of the filing date of US Provisional Application No. 61 / 971,388, filed March 27, 2014.

In addition, this application is 35 U.S. S. C. Under 119 (e), US Provisional Application Nos. 62 / 117,550 filed on February 18, 2015, US Provisional Application Nos. 61 / 993,783 filed on May 15, 2014, and It also claims the benefit of the filing date of US Provisional Application No. 61 / 971,388, filed March 27, 2014.

The entire contents of each of the above reference applications, including all of the drawings and sequence listing, are incorporated herein by reference.

Membrane proteins play a heavy role in all biological systems. Approximately 30% of all genes in almost all sequenced genomes encode membrane proteins. However, our in-depth understanding of their structure and function lags far behind our understanding of soluble proteins. As of March 2015, more than 100,000 types of structures are registered in the protein structure databank. However, only 945 membrane protein structures containing 28 G protein-coupled receptors and 530 unique structures containing no tetraspanin membrane protein have been registered.

Although membrane receptors are very important, there are several obstacles to elucidating the structure and function of membrane receptors as well as their recognition and ligand binding properties. The most serious and difficult task is that it is extremely difficult to produce milligrams of soluble and stable receptors. Inexpensive large-scale production methods are urgently needed and are therefore the focus of extensive research. Detailed structural studies can only be performed if these preceding obstacles are overcome.

Zhang et al. (US Pat. No. 8,637,452) (incorporated herein by reference) for water-soluble GPCRs in which certain hydrophobic amino acids located within the transmembrane region have been replaced by polar amino acids. Describes an improved method. However, this method requires a large labor force. Furthermore, although the modified transmembrane region meets the water solubility criteria, improvement in water solubility and ligand binding is desired. Therefore, there is a need for improved research methods for G protein-coupled receptors in the art.

The present invention relates to methods for designing, selecting and / or producing water-soluble membrane proteins and peptides, peptides designed, selected or produced from them (and transmembrane domains), compositions containing said peptides and methods of use thereof. In particular, the method uses water-insoluble amino acids (leucine, isoleucine, valine and phenylalanine or simple letter codes L, I, V, F) as nonionic water-soluble amino acids (glutamine, threonine and tyrosine or simple letter code Q). , T, Y), according to a method for designing a library of water-soluble membrane peptides such as GPCR variants and tetraspanine membrane proteins, using the "QTY principle". In addition, two additional nonionic amino acids, asparagine (N) and serine (S), may be used to replace L, I and V other than F. In the embodiments discussed below, asparagine (N) and serine (S) are substituted for Q and T (described as mutants) or L, I or V (described as native protein). It should be understood that it is supposed to be possible. However, for the sake of brevity, details of these other embodiments will be known to those skilled in the art by the teachings herein and will not be further detailed in this application.

The present invention is a water-soluble polypeptide comprising one or more modified, synthetic and / or non-naturally occurring α-helix domains and one or more such modified α-helix domains (eg, “sGPCR”). In this case, one or more modified α-helix domains are hydrophilic in which multiple hydrophobic amino acid residues (L, I, V, F) within the α-helix domain of a natural membrane protein are nonionic. Includes amino acid sequences substituted with sex amino acid residues (Q, T, T, Y or "Q, T, Y" respectively) and / or N and S. The present invention presents a plurality of hydrophobic amino acid residues (L, I, V, F) within one or more α-helix domains of a natural membrane protein with nonionic hydrophilic amino acid residues (Q / N / S, It also includes a method for preparing a water-soluble polypeptide, which comprises a step of substituting with T / N / S, Y). In the present invention, a plurality of hydrophobic amino acid residues (L, I, V, F) in the α-helix domain of a natural membrane protein are replaced with nonionic hydrophilic amino acid residues (Q / N / S and T / N, respectively). It further includes the polypeptide prepared by substituting with / S, Y). The variant can be characterized by a name (eg, CXCR4-QTY) in which the parent or native protein (eg, CXCR4) is followed by the abbreviation "QTY".

Therefore, one aspect of the invention is (1) a step of inputting a sequence of a membrane protein (eg, GPCR) for analysis and (2) a transmembrane (TM) domain α-helix of the membrane protein (eg, GPCR). A step of obtaining a variant of a membrane protein (eg, GPCR) in which a plurality of hydrophobic amino acids in a segment (“TM region”) is substituted, wherein (a) the hydrophobic amino acid is leucine (L). Selected from the group consisting of isoleucine (I), valine (V) and phenylalanine (F), (b) the leucine (L) is independently glutamine (Q), asparagine (N) or serine (S). Substituted, (c) the isoleucine (I) and the valine (V) are independently substituted with threonine (T), asparagine (N) or serine (S), and (d) the phenylalanine is a tyrosine, respectively. A step of being replaced with (Y), followed by (3) obtaining an α-helix secondary structure result for the variant and confirming the maintenance of the α-helix secondary structure in the variant. 4) It is characterized by including a step of obtaining a transmembrane region result for the variant and confirming the water solubility of the variant, thereby designing a water-soluble variant of a membrane protein (eg, GPCR). Provided is a method of operating a computer program to perform a scripting procedure for designing a water-soluble variant of a membrane protein (eg, a G protein-coupled receptor (GPCR)).

In a particular embodiment, step (3) is performed before, simultaneously with, or after step (4). Further steps described herein can be incorporated into the processing procedure. The processing preferably uses a calculation process preformed by the data processing system. The system utilizes an automated calculation system and a method for selecting protein variants.

In a particular embodiment, in step (2), one subset of the plurality of hydrophobic amino acids within the same TM region of the GPCR is replaced to make one member of the mutant candidate library. And one or more different subsets of the plurality of hydrophobic amino acids are replaced to make additional members of the library. In certain embodiments, the method may further include a step of ranking all members of the library based on a combination score, the combination score being an α-helix secondary structure prediction result and a transmembrane region prediction result. Is a weighted combination of. In certain embodiments, the method further comprises the step of ranking the variant using a ranking function. In certain embodiments, the ranking function may include a secondary structure component and a water soluble component. For example, the ranking function may include weighted values for secondary structure components and / or water-soluble components. In certain embodiments, the method further comprises the step of performing the method by a data processor, which may further include memory attached thereto.

In certain embodiments, the method may further include selecting the N species member having the highest combination score to form a first library of mutant candidates for said TM region. And N is a predetermined integer (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). is there. In certain embodiments, the method further comprises the step of creating a library of one mutant candidate for all three other TM regions of the GPCR. Good. In certain embodiments, the method may further comprise the step of substituting the two or more TM regions of the GPCR with the corresponding TM regions in the mutant candidate library to create a combined mutant library. In certain embodiments, the method further comprises the step of producing / expressing the combination variant. In certain embodiments, the method further comprises testing the combination variant for ligand binding (eg, by the yeast two-hybrid method), where the ligand binding is substantially the same as compared to the GPCR. Select what you have. In certain embodiments, the method further comprises testing the combination variant for the biological function of the GPCR, where it has substantially the same biological function as compared to the GPCR. Select.

Certain water-soluble polypeptides of the invention have the ability to bind ligands that normally bind wild-type or natural membrane proteins (eg, GPCRs). In certain embodiments, amino acids within potential ligand binding sites of a natural membrane protein (eg, GPCR) are not substituted and / or the extracellular and / or intracellular domain of the natural membrane protein (eg, GPCR). The sequence of is the same.

(Non-ionic) hydrophilic residues, which replace one or more hydrophobic residues within the α-helix domain of natural membrane proteins, are glutamine (Q), threonine (T), tyrosine (Y), It is selected from the group consisting of asparagine (N) and serine (S) and any combination thereof. In a further embodiment, the hydrophobic residues selected from leucine (L), isoleucine (I), valine (V) and phenylalanine (F) have been substituted. In certain embodiments, the phenylalanine residue in the α-helix domain of the protein is replaced with tyrosine, and the isoleucine and / or valine residues in the α-helix domain of the protein are independently treonine (or S or N). And / or the leucine residue of the α-helix domain of the protein is independently substituted with glutamine (or S or N).

In certain embodiments, substantially all of the leucine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with glutamine. In certain embodiments, substantially all of the threonine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with threonine. In certain embodiments, substantially all of the valine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with threonine. In certain embodiments, substantially all of the phenylalanine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with tyrosine. In certain embodiments, one or more (eg, 1, 2 or 3) leucines are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the isoleucines are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of said valine are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the phenylalanines are not substituted.

In certain embodiments, the combination mutant library comprises less than about 2 million members. In certain embodiments, the GPCR sequence contains information about the TM region of the GPCR. In certain embodiments, the GPCR sequence is obtained from a protein structure database (eg, PDB, UniProt). In certain embodiments, the TM region of the GPCR is predicted based on the sequence of the GPCR. For example, the TMHMM2.0 (Hidden Markov Model-based Transmembrane Prediction) software module / package can be used to predict the TM region of the GPCR. In certain embodiments, the TMHMM 2.0 software module / package utilizes a dynamic baseline for peak search.

In certain embodiments, the method further comprises providing a polynucleotide sequence for each variant of the GPCR. The polynucleotide sequence is a codon optimized for expression in a host (eg, a bacterium such as Escherichia coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cell, a non-human mammalian cell or a human cell). There may be.

In certain embodiments, the scripting procedure can include a VBA script. In certain embodiments, the scripting procedure can be run on a Linux® system (eg, Ubuntu 12.04 LTS), a Unix® system, a Microsoft Windows operating system, an Android operating system, or an Apple iOS operating system. With the implementation of the present invention, different programming languages can be used, including C ++, Javascript®, MATLAB, and the like. The coded instructions can be stored in a memory device such as a non-temporary computer-readable medium that can be used with computer systems known to those of skill in the art.

In certain embodiments, the α-helix domain is one of seven transmembrane α-helix domains within a natural membrane protein that is a G protein-coupled receptor (GPCR). In some embodiments, the GPCR is a purine receptor (P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ ), M ₁ and M ₃ muscarinic acetylcholine receptor, thrombin receptor (proteoactive receptor (PAR)). ) -1, PAR-2), thromboxane (TXA ₂ ), sphingosine 1-phosphate (S1P ₂ , S1P ₃ , S1P ₄ and S1P ₅ ), lysophosphatidic acid (LPA ₁ , LPA ₂ , LPA ₃ ), angiotensin II (AT ₁ ), serotonin (5-HT _2c and 5-HT ₄ ), somatostatin (sst ₅ ), endoserin (ET _A and ET _B ), cholesistkinin (CCK ₁ ), V _1a vasopressin receptor, D ₅ Dopamine receptor, fMLP formyl peptide receptor, GAL ₂ galanin receptor, EP ₃ prostanoid receptor, A ₁ adenosine receptor, α ₁ adrenalinergic receptor, BB ₂ bombesin receptor, B ₂ brazikinin receptor, calcium sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK ₁ tachykinin receptor, thyroid stimulating hormone (TSH) receptor, protease-activated receptor, neuropeptide receptors, the adenosine A2B receptor, P2Y purinergic receptors , Metalytic glutamate receptor, GRK5, GPCR-30 and CXCR4.

In other embodiments, the natural membrane protein or membrane protein is an essential membrane protein. In a further aspect, the natural membrane protein is a mammalian protein. The protein of the present invention is preferably a human protein. In certain embodiments, reference to a specific GPCR protein (eg, CXCR4) refers to a mammalian GPCR or human GPCR, such as a non-human mammalian GPCR.

In some embodiments, the α-helix domain is a G protein-coupled receptor modified, eg, in an extracellular or intracellular loop, to improve or alter ligand binding, as described elsewhere in the literature. It is one of seven transmembrane α-helix domains within a body (GPCR) mutant. For the purposes of the present invention, the term "natural" or "wild-type" shall refer to a protein (or α-helix domain) prior to water solubilization according to the methods described herein.

In certain embodiments, the membrane protein may be a tetraspanin membrane protein characterized by four transmembrane α-helices. Approximately 54 human tetraspanin membrane proteins have been reviewed and annotated. Many are known to mediate intercellular signaling events that play important roles in the regulation of cell development, activation, proliferation and motility. For example, the CD81 receptor plays an important role as a receptor for hepatitis C virus invasion and malaria parasite infection. The CD81 gene is localized in the tumor suppressor gene region and can be a candidate for transmitting cancer malignancies. CD151 is involved in increased cell motility, infiltration and metastasis of cancer cells. Expression of CD63 correlates with the invasiveness of ovarian cancer. The tetraspanin membrane protein is characterized by a second or large extracellular loop cysteine-cysteine-glycine motif.

Another aspect of the invention is that (1) a plurality of hydrophobic amino acids within the transmembrane (TM) domain α-helix segment (“TM region”) of the GPCR are substituted, wherein (a) said hydrophobic. The sex amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F), and (b) the leucine (L) is independently glutamine (Q) and asparagine. Substituted with (N) or serine (S), (c) said isoleucine (I) and said valine (V) were independently replaced with threonine (T), asparagine (N) or serine (S), respectively. And (d) the phenylalanine is each substituted with tyrosine (Y), and (2) the α-helix secondary structure is maintained by all seven TM regions of the variant. (3) Provided is a water-soluble variant of a G protein-coupled receptor (GPCR), which is characterized by the absence of a predicted transmembrane region.

In certain embodiments, the water-soluble variant is selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64. Contains one or more amino acid sequences. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57. In certain embodiments, the water-soluble variant binds to a CXCR4 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114 and 116-123. Contains the amino acid sequence of. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124. In certain embodiments, the water-soluble variant binds to a CX3CR1 ligand.

In certain embodiments, the water-soluble variants are SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-
Contains one or more amino acid sequences selected from the group consisting of 182. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183. In certain embodiments, the water-soluble variant binds to a CCR3 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234. Contains the amino acid sequence of. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235. In certain embodiments, the water-soluble variant binds to a CCR5 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290. Contains the amino acid sequence of. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. In certain embodiments, the water-soluble variant binds to a CXCR3 ligand.

In certain embodiments, the water-soluble mutant is SEQ ID NO: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317. Includes one or more transmembrane domains described in any one of 319, 321, 323 or 325. In certain embodiments, the variant is water soluble and binds to a ligand for a homologous natural transmembrane protein.

Another aspect of the present invention is (a) culturing the bacterium in a growth medium under conditions suitable for protein production, and (b) dividing the lysate of the bacterium into fractions for soluble and insoluble pellet fractions. A step of producing a portion and (c) a step of isolating the protein from a soluble fraction are included, wherein (1) the protein is a G-protein conjugated receptor according to any one of claims 29 to 46. It is a variant of the body (GPCR) and (2) the yield of the protein is at least 20 mg / L (eg, 30 mg / L, 40 mg / L, 50 mg / L or more) of the growth medium. Provided is a method for producing a protein in a bacterium (for example, Escherichia coli).

In certain embodiments, the bacterium is E. coli BL21 and the growth medium is LB medium. In certain embodiments, the protein is encoded by a plasmid within the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter, such as an inducible promoter inducible by IPTG. In certain embodiments, the lysate is produced by sonication. In certain embodiments, the lysate is centrifuged at 14,500 xg or greater to produce a soluble fraction.

Another aspect of the invention is the treatment of a disorder or disease mediated by the activity of a membrane protein in a subject in need thereof, wherein an effective amount of the water-soluble polypeptide described herein in the subject. A method comprising the step of administering a peptide is provided.

In certain embodiments, the water-soluble polypeptide retains the ligand-binding activity of the membrane protein. Examples of disorders and diseases that can be treated by administering the water-soluble peptides of the invention include, but are not limited to, cancers (eg, small cell lung cancer, melanoma, triple negative breast cancer), Parkinson's disease. , Cardiovascular disease, hypertension and bronchial asthma.

Another aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the water-soluble polypeptide of the invention and a pharmaceutically acceptable carrier or diluent.

In yet another aspect, the invention provides cells transfected with a water-soluble peptide of the subject containing a modified α-helix domain. In certain embodiments, the cells are animal cells (eg, humans, non-human mammals, insects, birds, fish, reptiles, amphibians or other cells), yeast or bacterial cells.

The present invention also includes computer implementation methods performed on a computer system, including one or more of the methods (or steps thereof) described herein. The computer system includes a non-temporary computer-readable medium in which the computer executable instructions are stored, and when the computer executable instructions are executed by the computer system, the computer system causes the computer system to execute this method, and the computer executable instructions are the relevant. When executed by a computer system, it causes the computer system to perform the methods envisioned herein. In addition, at least one memory for storing the sequence data and quantitative results described herein and connected to a memory configured to perform the methods described herein. A computer system with at least one processor is envisioned. A user interface, such as a graphical user interface (GUI), can be used with the electronic display device to select processing parameters that operate to control selection methods, including the calculation methods described herein.

Another aspect of the invention provides a non-transitory computer-readable medium in which a series of instructions for performing any of the methods of the invention is stored.

A further aspect of the invention comprises a data processor that operates to perform amino acid substitutions similar to any of the methods of the invention, ranking protein variants by a ranking function, and water-soluble G protein-coupled receptors. Provided is a data processing system that operates to select a sex variant.

Of course, all embodiments of the invention, including those described in only one aspect of the invention (eg, screening methods), are explicit, as should be readily understood by those skilled in the art. It should be construed as applicable to all aspects of the invention (eg, water-soluble proteins or methods of use) unless the claim is waived or otherwise inappropriate. Should be construed as being combinable with any one or more additional embodiments of.

The above and other objects, features and advantages of the present invention will be apparent from the following more detailed description of typical embodiments of the invention shown in the accompanying drawings in which similar reference numerals refer to the same parts throughout different drawings. Will be. These drawings are not necessarily on scale and instead focus on demonstrating the principles of the invention.

1A-1D are general examples of QTY codes that systematically replace the hydrophobic amino acids L, I, V and F with Q, T, T and Y, respectively. (Fig. 1A) The molecular shapes of the amino acids leucine and glutamine are similar, similarly, the molecular shapes of isoleucine and valine are similar to threonine, and the molecular shapes of phenylalanine and tyrosine are similar. Leucine, isoleucine, valine and phenylalanine are hydrophobic and cannot bind to water molecules. In contrast, glutamine can bind to four water molecules, two hydrogen donors and two hydrogen acceptors, and the -OH groups on treonin and tyrosine are three water molecules, i.e. one hydrogen donor and two Can be combined with an acceptor. FIG. 1B is a side view of the α helix. After applying the QTY code for systematic amino acid changes, the α-helix becomes water soluble. FIG. 1C is a top view of the α-helix before and after the QTY code substitution. The helix on the left is a natural membrane helix with predominantly hydrophobic amino acids, and the helix on the right is the same helix after applying the QTY code substitution. This helix has the most hydrophilic amino acids at this time. (FIG. 1D) Prior to the QTY code, the GPCR membrane proteins are surrounded by hydrophobic lipid molecules so that they are embedded within the lipid membrane (left portion of FIG. 1D). After applying the QTY code, the GPCR membrane protein becomes water soluble and no longer requires a detergent surrounding it for stabilization (right part of FIG. 1D). TMHMM prediction of the transmembrane domain region of CXCR4. This prediction shows seven distinguishable hydrophobic transmembrane segments. In contrast, the TMHMM prediction of the CXCR4 variant (CXCR4-QTY) used in the QTY substitution method of the present invention no longer has seven visible and identifiable hydrophobic transmembrane segments. The predicted α-helix wheel structure of the fully QTY-coded TM1 domain of CXCR4 is shown. It is an example of the mutant candidate in each of the seven TM regions of GPCR CXCR4. Sequence alignment of wild-type proteins and QTY variants of CXCR4, CXCR3, CCR3 and CCR5 respectively. The QTY code applies only to the seven hydrophobic transmembrane segments, not the extracellular and intracellular segments. Sequence alignment of wild-type proteins and QTY variants of CXCR4, CXCR3, CCR3 and CCR5 respectively. The QTY code applies only to the seven hydrophobic transmembrane segments, not the extracellular and intracellular segments. Sequence alignment of wild-type proteins and QTY variants of CXCR4, CXCR3, CCR3 and CCR5 respectively. The QTY code applies only to the seven hydrophobic transmembrane segments, not the extracellular and intracellular segments. Sequence alignment of wild-type proteins and QTY variants of CXCR4, CXCR3, CCR3 and CCR5 respectively. The QTY code applies only to the seven hydrophobic transmembrane segments, not the extracellular and intracellular segments. It is a flowchart of a typical embodiment of this method. It is another flowchart of the typical embodiment of this method. It is an example of the computer system of the present invention. It is the schematic of the flowchart which describes the processing process of the specific preferable embodiment of this invention. It is the schematic of the flowchart which describes the processing process of the specific preferable embodiment of this invention.

The description of the preferred embodiment of the present invention is as follows. The words "one (seed) (a)" or "one (seed) (an)" shall include one or more (species) unless otherwise specified.

In some embodiments, the invention comprises hydrophilic residues leucine (L), isoleucine (I), valine (V) and phenylalanine (F), which are the hydrophobic residues of the seven transmembrane α-helices of the natural protein. With respect to the use of QTY (glutamine, threonine and tyrosine) substitution (ie, "QTY code") methods (or "principles") to convert to glutamine (Q), threonine (T) and tyrosine (Y). In certain embodiments, asparagine (N) and serine (S) can also be used as substitution residues for L, I and / or V other than F, as described above. The present invention can convert a water-insoluble natural membrane protein into a more water-soluble counterpart that still retains some or substantially all the functions of the natural protein.

The present invention includes a method for designing a water-soluble peptide. First, the present method will be described with respect to the GPCR protein, using human CCR3, CCR5, CXCR4 and CX3CR1 as specific examples. However, the general principles of the present invention also apply to other proteins that have a transmembrane (α-helix) region.

GPCRs typically have seven transmembrane α-helices (7 TMs) and 8 loops (8 NTMs) connected by 7 TM regions. These transmembrane segments may be referred to as TM1, TM2, TM3, TM4, TM5, TM6 and TM7. Eight non-transmembrane loops are connected to four extracellular loops EL1, EL2, EL3 and EL4 and four intracellular loops IL1, IL2, IL3 and IL4, thus a total of eight loops (each connected to only one TM region). It is divided into N-terminal and C-terminal loops, each of which has a free end). In this way, the 7TM-GPCR protein can be divided into 15 fragments based on transmembrane and non-transmembrane characteristics.

One aspect of the invention is a method of causing a computer program to perform a scripting procedure to select or prepare a water-soluble variant of a membrane protein (eg, a G protein-coupled receptor (GPCR)).
(1) A step of inputting the sequence of the membrane protein (for example, GPCR) for analysis, and
(2) A variant of the membrane protein (eg, GPCR) in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helix segment (“TM region”) of the membrane protein (eg, GPCR) are substituted. Is the process of obtaining
(A) The hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F).
(B) The leucine (L) is independently replaced with glutamine (Q), asparagine (N) or serine (S).
(C) The isoleucine (I) and the valine (V) are independently substituted with threonine (T), asparagine (N) or serine (S), and (d) the phenylalanine is tyrosine (Y), respectively. A step characterized by being replaced with, followed by (3) a step of obtaining an α-helix secondary structure result for the variant and confirming the maintenance of the α-helix secondary structure in the variant.
(4) To obtain a transmembrane region result for the mutant and confirm the water solubility of the mutant, thereby selecting a water-soluble variant of the membrane protein (eg, GPCR). Provide a method characterized by.

The "water-soluble variant of a membrane (penetrating) protein" or "water-soluble membrane (penetrating) variant" used herein can be used interchangeably.

The exact order in which the steps of the present invention are performed may be variable. For example, in a particular embodiment, step (3) is performed before step (4). In a particular embodiment, step (3) is performed at the same time as step (4). In a particular embodiment, step (4) is followed by step (3).

In certain embodiments, the plurality of hydrophobic amino acids are randomly selected from all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. In certain embodiments, the plurality of hydrophobic amino acids is about 5%, 6%, 7%, 8% of all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% , 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the plurality of hydrophobic amino acids is at least about 10%, 15%, 20%, 25% of all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. , 30%, 35%, 40%, 45%, 50%. In certain embodiments, the plurality of hydrophobic amino acids is about 95%, 90%, 85%, 80% of all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. It is 75%, 70%, 65%, 60% or 50% or less. In certain embodiments, randomly selected hydrophobic amino acids L, I, V and F may be distributed approximately evenly over all TM regions, or 1, 2, 3, 4, 5 or 6 TMs. It may be preferentially or exclusively distributed in the region.

In certain embodiments, all hydrophobic amino acid candidates L, I, V and F on all TM regions of the protein have been substituted. For example, all Ls are independently substituted with Q (or S or N) and / or all I and V are independently substituted with T (or S or N) and / or All Fs are replaced by Y. In certain embodiments, all L's are replaced with Q, all I and V are replaced with T, and all F are replaced with Y.

In certain embodiments, instead of randomly substituting selected hydrophobic amino acids L, I, V and F within all TM regions, all substitutions are first made into any one of the TM regions (eg, most). It can be limited to the N-terminal or C-terminal TM region), and only the desired substitution mutants are selected as members of the mutant candidate library. The position being substituted (eg, the 3rd residue is substituted for the 10th residue in the TM region), or the identity of the substituted residue (eg, T for I or V substitution). With respect to S) or both, all members of the library differ in the substitutions within the selected TM region. The desired substitution mutant is selected based on predetermined criteria such as a scoring system that considers the α-helix secondary structure prediction result and / or the transmembrane region prediction result.

This method can be repeated for one, two, three, four, five, six additional TM regions of the protein or all remaining TM regions of the protein, each repetition storing in electronic memory or database. Create a mutant candidate library that can be used. Within the same library, all variants differ in substitutions within the selected TM territory (see above), but are otherwise the same within the remaining TM and non-TM regions.

Domain exchange or domain shuffling using sequences within two or more such libraries produces combinatorial variants with hydrophobic amino acids L, I, V, F substitutions in the two or more TM regions. Depending on the number of members in each library, the total number of possible combinations of combinatorial variants can approach millions with just a few members in each library. For example, the GPCR having seven TM regions, if there are eight members in each of the seven libraries, the total number of combinations variants based on the library of 8 ⁷ or about 2.1 million. In certain embodiments, the combination mutant library comprises members of approximately 5 million, 4 million, 3 million, 2 million, 1 million or less than 500,000 species.

Thus, in a particular embodiment, in step (2), one subset of the plurality of hydrophobic amino acids within the same TM region of the protein (eg, GPCR) is replaced with one of the variant candidate libraries. Species members are made and one or more different subsets of the plurality of hydrophobic amino acids are replaced to make additional members of the library.

In certain embodiments, the method further comprises a step of ranking all members of the library based on a combination score, where the combination score is a weighting of the α-helix secondary structure prediction result and the transmembrane region prediction result. It is a combination made.

Domains with different sequences will probably predict different water solubility and tendencies for α-helix formation, as will be appreciated by those skilled in the art. Assign a "score" to a particular predicted water-soluble or water-soluble range, a tendency or tendency range to form an α-helix structure. This score may be qualitative (0, 1), where 0 can represent a domain having, for example, unacceptable predicted water solubility, and 1 can be, for example, acceptable predicted water solubility. Can represent a domain having. This score can be based on, for example, a threshold. Alternatively, this score can be assessed on a scale of 1-10 established, for example, characterized by an increased degree of water solubility. Alternatively, this score may be quantitative, such as describing the expected solubility in mg / mL. Once the scores have been evaluated for each domain, domain variants are easily compared (or ranked) by one or preferably both of those scores to form both water-soluble and alpha-helices. You can select the domain mutant to be used. Therefore, a preferred embodiment can utilize a ranking function that can be used to calculate the ranking data. Water-soluble proteins prepared based on the systems currently described are analyzed and characterized, and computational models are constrained using their substitution combinations that are not effective in achieving a given biological function. It should also be noted that it is possible to obtain an input to the system that enables more efficient information processing.

For example, using the methods of the invention, one or more variants can be designed and produced in vitro and / or in vivo, based on any of the methods approved in many arts. One or more biological functions of the mutant can be determined. For GPCRs, for example, ligand binding and / or downstream signaling by the variant can be compared to that of wild-type GPCRs, and the QTY substitution pattern used to produce a particular variant is biological. It can be associated with increased, maintained or decreased activity. The methods of the invention can be used for machine learning with such structural / functional relationship information obtained based on one or more variants, or with additional constraints on the computational model of the invention. The mutants produced by can be ranked more efficiently. Thus, a new mutant candidate having a substitution pattern that closely matches the known successful mutant substitution pattern is a substitution pattern or known that does not closely match the known successful mutant substitution pattern. Can be ranked higher than other mutant candidates with a substitution pattern that closely matches the substitution pattern of the unsuccessful mutant of.

When the TMHMM program is run as a stand-alone software module / package (eg, for Linux® systems), 0 to 1 can be used to predict the tendency to form transmembrane regions / proteins. Generate a score. This score can be used as a quantitative prediction of water solubility in the methods of the invention.

Thus, in certain embodiments, the α-helix secondary structure component of the ranking function maintains 0.5 or 1 in the absence of the predicted α-helix secondary structure, and the predicted α-helix secondary structure. It may be a quantitative score such as 0 when it is. In certain embodiments, a TM region prediction program such as TMHMM 2.0 provides a number 0 to 1 where 0 has no predicted TM region and 1 has the strongest tendency to form one or more TM regions. The transmembrane region result can be obtained. Thus, the two scores can be combined directly or with weights to represent a secondary structure in which the combined score is maintained as well as an overall assessment of the predicted water solubility (measured by the tendency to form the TM region). .. For example, a combination score of 0 indicates that the mutant does not have the predicted TM region but maintains the predicted α-helix secondary structure and is therefore the desired mutant. On the other hand, mutants have a strong tendency to form TM regions (eg, due to the presence of many hydrophobic residues) and tend to have a larger combination score, which is therefore undesirable under this scoring scheme. ..

In certain embodiments, the method comprises removing a variant having an α-helix secondary structure prediction result that tends to indicate that the α-helix secondary structure is disrupted or disrupted. In certain embodiments, the method comprises removing a variant having a transmembrane region prediction result that tends to show a strong tendency to form a TM region. Therefore, the system can include a BEAMing module that can exclude variants by further selection processing.

In certain embodiments, weights of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% are α-helix secondary structure predictions. The ranking function can be selected to include a weighting scheme that assigns the results and the rest to the transmembrane region prediction results. Depending on the desired property, such as biological function, the user can manually select the weighted feature, or the software can automatically select the weighted feature.

In certain embodiments, the method further comprises selecting the N species member having the highest combination score to form a first library of mutant candidates for said TM region, wherein the method further comprises. N is a predetermined integer (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).

In certain embodiments, the method creates a library of one of the mutant candidates for 1, 2, 3, 4, 5, 6 or all remaining TM regions of the protein (eg, GPCR). Further includes the steps to be performed. Each entry in the library can contain fields used to define the attributes of the entry, including the ranking data generated by one or more ranking functions.

In certain embodiments, the method replaces two or more (eg, all) TM regions of the protein (eg, GPCR) with the corresponding TM regions in the mutant candidate library to create a combined mutant library. Further includes the steps to be performed. As used herein, the "corresponding TM region" refers to a TM region within a mutant candidate library that is the same as or homologous to the TM region of the protein (eg, GPCR) being combined. For example, when the second and third TM regions from the N-terminus of the GPCR are replaced, the TM region sequence in the library having the substitution only in the second TM region and the substitution only in the third TM region. The TM region sequence in the library to be possessed is imported / pasted / transferred to the second and third TM regions of the GPCR to produce a combination mutant.

In certain embodiments, substantially all of the leucine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with glutamine. In certain embodiments, substantially all of the isoleucine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with threonine. In certain embodiments, substantially all of the valine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with threonine. In certain embodiments, substantially all of the phenylalanine (eg, 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 95% are replaced with tyrosine. In certain embodiments, one or more (eg, 1, 2 or 3) leucines are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the isoleucines are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of said valine are not substituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the phenylalanines are not substituted.

In certain embodiments, the method further comprises the step of producing / expressing the combination variant. In certain embodiments, the method further comprises testing the combination variant for ligand binding (eg, in vitro or in a biological system such as the yeast two-hybrid method), wherein the GPCR ligand binding. Select those having substantially the same ligand binding in comparison. In certain embodiments, the method further comprises testing the combination variant for the biological function of the GPCR, wherein the biology is substantially the same as the biological function of the GPCR. Select one that has a specific function.

In certain embodiments, the sequence of a TM protein (eg, GPCR) comprises information about the TM region of the protein, eg, the location of one or more transmembrane regions of the TM protein, such as the location of all TM regions. .. Such a sequence may belong to a protein having a crystal structure elucidated by a defined TM region. Also, such sequences may belong to proteins with TM region information annotated based on previous studies, such information being published by PDB, UniProt, GenBank, EMBL, DBJ, etc. Or easily available from your own database

The Protein Data Bank (PDB) is a weekly updated repository for 3D structural data of large biomolecules such as proteins and nucleic acids. Data typically obtained by X-ray crystallography or NMR spectroscopy and submitted by biologists and biochemists around the world are posted on the Internet via the websites of their member tissues (PDBe, PDBj and RCSB). It is freely accessible. PDB is overseen by the World Protein Data Bank or www PDB. The PDB is an important resource in the area of structural biology such as structural genomics, and the most major scientific journals and some funding agencies are currently seeking scientists to submit their structural data to the PDB.

Considering the contents of a PDB as primary data, there are hundreds of derived (ie, secondary) databases that classify the data differently. For example, SCOP and CATH both classify structures according to type of structure and assumed evolutionary relationships, GO classifies structures based on genes, while crystallographic databases show 3D structures of proteins. I remember information about. All such publicly available databases may be used to obtain input sequence information, including information regarding the presence and location of transmembrane regions.

Another publicly available database that can provide the sequence information used in the methods of the invention is UniProt. UniProt is a comprehensive, high-quality, freely accessible database of protein sequence and functional information, with many entries derived from genome sequencing projects. It contains a lot of information about the biological function of proteins from the research literature. UniProt provides four core databases: UniProtKB (including the subdivisions Swiss-Prot and TREMBL), UniParc, UniRef and UniMes. Of these, UniProtKB / Swiss-Prot is a manually annotated non-redundant protein sequence database that combines information extracted from scientific literature and computer analysis evaluated by biocurators. The purpose of UniProtKB / Swiss-Prot is to provide all known relevant information about a particular protein. Annotations are regularly reviewed to keep up with current scientific findings. Manual annotation of the entry includes detailed analysis of protein sequences and scientific literature. Sequences from the same gene and the same species have been merged into the same database entry. Identify differences between sequences and document their causes (eg, alternative splicing, natural mutations, etc.). Manually evaluate computer predictions and select relevant results for inclusion in the entry. These predictions include post-translational modifications, transmembrane domains and topologies, signal peptides, domain identification and protein family classification, all of which are useful sequences associated with the TM region used in the methods of the invention. You may get information.

In certain embodiments, the sequence of the TM protein (eg, GPCR) does not contain information about the location of one or more (eg, optional) transmembrane regions. However, this one or more TM regions can be predicted based on sequence homology with related proteins having known TM regions. For example, the related protein may be a homologous protein in a different species.

In certain embodiments, the sequence of the TM protein (eg, GPCR) does not contain information about the location of one or more (eg, optional) transmembrane regions, such information being readily available based on known information. Not available to. In this embodiment, the invention is a method approved in the art, such as the TMHMM 2.0 (Hidden Markov Model) program developed by the Center for Biological Sequence Analysis. The TM area is calculated using. See further details on this below.

In certain embodiments, the method further comprises providing a polynucleotide sequence for each variant of the protein (eg, GPCR). Such a polynucleotide sequence can be easily generated based on the protein sequence of the protein (eg, GPCR) and the known genetic code. In certain embodiments, the polynucleotide sequence is a codon optimized for expression in the host. The host may be a bacterium such as Escherichia coli, a yeast such as budding yeast or fission yeast, an insect cell such as Sf9 cell, a non-human mammalian cell or a human cell.

In certain embodiments, the protein is GPCR, eg, purine receptors (P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ ), M ₁ and M ₃ muscarinic acetylcholine receptors, thrombin receptors (proteo-activated receptors). Body (PAR-1, PAR-2), thromboxane (TXA ₂ ), sphingosine 1-phosphate (S1P ₂ , S1P ₃ , S1P ₄ and S1P ₅ ), lysophosphatidic acid (LPA ₁ , LPA ₂ , LPA ₃₎ ), Angiotensin II (AT ₁ ), Serotonin (5-HT _2c and 5-HT ₄ ), Somatostatin (sst ₅ ), Endoserin (ET _A and ET _B ), Cholecystokinin (CCK ₁ ), V _1a vasopressin receptor , _{D 5} dopamine receptors, fMLP formyl peptide receptor, GAL ₂ galanin receptor, EP ₃ prostanoid receptors, _{A 1} adenosine receptor, alpha ₁ adrenergic receptor, BB ₂ bombesin receptor, _{B 2} bradykinin receptor body, calcium-sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK ₁ tachykinin receptor, thyroid stimulating hormone (TSH) receptor, protease-activated receptor, neuropeptide receptors, the adenosine A2B receptor, P2Y GPCR selected from the group consisting of purine receptor, metabolic glutamate receptor, GRK5, GPCR-30 and CXCR4.

In certain embodiments, the scripting procedure of the method comprises a VBA script.

In certain embodiments, the scripting procedure is operational on a Linux® system (eg, Ubuntu 12.04 LTS), Microsoft Windows operating system, or Apple iOS operating system.

In certain embodiments, the method is
(1) A step of predicting the α-helix structure of the protein (for example, GPCR), if necessary, to identify the first transmembrane region of the membrane (penetrating) protein.
(2) A step of modifying a plurality of hydrophobic amino acids according to the QTY code defined in the present specification to obtain a modified first transmembrane sequence.
(3) Tendency of the α-helix structure of the first modified transmembrane sequence of (2) (eg, in the context of a modified membrane (penetrating) protein having a first modified transmembrane sequence). The process of scoring and obtaining a structural score,
(4) Score the water solubility prediction of the first modified transmembrane sequence of (2) (eg, in the context of a modified membrane (penetration) protein having a first modified transmembrane sequence). To obtain a solubility score,
(5) A step of repeating (2) to (4) to obtain a first library of the first modified transmembrane mutant which is presumably water-soluble.
(6) The structural score and solubility score of each of the first modified transmembrane mutants, which are presumably water-soluble in the first library, are compared, preferably using the structural score and the solubility score. The step of ranking the first modified transmembrane mutant, which is presumably water-soluble.
(7) Multiple presumably water-soluble first modified transmembrane variants (where plural is an integer H or preferably less than 10, 9, 8, 7, 6, 5 or 4). To obtain a second library of presumably water-soluble first modified transmembrane variants,
(8) A step of repeating steps (1)-(7) for the second, third, fourth, fifth, sixth, seventh or preferably all transmembrane regions of the protein (modified by this method). The sum of the transmembrane regions is an integer n),
(9) A step of identifying the amino acid sequence of a protein that is not contained in any of the transmembrane regions modified in steps (1) to (8) and contains an arbitrary extracellular or intracellular domain of the protein. ,
(10) Producing a combination variant of a modified transmembrane protein that is presumably water-soluble (see above), and (11) Optionally, a modified transmembrane variant that is presumably water-soluble, respectively. Includes all or substantially all of the steps of identifying a nucleic acid sequence of.

The nucleic acid sequences identified in the above method were used to generate the nucleic acid sequences of each of the presumably water-soluble modified transmembrane variants and non-transmembrane domains (including extracellular and intracellular domains). , And can be expressed in combination to create a library of up to H ⁿ species, which are presumably water-soluble transmembrane protein variants. For example, if H is 8 and n is 7, a library of approximately 2 million water-soluble protein variants can be designed.

Another aspect of the invention relates to the expression of a water soluble mutant protein (eg, GPCR) designed based on the methods of the invention. This aspect of the invention is in vitro expressed in a cell-free expression system and commonly used in E. coli, etc., in part by water-soluble variant proteins (eg, GPCRs) designed according to the methods of the invention. It is based on the surprising finding that high levels of expression can be achieved in both expression systems by E. coli. Also, the expressed protein is highly soluble, such as insoluble fractions of E. coli culture lysates, as opposed to insoluble aggregates or pellets, where most membrane proteins are typically present internally. It can be easily purified from the soluble fraction of the expression system.

Therefore, one aspect of the present invention is
(A) A step of culturing bacteria in a growth medium under conditions suitable for protein production, and
(B) A step of dividing the lysate of this bacterium into fractions to produce a soluble fraction and an insoluble pellet fraction, and
(C) A step of isolating the protein from the soluble fraction.
(1) The protein is a variant protein of the subject of the invention (eg, G protein-coupled receptor (GPCR)), and (2) the yield of the protein is at least 20 mg / L (eg, 30 mg) of growth medium. Provided is a method of producing a protein in a bacterium (eg, E. coli), comprising a step characterized by (/ L, 40 mg / L, 50 mg / L or more).

In certain embodiments, the bacterium is E. coli BL21 and the growth medium is LB medium. In certain embodiments, the protein is encoded by a plasmid within the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter. For example, the inducible promoter may be inducible by IPTG. In certain embodiments, the lysate is produced by sonication. In certain embodiments, the lysate is centrifuged at 14,500 xg or greater to produce a soluble fraction.

Specific features or specific embodiments of the present invention will be further described below using the general aspects of the present invention.

Transmembrane Region Prediction The particular method of the invention comprises the step of predicting the transmembrane region of a protein, such as a GPCR. There are many programs and software known in the art for the TM domain, any of which may be used individually or in combination in the methods of the invention that require a TM domain prediction step. These programs typically have a very simple user interface that requires the user to provide an input sequence in a typically specified format (such as FASTA or text format), and / or text. Is used to give the prediction result. There are also programs that provide more advanced features such as allowing the user to specify specific parameters to fine-tune the prediction results. All such programs can be used in the methods of the invention.

One of the exemplary TM region prediction programs is TMHMM (provided by the Center for Biological Sequences, Technical University of Denmark), which accurately predicts 97-98% TM region helix. It uses a hidden Markov model to predict transmembrane helix within a protein. The input protein sequence may be in FASTA format and its output can be presented as an html page containing an image of the predicted position of the TM region. In a study by Moller et al. entitled "Evaluation of Methods for the Prediction of Membrane Spanning Regions" (Bioinformatics 17 (7): 646-653, 2001), TMHMM was used at the time of evaluation. It was judged to be the best program to perform the transmembrane prediction program.

The programs compared in the study include the following programs: TMHMM 1.0, 2.0 and 2.0 retrained versions (Sonnhammer et al., Int. Conf. Intell. Syst. Mol. Biol. AAAI Press, Montreal , Canada, pp.176-182, 1998; Krogh et al., J Mol Biol. 305 (3): 567-80, 2001), MEMSAT1.5 (Jones et al., Biochemistry 33: 3038-3049, 1994) , Eisenberg (Eisenberg et al., Nature 299: 371-374, 1982), Kyte / Dootrain (Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982), TMAP (Persson and Argos, J. Protein) Chem. 16: 453-457, 1997), DAS (Cserzo et al., Protein Eng. 10: 673-676, 1997), HMMTOP (Tusnady and Simon, J. Mol. Biol. 283: 489-506, 1998) , SOSUI (Hirokawa et al., Bioinformatics 14: 378-379, 1998), PHD (Rost et al., Int. Conf. Intell. Syst. Mol. Biol. AAAI Press, St. Louis, USA, pp.192- 200, 1996), TMpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374: 166, 1993), KKD (Klein et al., Biochim. Biophys. Acta. 815: 468-476, 1985), ALOM2 (Nakai) and Kanehisa, Genomics 14: 489-911, 1992) and Toppred 2 (Claros and Heijne, Comput. Appl. Biosci. 10: 685-686, 1994), all to predict the TM region in the method of the invention. to use Can be done. All references cited are incorporated herein by reference.

The principle of TMHMM is Krogh et al., Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 305 (3): 567-580, January 2001 (incorporated by reference) and Sonnhammer et al., A hidden Markov model for predicting transmembrane helices in protein sequences (Hidden Markov model for predicting transmembrane helices in protein sequences) Model) In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park , CA, 1998, AAAI Press (incorporated by reference).

DAS (Dense Alignment Surface, Cserzo et al., “Prediction of transmembrane alpha-helices in procariotic membrane proteins: the Dense Alignment Surface method),” Prot . Eng. 10 (6): 673-676, 1997, Stockholm University, Sweden) predicts transmembrane regions using a high-density alignment surface method. DAS is based on a low-rigidity dot plot of query sequences against a set of library sequences (unhomologous membrane proteins) using a special score matrix previously obtained. The method provides an accurate hydrophobic profile of the query from which the locations of transmembrane segment candidates can be obtained. The novelty of the DAS-TMfilter algorithm is a second prediction cycle for predicting TM segments in an array of TM libraries. To use the DAS server, the user can go to www. sbc. su. The protein sequence is input in se / ~ miklos / DAS /, and the DA server predicts the TM region of the input sequence.

HMMTOP (Hungarian Academy of Sciences, Budapest) is a member of the Institute of Enzymology. E. An automated server for predicting transmembrane helix and protein topologies using a hidden Markov model developed by Tusnady. The method used by this prediction server is GE Tusnady and I. Simon (1998) “Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction. ), "J. Mol. Biol. 283: 489-506 (incorporated by reference). New features of the HMMTOP 2.0 version are described in GE Tusnady and I. Simon (2001) “The HMMTOP transmembrane topology prediction server,” Bioinformatics 17: 849-850 (incorporated by reference). ing.

The MEMSAT2 transmembrane prediction page (www.sacs.ucsf.edu/cgi-bin/memsat.py) predicts transmembrane segments within proteins using FASTA format or text format as input. The related program, MEMSAT (1.5) software, is copyrighted by Dr. David Jones (Jones et al., Biochemistry 33: 3038-3049, 1994). The latest version of MEMSAT, MEMSAT V3, is a widely used method for predicting all-helix membrane proteins, MEMSAT. This method was benchmarked with a test set of transmembrane proteins of known topologies. MEMSAT was estimated from sequence data to have greater than 78% accuracy in predicting the structure of all-helix transmembrane proteins and the location of their constituent helix elements within the membrane. MEMSAT-SVM is a very accurate prediction of transmembrane helix topology. It can distinguish between signal peptides and identify cytoplasmic and extracellular loops. Both MEMSAT3 and MEMSAT-SVM are part of the PSIPRED Protein Sequence Analysis Workbench, which brings together several structure prediction methods at the University of London.

The Phobius server (phobius.sbc.su.se) is for predicting transmembrane topologies and signal peptides from the amino acid sequences of FASTA format proteins. For Phobius, see Lukas et al., “A Combined Transmembrane Topology and Signal Peptide Prediction Method,” Journal of Molecular Biology 338 (5): 1027-1036, 2004). Are listed. For PoyPhobian, Lukas et al., “An HMM posterior decoder for sequence feature prediction that includes homology information,” Bioinformatics, 21 (Suppl 1): i251- It is described in i257, 2005. And for Phobius web server, Lukas et al., "Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server", "Nucleic Described in Acids Res. 35: W429-32, 2007 (all cited technical content is incorporated by reference).

SOSUI is intended to distinguish between membrane and soluble proteins, as well as the prediction of transmembrane helix. SOSUI predicts the transmembrane region using Hydrophobicity Analysis for Topology and Probe Helix Method for Tertial Structure for tertiary structure. The accuracy of protein classification is said to be as high as 99%, and the corresponding value for transmembrane helix prediction is said to be about 97%. This SOSUI system is available on the Internet Access www. tuat. ac. It is available by jp / mitaka / sosui.

TMPred (European Molecular Biology Network, Swiss Node) predicts transmembrane regions and protein orientation in query sequences. Specifically, the TM Pred algorithm is based on a statistical analysis of TMbase (a database of naturally occurring transmembrane proteins). The prediction is made using a combination of several weight matrices for scoring. See Hofmann & Stoffel (1993) “TMbase --A database of membrane spanning proteins segments”, Biol. Chem. Hoppe-Seyler, 374: 166.

The SPLIT 4.0 server is a membrane protein secondary structure prediction server (split.pmfst.hr/split/4) that predicts the transmembrane (TM) secondary structure of a membrane protein in the SWISS-PROT format using the method of preference function. Is. Juretic et al., “Basic charge clusters and predictions of membrane protein topology,” J. Chem. Inf. Comput. Sci., 42: 620-632, 2002 (incorporated by reference) Please refer to).

PRED-TMR simply uses the protein sequence itself to predict the transmembrane domain within the protein. This algorithm refines standard hydrophobic analysis by detecting end (“end”, start and end) candidates for the transmembrane region. This allows the highly hydrophobic regions not separated by a well-defined start and end configuration to be discarded and the putative transmembrane segments indistinguishable by their hydrophobic composition. The accuracy obtained for a test set of 101 non-homologous transmembrane proteins with a reliable topology is well comparable to the accuracy of other common existing methods. When this algorithm was applied to all transmembrane proteins in the SwissProt database (Release 35), only a slight decrease in predictive accuracy was observed. Pasquier et al., “A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm for predicting transmembrane segments in proteins based on statistical analysis of the SwissProt database. New Method: PRED-TMR Algorithm), "Protein Eng., 12 (5): 381-385, 1999 (incorporated by reference).

In the relevant PRED-TMR2, its application is extended by pretreatment steps represented by artificial neural networks that can distinguish transmembrane proteins from soluble or fibrous proteins with high accuracy. When applied to several test sets of transmembrane proteins, the system gives a 100% perfect predictive rating by classifying all sequences within the transmembrane class. When applied to 995 non-transmembrane proteins extracted from the PDBselect database, neural networks mistakenly predict 23 of them to be transmembrane (97.7% accurate allocation). Pasquier and Hamodrakas, “An hierarchical artificial neural network system for the classification of transmembrane proteins,” Protein Eng., 12 (8): 631-634, See 1999 (incorporated by reference).

Predicting the α-helix secondary structure of a protein The specific method of the present invention comprises the step of predicting the α-helix secondary structure of a protein such as GPCR. Many such programs and software are known in the art and one of them is used individually or in combination in a method of the invention that requires an alpha helix secondary structure prediction step. May be good. All such programs can be used in the methods of the invention.

Early methods of secondary structure prediction were limited to the prediction of three predominant states: helices, sheets or random coils. These methods were based on the helix or sheet formation tendency of individual amino acids, which is sometimes associated with rules for estimating the free energy that forms secondary structure elements. Such a method was typically about 60% accurate in predicting which of the three states (helix / sheet / coil) the residue would take. The first technique widely used to predict protein secondary structure from amino acid sequences was the Chou-Fasman method.

A significant increase in accuracy (increased to approximately 80%) is made by utilizing the information provided by multiple sequence alignments, and through evolution, a position (and close, typically about 7 on one side). Knowing the complete distribution of the amino acids that occur at the residue) gives a very good image of the structural tendency near that position. For example, a given protein may have glycine at a given position, which itself may suggest a random coil. However, multiple sequence alignments may reveal that helix-loving amino acids occur at that position (and nearby positions) in 95% of homologous proteins throughout evolution. In addition, examining the average hydrophobicity at that location and nearby locations may also suggest a residue solvent exposure pattern in which the same alignment matches the α-helix. Taken together, these factors appear to suggest that the original protein, glycine, has an α-helical structure rather than a random coil. Therefore, in the method of the present invention, an α-helix secondary structure prediction program including a neural network, a hidden Markov model and a support vector machine may combine all available data to make predictions of three states. Such prediction methods also provide confidence scores for those predictions at all positions.

Benchmarks are continuously performed on the secondary structure prediction method (for example, EVA (benchmark)). EVA continuously runs benchmark projects to evaluate the quality of protein structure prediction and secondary structure prediction methods. Compare methods for predicting both secondary and tertiary structure, such as homology modeling, protein threading and contact order prediction, with results from weekly newly elucidated protein structures deposited in the Protein Data Bank (PDB). .. This project aims to determine the expected predictive accuracy for non-expert users of common publicly available predictive web servers.

According to these tests, the most accurate method at this time is Psipred, SAM (Karplus, "SAM-T08, HMM-based protein structure prediction", "Nucleic Acids Res. (2009) 37) (Web Server issue): W492-497. doi: 10.1093 / nar / gkp403), PORTER (Pollastri & McLysaght, "Porter: a new, accurate server for protein secondary structure prediction" Server), "Bioinformatics 21 (8): 1719-1720, 2005), PROF (Yachdav et al. (2014)." PredictProtein--an open resource for online prediction of protein structural and functional features (Structural and functional features of proteins) Open resource for online prediction of protein features), "Nucleic Acids Res. 42 (Web Server issue): W337-343. Doi: 10.1093 / nar / gku366) and SABLE (Adamczak et al. (2005)" Combining prediction of Secondary structure and solvent accessibility in proteins, "Proteins 59 (3): 467-475. Doi: 10.1002 / prot. 20441). Also, the standard method for assigning a secondary structure class (helix / strand / coil) to a PDB structure is DSSP (Kabsch W and Sander (1983) "Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical". Features (Dictionary of protein secondary structure: pattern recognition of hydrogen bonds and geometric features), "Biopolymers 22 (12): 2577-2637. Doi: 10.1002 / bip.360221211), benchmarking against these predictions Is done. All are incorporated by reference and all can be used in the methods of the invention.

The DSSP algorithm is a standard method for assigning secondary structures to amino acids in a protein, taking into account the atomic resolution coordinates of the protein. DSSP begins by simply identifying the intraskeletal hydrogen bonds of the protein using electrostatic definitions, assuming partial charges of -0.42e and +0.20e for carbonyl oxygen and amide hydrogen, respectively, of theirs. The reverse is assigned to carbonyl carbon and amide nitrogen. If E in the following equation is less than -0.5 kcal / mol, a hydrogen bond is specified.

と

との角度は７０°未満である）、空白（すなわちスペース）は他の規則が適用されない場合に使用され、ループを指す。これらの８種類は通常、３つのより大きなクラスすなわちヘリックス（Ｇ、ＨおよびＩ）、ストランド（ＥおよびＢ）ならびにループ（それ以外の全て）にグループ化される。 Based on this, eight types of secondary structures are assigned. 3 ₁₀ helix, α-helix and π-helix have the codes G, H and I, and their residues are recognized by having a repeating sequence of hydrogen bonds separated by 3, 4 or 5 residues, respectively. There are two types of β-sheet structures, the β-bridge has the symbol B, and the longer set of hydrogen bonds and β-bulges has the symbol E. T is used for rotations characterized by hydrogen bonds typical of helix, and S is used for regions of high curvature (where, here).

When

The angle with and is less than 70 °), blanks (ie spaces) are used when no other rules apply and refer to loops. These eight types are usually grouped into three larger classes: helices (G, H and I), strands (E and B) and loops (all others).

PSIPLED (secondary structure prediction by PSI-BLAST) is a technique used to investigate protein structures. It uses a neural network or machine learning method in its algorithm. It is a server-side program that features a website that provides services as a front-end interface and can predict protein secondary structures (β-sheets, α-helices, and coils) from primary sequences. bioinf. cs. ucl. ac. See uk / plex. The idea of this method is a machine learning method that uses information on evolutionarily related proteins to predict the secondary structure of new amino acid sequences. Specifically, PSI-BLAST is used to find relevant sequences and construct a position-specific score matrix. Process this matrix with a neural network constructed and trained to predict the secondary structure of the input array. This prediction method or algorithm is divided into three stages: sequence profile creation, first secondary structure prediction and predicted structure filtering. PSIPLED acts to normalize the sequence profile created by PSI-BLAST. The first secondary structure is then predicted by using neural networking. For each amino acid in the sequence, the neural network is given a window of 15 acids. Further information is attached to indicate whether these windows straddle the N- or C-terminus of the chain. As a result, a final input layer consisting of 315 input units divided into 15 groups consisting of 21 units is obtained. The network has a single hidden layer of 75 units and 3 output nodes (1 for each secondary structure element ie helix, sheet, coil). A second neural network is used to filter the predicted structure of the first network. Give this network a window at 15 locations. It also transfers an indicator of the possible position of this window at the end of the chain. As a result, 60 input units divided into 15 groups consisting of 4 are obtained. This network has results in a single hidden layer of 60 units and 3 output nodes (1 for each secondary structure element ie helix, sheet, coil). The three final output nodes send the score of each secondary structure element for the center position of this window. Using the secondary structure with the highest score, PSIPRED produces the protein prediction. The Q3 value is the percentage of residues accurately predicted in the secondary structure state ie helices, strands and coils.

Step-by-Step Description of an exemplary Embodiment Using the invention outlined above, with reference to a representative flowchart in the figure for a particular non-limiting but exemplary embodiment. This will be described below.

FIG. 9A shows a non-limiting embodiment of the present invention. This figure shows the method 200 of the invention as a whole, where selected hydrophobic amino acids L, I, V and F within the TM region of the protein (eg, GPCR) are placed in any particular TM region / domain. Substitution according to the "QTY code" of the present invention without limiting the substitution in.

In its specific embodiment, the method begins by obtaining or reading (204) an input of a protein sequence that may or may not be a transmembrane protein (202). This protein sequence is then subjected to an α-helix secondary based on either TM region prediction (206) (if such information is not yet available from the input protein sequence) and methods approved in the art. It can be used for structural prediction. For example, a TM region prediction can be performed using a program such as the TMHMM program (240). If no TM region is obtained in 242 by this prediction, one or more different TM region prediction programs such as SOSUI may be used to enable prediction of the presence / absence of (250) TM regions. If the TM region is not predicted in 252 based on such a program, the TM region may not be present in the protein (254) and the method ends (260).

On the other hand, if one or more TM regions are predicted by any of the suitable programs in 242, a TM region protein sequence is obtained (244) and within such one or more TM regions the QTY code of the invention. Can be applied to the hydrophobic amino acids L, I, V and F. More specifically, according to the QTY code, leucine in the TM region can be independently replaced with glutamine (Q), serine (S) or asparagine (N) or left unreplaced (212). Isoleucine and valine in the TM region can be independently replaced or left unchanged with threonine (T), serine (S) or asparagine (N), and phenylalanine in the TM region is tyrosine (Y), respectively. ) Can be replaced or left unreplaced. The results of such QTY substitutions produce one or more presumably water-soluble variants of the original transmembrane protein. The number of substitutions made for each amino acid in a region can be selected as a parameter.

Any program approved in the art, such as PORTER, can then be used to predict the α-helix secondary structure in each of the putatively water-soluble mutants (210). The results can be compared to the expected results of the original protein, preferably using the same program (eg, PORTER) (208). It should be noted that the α-helix secondary structure of the original protein can be predicted using any program approved in the art before, at the same time as, or after the TM region prediction step of the original protein.

If the α-helix secondary structure prediction results indicate at 214 that the water-soluble variant candidate retains or most of the same α-helix secondary structure as the original protein, it is in that variant. It is suggested that the specific pattern of QTY substitution does not affect or significantly affect the α-helix secondary structure in the original protein. This TM region can then be predicted (220), confirmed (222), and generate mutant sequences (224). Optionally, if this result indicates at 214 that one or more of the one or more α-helix secondary structures in the original protein have been disrupted, the variant is discarded as undesirable in this step. , The method can be terminated in this way.

On the other hand, the method of the present invention also needs to show that the predicted QTY mutants are less or less prone to form TM regions compared to the original protein. Therefore, a mutant that is presumably water-soluble can be subjected to TM region prediction using the same TM region prediction program or the like used for the initial TM region prediction in the original protein (if necessary). If the results indicate that a significant TM region is still present, the variant may be discarded. On the other hand, if the results show that the TM region is absent or less prone to form the TM region, the mutant has higher water solubility than the original protein but retains the α-helix secondary structure. And therefore it can probably be selected as the desired variant that retains the function of the original protein.

If desired, further characterization of the water-soluble mutants obtained by further stepping can be performed. Such further characterization may include calculating the pI of the variant (226), and this pI is compared to the pI of the original protein. This pI should be unchanged or very slight (ie less than 30% or preferably less than 20% or more preferably less than 10%). Other further characterizations may include creating a helix wheel model (eg, as shown in FIG. 3) (246) to indicate the location of QTY substitutions and any clustering in any particular TM region.

Another exemplary embodiment of the invention for designing a transmembrane region of a protein (eg, GPCR) according to the QTY code of the invention is a computer system using the representative method 10 described in FIG. 9B. Some of the detailed steps that can be performed above are described further below. Many of the steps are optional or can be combined according to the methods of the invention.

1: In step 1, the computer interface of the computer system receives the protein sequence, selects it for analysis, and inputs data describing the input protein (eg, the sequence) via the computer interface of the computer system. Upload or enter (12). The data entered may be a protein name, database reference or protein sequence. For example, the protein sequence can be uploaded via a computer interface.

2: In step 2, additional data about the protein, including its name or sequence, can be identified, determined, obtained and / or entered and can be entered via a computer interface. One source for obtaining protein data (20) is a database named UniProt (www.uniprot.org). Alternatively, the methods of the invention can store data about the protein or sequences associated with the protein for later retrieval by the user in this step. In embodiments, the program can prompt the user to select a database or file for retrieving additional data (eg, sequence data) about the protein selected for analysis.

3: In step 3, the user can input, upload or acquire data for identifying the transmembrane region. For example, users can be encouraged to retrieve data from public sources such as UniProt. The information can be confirmed (30) and collected from the database for use in step 5.

4: Alternatively or additionally, the transmembrane region can be established by any method approved in the art, even if TM region information is not readily available from the input protein sequence ( 40). The transmembrane region is generally characterized by an α-helix conformation. For example, using a software module / package named TMHMM2.0 (transmembrane prediction using a hidden Markov model) developed by the Biological Sequence Center (www.cbs.dtu.dk/services/TMHMM). Helix prediction can be performed. This software version may have problems with peak search and may fail to find the seven TM regions for GPCRs. Therefore, a modified version of this program with a dynamic baseline introduced into the peak search method performed by the computer system may be used as needed. Here, for example, in the case of GPCRs, the baseline can be changed to a lower value if all seven TM regions are not found using the first baseline value. For example, the default baseline may be set to 0.2. A baseline value can be set to 0.1 to identify the missing seventh transmembrane region. If eight or more TM regions are found, the baseline can be changed to a higher value, such as 0.15, to exclude irrelevant TM predictions. For example, when the amino acid sequence of CCR-2 was subjected to TMHMM 2.0 software, only six transmembrane regions were initially identified. However, setting the baseline value of TMHMM 2.0 to 0.07 identified a total of seven correct transmembrane regions. Next, the result of TM region prediction is given to step 5.

5: In step 5, after identifying the TM data by new prediction or by obtaining such information from the first sequence input, the GPCR sequence is sequenced according to the TM region information into a total of 15 fragments (ie, 7). Divided into transmembrane segments (7 TMs) (52) and 8 non-transmembrane segments (8 NTMs) (54) (50), ie 7 TMs and 8 for each typical GPCR. The NTM fragment must be present.

Of course, the system can perform one or more, eg, all, of the above steps using a computer interface for user input. Further, as a matter of course, this system can omit one or more of the above steps or combine two or more steps.

6: In step 6, QTY substitutions are partially performed on selected subsets of the hydrophobic amino acids L, I, V and F within a given TM region of the protein (60). Specifically, the first transmembrane region (typically, but not essential, the most proximal transmembrane region to the N-terminus of the protein) is selected first for mutation. Then, some or all of the hydrophobic amino acids (L, I, V and F) in the first transmembrane region are replaced with the corresponding nonionic hydrophilic amino acids (Q / S / N, T / S / N). , T / S / N or Y). Of course, in this case this amino acid is not actually substituted in the protein. Rather, this amino acid designation is replaced in the modeling sequence. Therefore, the term "array" shall include "array data". Typically, most or all of the hydrophobic amino acids are selected for substitution. If less than all amino acids are selected, it may be desirable to select internally hydrophobic amino acids while leaving one or more N and / or C-terminal amino acids in the transmembrane region hydrophobic. In addition or as an alternative, it may be desirable to choose to replace all of the leucine (L) in the transmembrane region. As an addition or alternative, it may be desirable to selectively replace all isoleucine (I) within the transmembrane region. As an addition or alternative, it may be desirable to selectively replace all valine (V) in the transmembrane region. As an addition or alternative, it may be desirable to selectively replace all of the phenylalanine (F) in the transmembrane region. In addition or as an alternative, it may be advantageous to retain one or more phenylalanines within the transmembrane region. In addition or as an alternative, it may be advantageous to retain one or more valines in the transmembrane region. In addition or as an alternative, it may be advantageous to retain one or more leucines in the transmembrane region. In addition or as an alternative, it may be advantageous to retain one or more isoleucines within the transmembrane region. In addition or as an alternative, it may be advantageous for the wild-type sequence to retain one or more hydrophobic amino acids within a transmembrane region characterized by three or more contiguous hydrophobic amino acids.

7: In step 7, the transmembrane region so designed is returned to the original protein sequence context. That is, since each set of substitutions produces one particular putative variant for that TM region, the mutation or redesigned TM region (62) with the QTY substitution is associated with the original protein. It exchanges with the TM region to generate a transmembrane mutant, or "estimated mutant" (70). At the same time, these related putative variants form a first library of putative variants.

8: Then, in steps 82 and 84, each putative variant is subjected to the transmembrane region prediction method (84) described herein (eg, predicted loss of TM region). The variant is also evaluated for a score of propensity to form an α-helix for that sequence (82). The mutant is also used in the water solubility prediction method described herein. For example, the mutant is evaluated for its sequence's tendency to be water soluble. Such scores may be based on the expected tendency to form TM regions, the strength of the tendency to form TM regions is associated with low water solubility, the low tendency to form TM regions or its The lack of tendency is associated with high water solubility. Of course, complete water solubility at all concentrations is not required for most commercial purposes. Water solubility is preferably determined to be necessary for functionality under the expected conditions of use (eg, ligand binding assay).

9: In step 9, the putative variants predicting loss of α-helix structure and / or "water insoluble" (predicted under expected conditions of use) are discarded. For example, the α-helix structure and water solubility are predicted using a combination score or rank (90), which is a weighted combination based on the ranking function of the α-helix secondary structure prediction result and the TM region / water solubility prediction result. It is possible to select a putative variant to be used. For example, if it is possible to predict that the α-helix structure can be compromised, then either highly water-soluble or 0, 1, 2 or 3 hydrophobic amino acids (eg, higher weights for water-soluble prediction results). Characteristic transmembrane variants can be selected. Alternatively or additionally, advanced α-helical structures characterized by 3, 4, 5 or 6 hydrophobic amino acids (eg, higher weights for α-helix secondary structure prediction results) can be selected.

10: In step 10, putative variants within the same library can be sorted or ranked based on the scoring scheme (94) outlined above (100). A predetermined number of putative variants can then be selected as the final member of the library of first putative variants. For example, in the above combination score, a score of 0 means that there is no tendency to form TM regions and that the original α-helix secondary structure is completely maintained, and thus is the most desired putative variant. .. A slightly higher score may indicate a slight tendency to form TM regions (or a low tendency to be water soluble). Therefore, this putative variant is less desirable, but can still be selected based on its superior combination score compared to other putative variants in the library.

In certain embodiments, a predetermined number of desired putative variants, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1, can be selected.

Repeating these steps (eg, steps 6-10) for the second, third, fourth, fifth, sixth and / or seventh (or higher) transmembrane regions or domains, such A library of one putative variant can be created for each TM region or domain.

11: In step 11, a combination of a TM region or domain with a putative variant and an unreplaced non-TM region can be selected (110). For example, one, two, three or four domains containing a putative variant with a high α-helix structure score and one, two, three containing a putative variant with a high water solubility score. You can combine one, four, five or six domains. In another example, in the selection of multiple variants, the domain / TM region is characterized in that all hydrophobic amino acids are replaced with hydrophilic amino acids and thus maximize their water solubility score, and three. It can be combined with a second domain / TM region that holds four or five hydrophobic amino acids. As is known in the art, the first combinatorial library of protein variants that are putatively water-soluble by "recombining" such selected putative variants with extracellular and intracellular domains. Can be made.

In certain embodiments, all or fragments of a putatively water-soluble protein variant of the first combinatorial library designed as described herein are prepared (produced or expressed in vitro or in a host cell). ), And preferably high-throughput screening can be screened for water solubility and / or ligand binding. For example, amplification of the library can express less than 100% presumably water-soluble protein combination variants. As is well known in the art, a reporter system can be used to screen for ligand binding. Using the methods of the invention, a library of presumably water-soluble modified transmembrane combination variants containing functionally combined extracellular and intracellular domains was rapidly identified and suitable for wild-type proteins. It is possible to produce a water-soluble protein mutant having a three-dimensional structure and retaining a ligand-binding function (including binding affinity) or other functions. The software can include learning modules that use the confirmed functionality of protein variants to exclude specific variants or rank them differently.

In certain embodiments, the first combinatorial library has approximately 2 million potentially water-soluble GPCRs or CXCR4 variants to make it experimentally practical. Of course, a library of more or less mutants can also be designed. In certain embodiments, smaller libraries may be preferred because they can be optimized based on the analysis of the research results described herein. Analysis of the study results will probably establish a tendency to optimize the number of domain variants to be recombined and estimates for selecting domain variants.

In certain embodiments, the TM of the transmembrane protein is due to a modification based on the helix-forming tendency, also known as the "helix prediction score" (see www.proteopedia.org/wiki/index.php/Main_Page). Select specific hydrophobic amino acids within the region. Various fragments combined randomly to form about 2 million one million (8 ⁷⁾ species variant of the full-length GPCR gene. The expected number of mutants is generally the formula H ⁿ (in the formula n = number of transmembrane regions modified and / or modified by the method (n = 7 in the GPCR example) and H = combination mutants. It can be characterized by the estimated number of variants in each transmembrane region available to produce.

Once the first combinatorial library or set of domain variants to be recombined is selected, the nucleic acid molecule or DNA or cDNA molecule encoding the protein in the first combinatorial library can be designed. These nucleic acid molecules are preferably designed to undergo codon optimization and intron deletion for the expression system selected to create a library of coding sequences. For example, if the expression system is E. coli, codons optimized for E. coli expression can be selected. www. dna20. com / resources / genesigner. In addition, a promoter region such as a promoter suitable for expression in an expression system (for example, Escherichia coli) is selected and functionally linked to a coding sequence in a library of coding sequences.

The first library of coding sequences or a portion thereof is then expressed to create a library of presumably water-soluble GPCRs. The library is then subjected to a ligand binding assay. In the binding assay, a putatively water-soluble GPCR is preferably contacted with the ligand in an aqueous medium to detect ligand binding.

The present invention includes transmembrane domain variants obtained or obtained from the methods described herein and nucleic acid molecules encoding them.

The present invention relates to at least 50%, preferably at least about 60%, more preferably at least about 70% or 80%, eg, a natural transmembrane protein (eg, GPCR) substituted by Q, T, T or Y, respectively. Water-soluble GPCR variants (“sGPCRs”) characterized by multiple transmembrane domains independently characterized by at least about 90% of hydrophobic amino acid residues (L, I, V and F) are also envisioned. .. The sGPCRs of the present invention are characterized by water solubility and ligand binding. In particular, the sGPCR binds to the same natural ligand as the corresponding natural GPCR.

The present invention is a method of treating the disorder or disease comprising the use of a water-soluble polypeptide to treat a disorder and disease mediated by the activity of a membrane protein, wherein the water-soluble polypeptide is a modified α-helix. It further includes a method comprising a domain and characterized in that the water-soluble polypeptide retains the ligand-binding activity of its natural membrane protein. Examples of such disorders and diseases include, but are not limited to, cancer, small cell lung cancer, melanoma, breast cancer, Parkinson's disease, cardiovascular disease, hypertension and asthma.

As described herein, the water-soluble peptides described herein can be used for the treatment of diseases or disorders mediated by the activity of membrane proteins. In certain embodiments, the water-soluble peptide can act as a "decoy" for the membrane receptor and otherwise bind to a ligand that activates the membrane receptor. Therefore, the water-soluble peptides described herein can be used to reduce the activity of membrane proteins. These water-soluble peptides remain in the circulatory system and can competitively bind to specific ligands, thereby reducing the activity of membrane-bound receptors. For example, GPCR CXCR4 is overexpressed in small cell lung cancer and promotes tumor cell metastasis. Binding to this ligand by a water-soluble peptide as described herein can significantly reduce metastasis.

The chemokine receptor CXCR4 is known in viral studies as a major co-receptor for the invasion of T-cell line-directed HIV (Feng et al. (1996) Science 272: 872-877; Davis et al. ( 1997) J Exp Med 186: 1793-1798; Zaitseva et al. (1997) Nat Med 3: 1369-1375; Sanchez et al. (1997) J Biol Chem 272: 27529-27531). Stromal cell-derived factor 1 (SDF-1) is a chemokine that specifically interacts with CXCR4. When SDF-1 binds to CXCR4, CXCR4 is a downstream kinase pathway such as Ras / MAP kinase and phosphatidylinositol 3-kinase (PI3K) / Akt in lymphocytes, megakaryocytes and hematopoietic stem cells (Bleul et al. (1996)). ) Nature 382: 829-833; Deng et al. (1997) Nature 388: 296-300; Kijowski et al. (2001) Stem Cells 19: 453-466; Majka et al. (2001) Folia. Histochem. Cytobiol. 39: 235-244; Sotsios et al. (1999) J. Immunol. 163: 5954-5963; Vlahakis et al. (2002) J. Immunol. 169: 5546-5554) Gαi protein-mediated signaling (pertussis) Activates toxin sensitivity) (Chen et al. (1998) Mol Pharmacol 53: 177-181). In mice transplanted with human lymph nodes, SDF-1 induces CXCR4-positive cell migration to the transplanted lymph nodes (Blades et al. (2002) J. Immunol. 168: 4308-4317).

Recent studies have shown that CXCR4 interactions can regulate the migration of metastatic cells. Hypoxia, or reduced oxygen partial pressure, is a microenvironmental change that occurs in most solid tumors and is a major inducer of tumor angiogenesis and treatment resistance. Hypoxia raises CXCR4 levels (Staller et al. (2003) Nature 425: 307-311). Microarray analysis of subpopulations of cells from bone metastatic models with increased metastatic activity revealed that one of the increased genes in the metastatic phenotype was CXCR4. In addition, overexpression of CXCR4 in isolated cells significantly increased metastatic activity (Kang et al. (2003) Cancer Cell 3: 537-549). In samples taken from various breast cancer patients, Muller et al. (Muller et al. (2001) Nature 410: 50-56) found that CXCR4 expression levels were higher in primary tumors relative to normal mammary or epithelial cells. I found it. In addition, CXCR4 antibody therapy was found to inhibit metastasis to regional lymph nodes as compared to control isotypes, which all metastasized to lymph nodes and lungs (Muller et al. (2001)). Therefore, the decoy treatment model is suitable for treating CXCR4-mediated diseases and disorders.

In another embodiment, the invention relates to the treatment of a disease or disorder associated with CXCR4-dependent chemotaxis with leukocyte mobilization or abnormal activation. The diseases include arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, allergy, asthma, AIDS-related encephalitis, AIDS-related patchy rash-like rash, AIDS-related interstitial pneumonia, AIDS-related intestinal disease, AIDS-related It is selected from the group consisting of peri-moneral pneumonia and AIDS-related glomerulonephritis.

In another aspect, the invention comprises arthritis, lymphoma, non-small cell lung cancer, lung cancer, breast cancer, prostate cancer, multiple sclerosis, central nervous system developmental disorders, dementia, Parkinson's disease, Alzheimer's disease, tumors, fibromas, It relates to the treatment of diseases or disorders selected from astrocytoma, myeloma, glioblastoma, inflammatory disease, organ transplant rejection, AIDS, HIV infection or angiogenesis.

The present invention also includes pharmaceutical compositions containing the water-soluble polypeptide and a pharmaceutically acceptable carrier or diluent.

The composition is a pharmaceutically acceptable non-toxic carrier defined as an excipient commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. Alternatively, a diluent can also be included. The diluent is selected so as not to affect the biological activity of the drug or pharmacological composition. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hanks solution. The pharmaceutical composition or formulation may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Pharmaceutical compositions include proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acids and copolymers (eg, latex functionalized SEPHAROSE ™, agarose, cellulose, etc.), polymerized amino acids, amino acid copolymers and lipids. Large, slowly metabolized macromolecules such as aggregates (eg, oil droplets or liposomes) can also be included.

The composition can be administered parenterally, for example, by intravenous, intramuscular, intrasubarachnoid space, or subcutaneous injection. Parenteral administration can be achieved by incorporating the composition into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Parenteral preparations may also include antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffering agents such as acetates, citrates or phosphates and tension regulators such as sodium chloride or dextrose may also be added. The parenteral formulation can be encapsulated in glass or plastic ampoules, disposable syringes or multi-dose vials.

In addition, auxiliary substances such as infiltrators, emulsifiers, surfactants, pH buffers and the like may be present in the composition. Other components of the pharmaceutical composition are petroleum, animal, plant or synthetic oils such as peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions.

Injectable formulations can be prepared as either liquid solutions or suspensions, and solids suitable for dissolution in solutions or suspensions or liquid excipients prior to injection can also be prepared. The formulation may also be emulsified or encapsulated in liposomes or microparticles such as polylactic acid, polyglycolide or copolymers to enhance the adjuvant effect as described above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The compositions and agents described herein can be administered in the form of depot injections or implantable formulations that can be formulated to allow sustained or pulsed release of the active ingredient.

Transdermal administration comprises transdermal absorption of the composition through the skin. Percutaneous preparations include patches, ointments, creams, gels, ointments and the like. Transdermal delivery can be achieved using skin patches or transfersomes. See Paul et al., Eur. J. Immunol. 25: 3521-24, 1995 and Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

"Treatment" or "treatment" means preventing or delaying the onset of symptoms, complications or biochemical signs of the disease, reducing or ameliorating the symptoms, or preventing or preventing further progression of the disease, illness or disorder. Including to inhibit. A "patient" is a human subject in need of treatment.

An "effective amount" is a therapeutic agent sufficient to ameliorate one or more symptoms of a disease and / or prevent the progression of the disease, cause recovery of the disease and / or achieve the desired effect. Refers to the quantity.

Computer Systems The various aspects and functions described herein may be implemented as dedicated hardware or software components running in one or more computer systems. There are many examples of computer systems currently in use. Examples of these include, among others, net appliances, personal computers, workstations, mainframes, networked clients, servers, media servers, application servers, database servers and web servers. Other examples of computer systems include mobile computing devices such as mobile phones and personal digital assistants, and network devices such as load balancers, routers and switches. Further, the embodiments may be located on a single computer system or may be distributed among multiple computer systems connected by one or more communication networks.

For example, various aspects, functions, and methods are distributed among one or more computer systems configured to serve one or more client computers or to perform the entire task as part of a distributed system. May be good. Further, the embodiment may be performed on a client server, that is, a multi-layer system, which includes components distributed among one or more server systems that perform various functions. Therefore, embodiments are not limited to execution on any particular system or group of systems. Further, aspects, functions and methods may be implemented in software, hardware or firmware or any combination thereof. Thus, aspects, functions and methods may be implemented within methods, behaviors, systems, system elements and components using various hardware and software configurations, and these examples are of any particular distributed type. Not limited to architecture, network or communication protocol.

With reference to FIG. 10, a block diagram of a distributed computer system 300 in which various aspects and functions are implemented is shown. As shown, the distributed computer system 300 includes one or more computer systems that exchange information. More specifically, the distributed computer system 300 includes computer systems 302, 304 and 306. As shown, the computer systems 302, 304 and 306 are interconnected via the communication network 308 and data can be exchanged via the communication network 308. The network 308 may include any communication network through which the computer system can exchange data. Computer systems 302, 304 and 306 and network 308 may use various methods, protocols and standards for exchanging data using network 308. Examples of these protocols and standards include NAS, Web, storage and other data movement protocols suitable for use in big data environments. To ensure that the data transfer is secure, the computer systems 302, 304 and 306 may transmit data over the network 308 using various security measures such as SSL or VPN technology. The decentralized computer system 300 represents three networked computer systems, but the decentralized computer system 300 is not so limited, and any number of networks networked using any medium and communication protocol. It may include a computer system and a computing device.

As shown in FIG. 10, the computer system 302 includes a processor 310, a memory 312, an interconnect element 314, an interface 316 and a data storage element 318. To implement at least some of the embodiments, functions and methods disclosed herein, processor 310 issues a series of instructions to obtain the processed data. The processor 310 may be any type of processor, multiprocessor or control device. Examples of processors include commercially available processors such as Intel Xeon, Itanium, Core, Celeron or Pentium processors, AMD Opteron processors, Apple A4 or A5 processors, Sun UltraSPARC processors, IBM Power5 + processors, IBM mainframe chips or quantum computers. Can be mentioned. Processor 310 is connected by interconnect elements 314 to other system components, including one or more memory devices 312.

The memory 312 stores a program (eg, a series of instructions encoded by the processor 310) and data during the operation of the computer system 302. Therefore, the memory 312 may be a relatively high performance volatile RAM such as a dynamic RAM (“DRAM”) or a static memory (“SRAM”). However, the memory 312 may include any device for storing data, such as a disk drive or other non-volatile storage device. By various examples, the memory 312 may be personalized and, in some cases, organized into a unique structure to perform the functions disclosed herein. These data structures may be sized and organized to store specific data values and data types.

The components of the computer system 302 are connected by interconnect elements such as the interconnect element 314. The interconnect element 314 may include any communication coupling between system components such as one or more physical buses according to dedicated or standard computing bus technology such as IDE, SCSI, PCI and InfiniBand. The interconnect element 314 allows communication, including instructions and data, to be exchanged between system components of computer system 302.

The computer system 302 also includes one or more interface devices 316, such as an input device, an output device, and a combination of input / output devices. The interface device may receive an input or give an output. More specifically, the output device may provide information for external presentation. The input device may receive information from an external source. Examples of interface devices include keyboards, mouse devices, trackballs, microphones, touch screens, printing devices, display screens, speakers, network interface cards, and the like. The interface device allows the computer system 302 to exchange information and communicate with external entities such as users and other systems.

The data storage element 318 comprises a non-volatile or non-temporary data storage medium that can be read and written by a computer that stores instructions that define a program or other object executed by the processor 310. The data storage element 318 may also contain information that is recorded on or in the medium and processed by the processor 310 during program execution. More specifically, the information may be stored in one or more data structures specifically configured to save storage space or enhance data exchange performance. The instruction may be permanently stored as an encoded signal, and the instruction may cause the processor 310 to perform the functions described herein. The medium may be, for example, an optical disk, a magnetic disk or a flash memory, among others. During operation, processor 310 or some other controller allows faster access to information from the non-volatile recording medium by processor 310 than the storage medium contained in the data storage element 318. Load it into another memory such as 312. However, the memory may be located in the data storage element 318 or the memory 312, the processor 310 processes the data in the memory, and then the data is stored in the storage medium associated with the data storage element 318 after the processing is completed. Copy to. The various components may manage the movement of data between the storage medium and other memory elements, and these examples are not limited to specific data management components. Moreover, these examples are not limited to a particular memory system or data storage system.

The computer system 302 is shown, for example, as a type of computer system capable of performing various aspects and functions, but the aspects and functions are not limited to execution on the computer system 302 shown in FIG. The various aspects and functions can be performed on one or more computers having architectures or components different from those shown in FIG. For example, the computer system 302 comprises specially programmed, special purpose hardware such as an application specific integrated circuit (“ASIC”) designed to perform a particular operation as disclosed herein. You may be. On the other hand, another example uses a grid of some general purpose computing devices running MAC OS System X with a Motorola PowerPC processor and some dedicated computing devices running its own hardware and operating system. Can perform the same function.

The computer system 302 may be a computer system including an operating system that manages at least a portion of the hardware elements contained in the computer system 302. In some examples, a processor or controller, such as processor 310, runs an operating system. Examples of specific operating systems that can be run are Windows operating systems such as Windows NT, Windows 2000 (Windows ME), Windows XP, Windows Vista or Windows 7 operating systems available from Microsoft, Apple Computer. Available MAC OS System X or OS operating systems, one of many Linux® operating system distributions, such as the Enterprise Linux® operating system available from Red Hat. Examples include the Solaris operating system available from Oracle or the UNIX® operating system available from various sources. Many other operating systems may be used and these examples are not limited to any particular operating system.

The processor 310 and the operating system together define a computer platform on which application programs are written in a high-level programming language. These component applications may be executable intermediate bytecode or code that is interpreted and executed, communicating over a communication network, such as the Internet, using a communication protocol, such as TCP / IP. Similarly, the aspect is. It may be implemented using an object-oriented programming language such as Net, SmallTalk, Java®, C ++, Ada, C # (C Sharp), Python or Javascript®. Other object-oriented programming languages may also be used. Alternatively, you may use a function, script or logic programming language.

In addition, various aspects and functions may be implemented in an unprogrammed environment. For example, a document created in HTML, XML or other format can give a graphical user interface aspect or perform other functions when viewed in a browser program window. In addition, the various examples may be implemented as programmed or unprogrammed elements or any combination thereof. For example, the web page may be implemented using HTML, but the necessary data objects in the web page may be written in C ++. Therefore, the example is not limited to a specific programming language, and any suitable programming language can be used. Accordingly, the functional components disclosed herein include a wide variety of elements (eg, dedicated hardware, executable code, data structures) configured to perform the functions described herein. Or an object).

In some examples, the components disclosed herein may read parameters that affect the functionality performed by the component. These parameters may be physically stored in any form of suitable memory, including volatile memory (such as RAM) or non-volatile memory (such as magnetic hard drive). In addition, the parameters are logically stored in their own data structures (such as databases or files defined by userspace applications) or commonly shared data structures (such as application registries defined by operating systems). May be good. Also, some examples provide both a system and a user interface that allows an external entity to modify parameters and thereby configure the behavior of the component.

The software for performing the calculation method is shown as a whole in FIG. 11A, where the user selects the operating parameters to perform the procedure on the computer, as described earlier herein ( 402), where one or more sequences are input (404) and substitutions are made (408). The system is capable of confirming secondary structure (408) and confirming the water solubility of one or more mutants. As shown in FIG. 11B, the program can include additional processing options in addition to those described above, where it can store one or more ranking functions (442) and is used by the user. The ranking function to be selected can be selected or the system can automatically select it (444). The system then generates ranks as described herein (446), then the user produces selected variants (448) and measures function (448), followed by Functional data can be entered and the processing procedure modified (450) based on it.

The present invention will be better understood in the context of the following examples, which are intended as illustrative only and do not limit the scope of the invention. Various changes and amendments to the disclosed embodiments will be apparent to those skilled in the art and such changes can be made without departing from the spirit of the invention and the appended claims.

である。 Example 1: CXC chemokine receptor type 4 isotype a (CXCR4)
CXCR4 is a 356 amino acid long chemokine receptor. It has a molecular weight of about 8.61 pI and 40221.19 Da. The sequence of CXCR4 published in the literature is

Is.

This sequence is subjected to TMHMM to identify the transmembrane domain shown in FIG.

Substituting all or substantially all of the hydrophobic amino acids L, I, V and F with Q, T and Y (respectively) gives the following sequences.

The expected pI of this protein is 8.54 and the molecular weight is 40551.64 Da. Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the present invention includes a transmembrane domain (TM1) containing amino acids 47-70 of SEQ ID NO: 2 and a protein containing it. As an example, FIG. 3 shows an α-helix prediction of the TM1 sequence. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of the extracellular and intracellular loop sequences of SEQ ID NO: 2 (ununderlined sequences). In addition or as an alternative, the proteins containing TM1 herein are one, two, three or optionally of the native L, I, V and F amino acids set forth in SEQ ID NO: 2 or SEQ ID NO: 1. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding four or more.

The natural protein sequence of CXCR4 (different in N-terminal amino acids) is again subjected to the method. The program output divided the native sequences into extracellular and intracellular regions and selected eight transmembrane domain variants for each of the transmembrane domains. The results are shown in FIG. 4 and the table below.

MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNK (SEQ ID NO: 3; EC1)

TM1 mutant:
IFLPTTYSTTFQTGTTGNGQVTQVM (SEQ ID NO: 4)
IFQPTTYSTTFQTGTTGNGQVTQVM (SEQ ID NO: 5)
IFQPTTYSTTFQTGTTGNGQVTQTM (SEQ ID NO: 6)
IFQPTTYSTTYQTGTTGNGQVTQTM (SEQ ID NO: 7)
IFQPTTYSTTYQTGTTGNGQTTQVM (SEQ ID NO: 8)
IFQPTTYSTTYQTGTTGNGQTIQTM (SEQ ID NO: 9)
IFQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO: 10)
TYQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO: 11)

GYQKKLRSMTDKYR (SEQ ID NO: 12; IC1)

TM2 mutant:
LHLSTADQQFTTTQPFWAVDAV (SEQ ID NO: 13)
LHLSVADQQYTTTQPFWATDAV (SEQ ID NO: 14)
LHQSVADQQYVTTQPFWATDAT (SEQ ID NO: 15)
QHQSVADQQFTTTQPFWATDAT (SEQ ID NO: 16)
LHQSVADQQYTITQPYWATDAT (SEQ ID NO: 17)
QHLSVADQQYTITQPYWATDAT (SEQ ID NO: 18)
QHLSTADQQYVTTQPYWATDAT (SEQ ID NO: 19)
QHQSTADQQYTTTQPYWATDAT (SEQ ID NO: 20)

ANWYFGNFLCK (SEQ ID NO: 21; EC2)

TM3 mutant:
AVHVTYTVNQYSSVQIQAFT (SEQ ID NO: 22)
AVHTTYTVNQYSSVQIQAFT (SEQ ID NO: 23)
AVHTTYTVNQYSSVQTQAFT (SEQ ID NO: 24)
ATHTTYTVNQYSSVQTQAFT (SEQ ID NO: 25)
ATHTIYTTNQYSSVQTQAFT (SEQ ID NO: 26)
AVHTTYTTNQYSSVQTQAFT (SEQ ID NO: 27)
ATHTTYTTNQYSSVQTQAFT (SEQ ID NO: 28)
ATHTTYTTNQYSSTQTQAYT (SEQ ID NO: 29)

SLDRYLAIVHATNSQRPRKLLAEK (SEQ ID NO: 30; IC2)

TM4 mutant:
VTYTGVWTPAQQQTIPDFIF (SEQ ID NO: 31)
TTYTGTWIPAQQQTIPDFIF (SEQ ID NO: 32)
TTYTGTWTPAQQQTIPDFIF (SEQ ID NO: 33)
TTYTGTWTPAQQQTIPDFIY (SEQ ID NO: 34)
TTYVGTWTPAQQQTTPDYIF (SEQ ID NO: 35)
TTYVGTWTPAQQQTTPDFIY (SEQ ID NO: 36)
TTYTGVWTPAQQQTTPDYTF (SEQ ID NO: 37)
TTYTGTWTPAQQQTTPDYTY (SEQ ID NO: 38)

ANVSEADDRYICDRFYPNDLW (SEQ ID NO: 39; EC3)

TM5 mutant:
VVVFQFQHTMVGQTQPGTTTQ (SEQ ID NO: 40)
VVVFQFQHTMTGQTQPGTTTQ (SEQ ID NO: 41)
VVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 42)
VVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO: 43)
TVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 44)
VVTFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 45)
TVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO: 46)
TTTYQYQHTMTGQTQPGTTTQ (SEQ ID NO: 47)

SCYCIIISKLSHSKGHQKRKALKTT (SEQ ID NO: 48; IC3)

TM6 mutant:
VTQIQAFFACWQPYYTGTST (SEQ ID NO: 49)
VIQIQAYFACWQPYYTGTST (SEQ ID NO: 50)
VIQIQAYYACWQPYYTGTST (SEQ ID NO: 51)
VIQTQAFYACWQPYYTGTST (SEQ ID NO: 52)
VIQTQAYFACWQPYYTGTST (SEQ ID NO: 53)
VTQIQAFYACWQPYYTGTST (SEQ ID NO: 54)
VIQTQAYYACWQPYYTGTST (SEQ ID NO: 55)
TTQTQAYYACWQPYYTGTST (SEQ ID NO: 56)

DSFILLEIIKQGCEFENTVHK (SEQ ID NO: 57; EC4)

TM7 mutant
WISITEAQAFFHCCLNPIQY (SEQ ID NO: 58)
WISITEAQAFYHCCLNPIQY (SEQ ID NO: 59)
WISITEAQAYFHCCQNPTLY (SEQ ID NO: 60)
WISTTEALAFYHCCQNPTQY (SEQ ID NO: 61)
WISTTEALAYFHCCQNPTQY (SEQ ID NO: 62)
WISITEALAYYHCCQNPTQY (SEQ ID NO: 63)
WISTTEALAYYHCCQNPTQY (SEQ ID NO: 64)
WTSTTEAQAYYHCCQNPTQY

AFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 65; IC4)

The pre, inter, and post-sequences (SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57 and 65) of each list of transmembrane domain variants are N', intermediate and C'extracellular and intracellular, respectively. It seems clear from the above that it is a region.

The above sequences were then used to generate coding sequences suitable for expression in the expression system, in this case yeast, as is known in the art. This coding sequence is then rearranged and expressed, each containing one transmembrane domain variant within each variant list between the intracellular and extracellular domains of SEQ ID NOs: 3, 12, 21, 30. , 39, 48, 57 and 65, to create a library containing multiple proteins.

The library thus prepared was then assayed for the CXCR4 homologous ligand, SDF1a (or CCL12), on a plasmid expressed in yeast that binds within living yeast cells. Ligand binding was detected by gene activation with a yeast-to-hybrid system, and then samples were sequenced. Nineteen CXCR4 mutants were sequenced. The result is shown in FIG.

Example 2: CXC chemokine receptor type 3 isotype b (CX3CR1)
CX3CR1 is a 355 amino acid long chemokine receptor. It has a molecular weight of about 6.74 pI and 40396.4 Da. This sequence is submitted to TMHMM to identify its transmembrane domain. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain were (respectively) substituted with Q, T and Y and aligned to the wild type (upper line): Obtain an array (lower line).

The predicted pI for this protein variant is 6.74 and the molecular weight is 41027.17 Da. Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the present invention includes a transmembrane domain containing an underlined amino acid of SEQ ID NO: 67. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of the extracellular and intracellular loop sequences of SEQ ID NO: 66 (ununderlined sequences). In addition or as an alternative, the protein containing TM1 herein is one, two, three or one of the native V, L, I and F amino acids set forth in SEQ ID NO: 67 or SEQ ID NO: 66. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

The natural protein sequence of CX3CR1 is again subjected to this method. The program output divided the native sequences into extracellular and intracellular regions and selected eight transmembrane domain variants for each of the transmembrane domains. The results are shown in the table below.

MDQFPESVTENFEYDDLAEACYIGDIVVFGT (SEQ ID NO: 68)

TM1 mutant:
TYQSTYYSTTFATGQVGNQQVVFALTNS (SEQ ID NO: 69)
TYQSTYYSTTYATGQVGNQQVVFALTNS (SEQ ID NO: 70)
TYQSTYYSTTYATGQVGNQQVVFAQTNS (SEQ ID NO: 71)
TYQSTYYSTTYATGQTGNLQVTFAQTNS (SEQ ID NO: 72)
TYQSTYYSTTYATGQTGNQLVTFAQTNS (SEQ ID NO: 73)
TYQSTYYSTTYATGQTGNQQVVFAQTNS (SEQ ID NO: 74)
TYQSTYYSTTYATGQTGNLQVTYAQTNS (SEQ ID NO: 75)
TYQSTYYSTTYATGQTGNQQTTYAQTNS (SEQ ID NO: 76)

KKPKSVTDIY (SEQ ID NO: 77)

TM2 mutant
LLNQAQSDQLFVATQPFWTHY (SEQ ID NO: 78)
LLNQAQSDQQFVATQPFWTHY (SEQ ID NO: 79)
QQNLAQSDQQFVATQPFWTHY (SEQ ID NO: 80)
LQNLAQSDQQYTATQPFWTHY (SEQ ID NO: 81)
QLNLAQSDQQYTATQPFWTHY (SEQ ID NO: 82)
LLNQAQSDQQFTATQPYWTHY (SEQ ID NO: 83)
QQNLAQSDQQFTATQPYWTHY (SEQ ID NO: 84)
QQNQAQSDQQYTATQPYWTHY (SEQ ID NO: 85)

LINEKGLHNAMCK (SEQ ID NO: 86)

TM3 mutant:
YTTAYYYTGYYGSTYYTTTTST (SEQ ID NO: 87)

DRYLAIVLAANSMNNRT (SEQ ID NO: 88)

TM4 mutant:
VQHGTTTSQGTWAAATQVAAPQFMF (SEQ ID NO: 89)
VQHGVTTSQGTWAAATQTAAPQFMF (SEQ ID NO: 90)
VQHGTTTSQGVWAAATQTAAPQFMY (SEQ ID NO: 91)
VQHGTTTSQGTWAAAIQTAAPQFMY (SEQ ID NO: 92)
VQHGTTTSQGTWAAATQTAAPQFMF (SEQ ID NO: 93)
VQHGTTISQGTWAAATQTAAPQYMF (SEQ ID NO: 94)
VQHGTTTSQGTWAAATQTAAPQFMY (SEQ ID NO: 95)
TQHGTTTSQGTWAAATQTAAPQYMY (SEQ ID NO: 96)

TKQKENECLGDYPEVLQEIWPVLRNVET (SEQ ID NO: 97)

TM5 mutant:
NFLGFQQPQQIMSYCYFRIT (SEQ ID NO: 98)
NFQGFLQPQQTMSYCYFRIT (SEQ ID NO: 99)
NFQGFLQPQQTMSYCYFRTT (SEQ ID NO: 100)
NFQGFQQPQQTMSYCYYRIT (SEQ ID NO: 101)
NFQGFLQPQQTMSYCYYRTT (SEQ ID NO: 102)
NFQGYLQPQQTMSYCYFRTT (SEQ ID NO: 103)
NYQGFQQPQQTMSYCYFRTT (SEQ ID NO: 104)
NYQGYQQPQQTMSYCYYRTT (SEQ ID NO: 105)

QTLFSCKNHKKAKAIK (SEQ ID NO: 106)

TM6 mutant:
LIQQTTTTFYQFWTPYNTMTFQETL (SEQ ID NO: 107)
LIQQTTTTFYQYWTPYNVMTFQETQ (SEQ ID NO: 108)
LIQQTTTTYYQFWTPYNTMTFQETQ (SEQ ID NO: 109)
QIQQTTTTFYQYWTPYNTMTFQETQ (SEQ ID NO: 110)
LTQQTTTTYYQFWTPYNTMTFQETQ (SEQ ID NO: 111)
QIQQTTTTFFQYWTPYNTMTYQETQ (SEQ ID NO: 112)
QIQQTTTTFYQYWTPYNTMTYQETQ (SEQ ID NO: 113)
QTQQTTTTYYQYWTPYNTMTYQETQ (SEQ ID NO: 114)

KLYDFFPSCDMRKDLRL (SEQ ID NO: 115)

TM7 mutant:
ALSVTETVAFSHCCQNPQIYAFAG (SEQ ID NO: 116)
AQSVTETTAFSHCCQNPLIYAFAG (SEQ ID NO: 117)
ALSVTETVAFSHCCQNPQTYAYAG (SEQ ID NO: 118)
AQSVTETTAFSHCCQNPQIYAYAG (SEQ ID NO: 119)
ALSVTETTAFSHCCQNPQTYAYAG (SEQ ID NO: 120)
ALSTTETTAYSHCCQNPQIYAFAG (SEQ ID NO: 121)
ALSVTETTAYSHCCQNPQTYAYAG (SEQ ID NO: 122)
AQSTTETTAYSHCCQNPQTYAYAG (SEQ ID NO: 123)

EKFRRYLYHLYGKCLAVLCGRSVHVDFSSSESQRSRHGSVLSSNFTYHTSDGDALLLL (SEQ ID NO: 124)

Similar to Example 1 above, the anterior, interstitial and posterior sequences of each list of transmembrane domain variants are N', intermediate and C'intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in the expression system, in this case yeast, as is known in the art. This coding sequence is then rearranged and expressed, each containing one transmembrane domain variant within each variant list between the intracellular and extracellular domains of SEQ ID NOs: 68, 77, 86, 88. , 97, 106 and 115 were made to contain multiple proteins.

The library thus prepared was then assayed for binding to the CX3CR1 homologous ligand (CXCL1) in an aqueous medium as described in Example 1. Ligand binding was detected and then the sample was sequenced. Seven mutants were sequenced. The result is shown in FIG.

Example 3: CCR3 mutant The method of Example 1 was repeated for chemokine receptor type 3 isotype 3.

All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain were (respectively) substituted with Q, T and Y and aligned to the wild type (upper line): Obtain an array (lower line).

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain containing an underlined amino acid of SEQ ID NO: 126. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of the extracellular and intracellular loop sequences (ununderlined sequences) of SEQ ID NO: 126. As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the native V, L, I and F amino acids set forth in SEQ ID NO: 126 or SEQ ID NO: 125. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

The natural protein sequence of CCR3 is subjected to the present method again (note the difference in the N-terminal sequence). The program output divided the native sequences into extracellular and intracellular regions and selected eight transmembrane domain variants for each of the transmembrane domains. The results are shown in the table below.

MTTSLDTVETFGTTSYYDDVGLLCEKADTRALMA (SEQ ID NO: 127)

TM1 mutant:
QFVPPQYSQTFTTGQQGNVTVTMTQIKY (SEQ ID NO: 128)
QFVPPQYSQTFTTGQQGNTTVTMTQIKY (SEQ ID NO: 129)
QFVPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 130)
QFTPPQYSQTYTTGQQGNVTTTMTQIKY (SEQ ID NO: 131)
QFTPPQYSQTYTTGQQGNTVTTMTQIKY (SEQ ID NO: 132)
QFTPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 133)
QFTPPQYSQTYTTGQQGNTTTTMTQIKY (SEQ ID NO: 134)
QYTPPQYSQTYTTGQQGNTTTTMTQTKY (SEQ ID NO: 135)

RRLRIMTNIY (SEQ ID NO: 136)

TM2 mutant:
LLNQATSDQQFQVTQPFWIHY (SEQ ID NO: 137)
LQNQAISDQLFQTTQPFWTHY (SEQ ID NO: 138)
QQNLAISDQQFQTTQPFWTHY (SEQ ID NO: 139)
QLNQAISDQQFQTTQPYWTHY (SEQ ID NO: 140)
QQNLAISDQQYQVTQPYWTHY (SEQ ID NO: 141)
LQNQATSDQLFQTTQPYWTHY (SEQ ID NO: 142)
QQNQAISDQQYQVTQPYWTHY (SEQ ID NO: 143)
QQNQATSDQQYQTTQPYWTHY (SEQ ID NO: 144)

VRGHNWVFGHGMCK (SEQ ID NO: 145)

TM3 mutant:
LQSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 146)
QLSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 147)
QLSGFYHTGQYSETFYTTQQTT (SEQ ID NO: 148)
QLSGFYHTGQYSETYFTTQQTT (SEQ ID NO: 149)
QLSGYYHTGQYSETFFTTQQTT (SEQ ID NO: 150)
QQSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 151)
QQSGFYHTGQYSETFYTTQQTT (SEQ ID NO: 152)
QQSGYYHTGQYSETYYTTQQTT (SEQ ID NO: 153)

DRYLAIVHAVFALRART (SEQ ID NO: 154)

TM4 mutant:
TTFGTTTSTVTWGQAVQAAQPEFIF (SEQ ID NO: 155)
TTFGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO: 156)
TTYGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO: 157)
TTYGTTTSTTTWGQAVQAAQPEFTF (SEQ ID NO: 158)
TTYGTTTSTTTWGQATQAAQPEFIF (SEQ ID NO: 159)
TTFGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO: 160)
TTYGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO: 161)
TTYGTTTSTTTWGQATQAAQPEYTY (SEQ ID NO: 162)

YETEELFEETLCSALYPEDTVYSWRHFHTLRM (SEQ ID NO: 163)

TM5 mutant:
TIFCQVQPQQTMATCYTGTT (SEQ ID NO: 164)
TIFCQTQPQQVMATCYTGTT (SEQ ID NO: 165)
TIFCQTQPQQTMATCYTGIT (SEQ ID NO: 166)
TIFCQTQPQQTMATCYTGTI (SEQ ID NO: 167)
TTFCQVQPQQVMATCYTGTT (SEQ ID NO: 168)
TIYCQVQPQQVMATCYTGTT (SEQ ID NO: 169)
TIFCQTQPQQTMATCYTGTT (SEQ ID NO: 170)
TTYCQTQPQQTMATCYTGTT (SEQ ID NO: 171)

KTLLRCPSKKKYKAIR (SEQ ID NO: 172)

TM6 mutant:
QTYTTMATYYTYWTPYNTATQQSSY (SEQ ID NO: 173)

QSILFGNDCERSKHLDL (SEQ ID NO: 174)

TM7 mutant:
VMQVTEVTAYSHCCMNPVTYAFTG (SEQ ID NO: 175)
VMQVTEVTAYSHCCMNPTTYAYVG (SEQ ID NO: 176)
VMLTTEVTAYSHCCMNPTTYAFTG (SEQ ID NO: 177)
VMQVTETTAYSHCCMNPVTYAYTG (SEQ ID NO: 178)
TMQVTETIAYSHCCMNPTTYAFTG (SEQ ID NO: 179)
TMQVTETTAYSHCCMNPTTYAFVG (SEQ ID NO: 180)
VMQTTETIAYSHCCMNPTTYAYTG (SEQ ID NO: 181)
TMQTTETTAYSHCCMNPTTYAYTG (SEQ ID NO: 182)

ERFRKYLRHFFHRHLLMHLGRYIPFLPSEKLERTSSVSPSTAEPELSIVF (SEQ ID NO: 183)

Similar to Example 1 above, the anterior, interstitial and posterior sequences of each list of transmembrane domain variants are N', intermediate and C'intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in the expression system, in this case yeast, as is known in the art. This coding sequence is then rearranged and expressed, each containing one transmembrane domain variant within each variant list between the intracellular and extracellular domains of SEQ ID NO: 127, 136, 145, 154. , 163, 172, 174 and 183.

The library thus prepared was then assayed for binding to the CCR3 homologous ligand, CCL3, in an aqueous medium as described in Example 1. Ligand binding was detected and then the sample was sequenced. Eleven mutants were sequenced. The result is shown in FIG.

Example 4: CCR5 mutant The method of Example 1 was repeated for chemokine receptor type 5 isotype 3.

All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain were (respectively) substituted with Q, T and Y and aligned to the wild type (upper line): Obtain an array (lower line).

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the present invention includes a transmembrane domain containing an underlined amino acid of SEQ ID NO: 185. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of the extracellular and intracellular loop sequences (ununderlined sequences) of SEQ ID NO: 185. In addition or as an alternative, the protein containing TM1 herein is one, two, three or one of the native V, L, I and F amino acids set forth in SEQ ID NO: 185 or SEQ ID NO: 184. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

The natural protein sequence of CCR5 is subjected to the present method again (note the difference in the N-terminal sequence). The program output divided the native sequences into extracellular and intracellular regions and selected eight transmembrane domain variants for each of the transmembrane domains. The results are shown in the table below.

MDYQVSSPIYDINYYTSEPCQKINVKQIAA (SEQ ID NO: 186)

TM1 mutant:
RLQPPQYSQTFTFGFTGNMQVTQTQINC (SEQ ID NO: 187)
RLQPPQYSQTFTFGYTGNMQVTQTQINC (SEQ ID NO: 188)
RQQPPQYSQTFTFGFTGNMQTTQTQINC (SEQ ID NO: 189)
RQQPPQYSQTFTYGFTGNMQTTQTQINC (SEQ ID NO: 190)
RQQPPQYSQTYTFGFTGNMQTTQTQINC (SEQ ID NO: 191)
RQQPPQYSQTFTFGYTGNMQTTQTQINC (SEQ ID NO: 192)
RQQPPQYSQTYTFGYTGNMQTTQTQINC (SEQ ID NO: 193)
RQQPPQYSQTYTYGYTGNMQTTQTQTNC (SEQ ID NO: 194)

KRLKSMTDIY (SEQ ID NO: 195)

TM2 mutant:
LQNQAISDQFFQQTVPFWAHY (SEQ ID NO: 196)
LQNQAISDQFFQQTTPFWAHY (SEQ ID NO: 197)
LQNQAISDQFFQQTTPYWAHY (SEQ ID NO: 198)
LQNQAISDQFYQQTTPYWAHY (SEQ ID NO: 199)
LQNQAISDQYFQQTTPYWAHY (SEQ ID NO: 200)
LQNQATSDQFFQQTTPYWAHY (SEQ ID NO: 201)
LQNQAISDQYYQQTTPYWAHY (SEQ ID NO: 202)
QQNQATSDQYYQQTTPYWAHY (SEQ ID NO: 203)

AAAQWDFGNTMCQ (SEQ ID NO: 204)

TM3 mutant:
QQTGQYFTGYYSGTYYTTQQTT (SEQ ID NO: 205)
QQTGQYYTGYYSGTYYTTQQTT (SEQ ID NO: 206)

DRYLAVVHAVFALKART (SEQ ID NO: 207)

TM4 mutant:
TTYGTTTSTTTWTTATYASQPGTTY (SEQ ID NO: 208)

TRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKI (SEQ ID NO: 209)

TM5 mutant:
VIQGQVQPQQVMVTCYSGIQ (SEQ ID NO: 210)
VIQGQVQPQQVMTTCYSGIQ (SEQ ID NO: 211)
VIQGQVQPQQTMTTCYSGIQ (SEQ ID NO: 212)
VTQGQVQPQQTMVTCYSGTQ (SEQ ID NO: 213)
TIQGQVQPQQVMTTCYSGTQ (SEQ ID NO: 214)
TIQGQVQPQQTMVTCYSGTQ (SEQ ID NO: 215)
TTQGQVQPQQVMTTCYSGTQ (SEQ ID NO: 216)
TTQGQTQPQQTMTTCYSGTQ (SEQ ID NO: 217)

KTLLRCRNEKKRHRAVR (SEQ ID NO: 218)

TM6 mutant:
QTFTTMTTYYQFWAPYNIVQQLNTF (SEQ ID NO: 219)
QTFTTMTTYYQFWAPYNTVQQLNTF (SEQ ID NO: 220)
QTFTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO: 221)
QTFTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO: 222)
QTYTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO: 223)
QTFTTMTTYYQYWAPYNTTQQLNTF (SEQ ID NO: 224)
QTYTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO: 225)
QTYTTMTTYYQYWAPYNTTQQQNTY (SEQ ID NO: 225)

QEFFGLNNCSSSNRLDQ (SEQ ID NO: 226)

TM7 mutant:
AMQVTETQGMTHCCINPIIYAFVG (SEQ ID NO: 227)
AMQVTETLGMTHCCTNPIIYAFTG (SEQ ID NO: 228)
AMQVTETQGMTHCCINPTIYAYVG (SEQ ID NO: 229)
AMQTTETQGMTHCCINPITYAFTG (SEQ ID NO: 230)
AMQTTETQGMTHCCINPTIYAFTG (SEQ ID NO: 231)
AMQVTETQGMTHCCTNPTIYAYVG (SEQ ID NO: 232)
AMQTTETQGMTHCCINPTTYAYVG (SEQ ID NO: 233)
AMQTTETQGMTHCCTNPTTYAYTG (SEQ ID NO: 234)

EKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL (SEQ ID NO: 235)

Similar to Example 1 above, the pre-, inter- and post-sequences of each list of transmembrane domain variants are N', intermediate and C'intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in the expression system, in this case yeast, as is known in the art. This coding sequence is then rearranged and expressed, each containing one transmembrane domain variant within each variant list between the intracellular and extracellular domains of SEQ ID NO: 186, 195, 204, 207. , 209, 218, 226 and 235, and a library containing multiple proteins was prepared.

The library thus prepared was then assayed in an aqueous medium for binding to the CCR5 homologous ligand, CCL5, as described in Example 1. Ligand binding was detected and then the sample was sequenced. One mutant was sequenced. The result is shown in FIG.

Example 5: CXCR3 mutant The method of Example 1 was repeated for CXC chemokine receptor type 3 isotype 2. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y and aligned to the wild type (SEQ ID NO: 324, upper line). The following sequence (SEQ ID NO: 325, lower line) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the native V, L, I and F amino acids set forth in SEQ ID NO: 325 or SEQ ID NO: 324. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

As described above, the natural protein sequence of CXCR3 was subjected to this method. The program output divided the native sequences into extracellular and intracellular regions and selected eight transmembrane domain variants for each of the transmembrane domains. The results are shown in the table below.

MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR (SEQ ID NO: 235)

TM1 mutant:
AFLPALYSQQFQQGQQGNGAVAATQLS (SEQ ID NO: 236)
AFQPALYSQQFQQGQQGNGAVAAVQQS (SEQ ID NO: 237)
AFQPAQYSQQFLQGQQGNGAVAATQQS (SEQ ID NO: 238)
AYQPALYSLQYQQGQQGNGATAAVQQS (SEQ ID NO: 239)
AYQPALYSQLFQQGQQGNGATAATQQS (SEQ ID NO: 240)
AFQPALYSLQYQQGQQGNGATAATQQS (SEQ ID NO: 241)
AYQPAQYSLQYQQGQQGNGATAAVQQS (SEQ ID NO: 242)
AYQPAQYSQQYQQGQQGNGATAATQQS (SEQ ID NO: 243)

RRTALSSTD (SEQ ID NO: 244)

TM2 mutant:
TFLQHLAVADTQQVQTLPQWA (SEQ ID NO: 245)
TFLQHQAVADTQLVQTQPQWA (SEQ ID NO: 246)
TFQQHLAVADTQQVQTQPQWA (SEQ ID NO: 247)
TYLQHQAVADTQQVQTQPQWA (SEQ ID NO: 248)
TYQLHQAVADTQQVQTQPQWA (SEQ ID NO: 249)
TYQQHLAVADTQVQTQPQWA (SEQ ID NO: 250)
TYQQHQAVADTQQVQTQPQWA (SEQ ID NO: 251)
TYQQHQATADTQQTQTQPQWA (SEQ ID NO: 252)

VDAAVQWVFGSGLCK (SEQ ID NO: 253)

TM3 mutant:
TAGAQYNTNFYAGAQQQACISF (SEQ ID NO: 254)
TAGAQYNTNFYAGAQLQACTSF (SEQ ID NO: 255)
TAGAQYNTNFYAGAQQLACTSF (SEQ ID NO: 256)
TAGAQFNTNYYAGAQQQACISF (SEQ ID NO: 257)
TAGAQYNTNYYAGAQQQACISF (SEQ ID NO: 258)
TAGAQYNTNYYAGAQLQACTSF (SEQ ID NO: 259)
TAGAQYNTNYYAGAQQLACTSF (SEQ ID NO: 260)
TAGAQYNTNYYAGAQQQACTSY (SEQ ID NO: 261)

DRYLNIVHATQLYRRGPPARVT (SEQ ID NO: 262)

TM4 mutant:
LTCQAVWGQCQQFAQPDFIF (SEQ ID NO: 263)
QTCQAVWGQCQQFAQPDFIF (SEQ ID NO: 264)
QTCQATWGQCQQFAQPDFIF (SEQ ID NO: 265)
QTCQATWGQCQQYAQPDFIF (SEQ ID NO: 266)
QTCQATWGQCQQFAQPDFTF (SEQ ID NO: 267)
QTCQATWGQCQQFAQPDYIF (SEQ ID NO: 268)
QTCQATWGQCQQYAQPDYIF (SEQ ID NO: 269)
QTCQATWGQCQQYAQPDYTY (SEQ ID NO: 270)

LSAHHDERLNATHCQYNFPQVGR (SEQ ID NO: 271)

TM5 mutant:
TAQRTQQQTAGYQQPQQTMAY (SEQ ID NO: 272)

CYAHILAVLLVSRGQRRLRAMR (SEQ ID NO: 273)

TM6 mutant:
QVTTTTVAFAQCWTPYHQVVQV (SEQ ID NO: 274)
QVTTTTVAFAQCWTPYHQTVQV (SEQ ID NO: 275)
QVTTTTTAFAQCWTPYHQTVQV (SEQ ID NO: 276)
QVTTTTTAYAQCWTPYHQTVQV (SEQ ID NO: 277)
QVTTTTTAFAQCWTPYHQTTQV (SEQ ID NO: 278)
QTTTTTVAFAQCWTPYHQTTQV (SEQ ID NO: 279)
QVTTTTTAYAQCWTPYHQTTQV (SEQ ID NO: 280)
QTTTTTTAYAQCWTPYHQTTQT (SEQ ID NO: 281)

DILMDLGALARNCGRESRVDV (SEQ ID NO: 282)

TM7 mutant:
AKSVTSGQGYMHCCLNPLQYAFV (SEQ ID NO: 283)
AKSVTSGQGYMHCCLNPQLYAFT (SEQ ID NO: 284)
AKSVTSGQGYMHCCLNPLQYAFT (SEQ ID NO: 285)
AKSTTSGQGYMHCCLNPQQYAFV (SEQ ID NO: 286)
AKSTTSGQGYMHCCQNPLQYAFV (SEQ ID NO: 287)
AKSTTSGQGYMHCCQNPQLYAFV (SEQ ID NO: 288)
AKSTTSGQGYMHCCQNPLQYAFT (SEQ ID NO: 289)
AKSTTSGQGYMHCCQNPQQYAYT (SEQ ID NO: 290)

GVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO: 291)

The above sequences can be used to generate coding sequences suitable for expression in expression systems, in this case yeast, as is known in the art. The coding sequences are then rearranged and expressed, each having an intracellular and extracellular loop, each containing one transmembrane domain variant within each variant list between its intracellular and extracellular domains. A library containing the above proteins was prepared.

The library thus prepared can then be assayed for binding to a cognate ligand in an aqueous medium as described in Example 1.

Example 6: (CCR-1) CC Chemokine Receptor Type 1
Example 1 was repeated for the title protein.

All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 292). The following sequence (lower line, SEQ ID NO: 293) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, or three of the proteins depicted or the native V, L, I, and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding one or optionally four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 7: (CCR-2) CC Chemokine Receptor Type 2 Isotype A
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 294) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 295) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, or three of the proteins depicted or the native V, L, I, and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding one or optionally four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 8: (CCR-4) CC Chemokine Receptor Type 4
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 296). The following sequence (lower line, SEQ ID NO: 297) was obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, or three of the proteins depicted or the native V, L, I, and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding one or optionally four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 9: (CCR-6) CC Chemokine Receptor Type 6
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 298). The following sequence (lower line, SEQ ID NO: 299) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 10: (CCR-7) CC Chemokine Receptor Type 7 Precursor Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 300). The following sequence (lower line, SEQ ID NO: 301) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 11: (CCR-8) CC Chemokine Receptor Type 8
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 302). The following sequence (lower line, SEQ ID NO: 303) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 12: (CCR-9) CC Chemokine Receptor Type 9 Isotype B
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 304). The following sequence (lower line, SEQ ID NO: 305) was obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 13: (CCR-10) CC Chemokine Receptor Type 10
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 306) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 307) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more. Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 14: (CXCR1) Chemokine Receptor Type 1
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 308). The following sequence (lower line, SEQ ID NO: 309) was obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 15: (CXR1) CXR chemokine receptor type 1
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 310) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 311) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, or three of the proteins depicted or the native V, L, I, and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding one or optionally four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 16: (CXCR2) Chemokine Receptor Type 2
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 312). The following sequence (lower line, SEQ ID NO: 313) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 17: (CCR-10) CC Chemokine Receptor Type 10
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 314) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 315) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 18: (CXCR6) Chemokine Receptor Type 6
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 316) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 317) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 19: (CXCR7) Chemokine Receptor Type 7
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 318). The following sequence (lower line, SEQ ID NO: 319) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 20: (CLR-1a) chemokine-like receptor 1 isotype a
Example 1 was repeated for the title protein. All or substantially all of the hydrophobic amino acids L, I, V and F within the transmembrane domain are (respectively) substituted with Q, T and Y to align to the wild type (upper line, SEQ ID NO: 320). The following sequence (lower line, SEQ ID NO: 321) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 21: DARIA Duffy antigen / chemokine receptor isotype a
Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 322) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 323) is obtained.

Each of the predicted transmembrane regions is underlined, exemplifying a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains, each containing an underlined domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, three or one of the proteins depicted or the native L, I, V and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more. Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 22: CD81 antigen CD81 may play an important role in the regulation of lymphoma cell proliferation and interacts with the 16 kDa Leu-13 protein to optionally form a signal transduction complex. CD81 may function as a viral receptor for HCV.

Example 1 was repeated for the title protein. The following sequences are aligned to the wild type (upper line, SEQ ID NO: 324) by substituting each of the hydrophobic amino acids L, I, V and F in the transmembrane domain with Q, T and Y (respectively). (Lower line, SEQ ID NO: 325) is obtained.

The predicted transmembrane regions exemplify the modified domains of the invention, including (SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, respectively):

Thus, for example, the present invention includes transmembrane domains, each containing a modified domain or "mt" domain. Preferably, the TM1 -containing protein herein comprises one or more (eg, all) of extracellular and intracellular loop sequences (ununderlined sequences). As an addition or alternative, the protein containing TM1 herein is one, two, or three of the proteins depicted or the native V, L, I, and F amino acids listed in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence holding one or optionally four or more.

Additional transmembrane domain variants can be selected as described in Example 1 by using the wild-type sequence as described above. The coding sequence can be designed and recombined to express the protein. The expressed protein can be assayed for ligand binding as described herein.

Example 23: E. coli expression of QTY and CXCR4-QTY mutants

1. 1. Large-scale production of CXCR4-QTY in E. coli BL21 (DE3) Water-soluble GPCR CXCR4 was produced in E. coli in an estimated yield of approximately 20 mg of purified protein per liter of routinely used LB medium. .. The estimated production cost is about $ 0.25 per milligram. It is advantageous that grams of water-soluble GPCRs can be readily obtained using this technique, which in turn facilitates their structural determination.

2. 2. Determining the location of water-soluble CXCR4-QTY produced in E. coli cells Water-soluble CXCR4-QTY was cloned into a pET vector. We first conducted a small-scale E. coli culture study to assess the location of the CXCR4-QTY protein (150 ml culture) produced. After culturing cells induced at 24 ° C. for 4 hours using IPTG, we harvested and sonicated these cells and centrifuged at 14,637 xg (12,000 mp) to create two images. Divided into minutes. We then detected the location of the CXCR4-QTY protein using Western blot analysis of specific anti-rhho-tagged monoclonal antibodies. We observed that the CXCR4-QTY protein was in the supernatant fraction and the protein was not in the pellet fraction, thus suggesting that the protein was completely water soluble.

3. 3. Estimated Yield of CXCR4-QTY Produced in Soluble Fraction of E. Coli Cells Next, we separately cultured 150 ml to obtain CXCR4-QTY purified with about 6 mg of 1D4 monoclonal antibody. Because we underestimated the yield (we did not expect surprisingly high yields), we to capture the CXCR4-QTY produced. Sufficient affinity rho-1D4 tag monoclonal antibody beads were not used. Therefore, a significant amount of CXCR4-QTY protein did not bind to the beads because sufficient beads were not added during purification, the protein was in the outflow lane, and was further washed away. Despite the significant loss, we can still obtain about 6 mg for 150 ml of culture, as can be seen from lanes 8-10 (elution fraction).

4. Measurement of Thermal Stability of Purified Water Soluble CXCR4-QTY Protein In most cases, structure determines function in the protein. Therefore, it is important to know if the purified CXCR4-QTY protein produced in E. coli is still accurately folded into a typical α-helix structure with an α-helix of about 50%. We performed secondary structure measurements using circular dichroism (CD). The present invention observed the CD spectra of CXCR4-QTY proteins purified at various temperatures. The present invention measured the thermal stability of the purified CXCR4-QTY protein. We have observed that the purified CXCR4-QTY protein is relatively stable up to 55 ° C., the protein is only partially denatured gradually and the CD signal reduction is about 15%. At 55 ° C to 65 ° C, the denaturation increased towards 65 ° C, a denaturing transition occurred at 65 ° C to 75 ° C, and the protein was almost completely denatured at 75 ° C.

The present invention plotted the temperature against ellipticity at 222 nm to obtain the melting temperature (Tm) of the purified water-soluble CXCR4-QTY protein. From this plot, we estimated that the Tm of the purified CXCR4-QTY protein was about 67 ° C. This Tm suggests that the purified water-soluble CXCR4-QTY protein is very stable compared to many other soluble proteins. It is known that the better the thermal stability, the better the crystal lattice filling, and therefore the more chances of obtaining a structure. Therefore, this thermal stability property makes it easy to obtain a diffracted crystal.

5. Further G Protein-coupled Receptors We selected 10 G protein-coupled receptors (GPCRs) from Zhang et al., "Water Soluble Membrane Proteins and Methods for the Preparation and Use Thereof". The water-soluble form was designed using the QTY method described in US Patent Publication No. 2012/0252719A (“Zhang”) entitled “Preparation and Usage)”. Alternatively, the proteins described herein can be selected.

6. Molecular Cloning of Genes We have succeeded in confirming the natural and QTY genes of GPCR in the cell-free protein expression plasmid vector pIVex2.3d and the Escherichia coli pET28a and pET-duet-1 plasmid vectors.

7. Production of Water-Soluble GPCRs We have produced several natural and QTY proteins. When producing a native GPCR in a cell-free system, the detergent Brij35 is required, and in the absence of the detergent, the protein precipitates as soon as it is produced. On the other hand, we tested QTY variants in the presence and absence of detergents. In the absence of detergent, the cell-free system produced soluble protein.

We cloned QTY mutants into E. coli in vivo expression systems pET28a and pET-duet-1 plasmid vectors for E. coli cell protein production in E. coli BL21 (DE3) strains. We purified several water-soluble GPCR proteins, including CXCR4 and CCR5, and we used them for secondary structure analysis. The present invention conducted a ligand binding study on CXCR4 using its natural ligand CCL12 (SDF1a). The present inventions produced and purified Escherichia coli of a water-soluble GPCR CCR5e mutant. The CCR5e variant had 58 amino acid changes (about 18% change). Water-soluble GPCR CCR5e variants were purified using a specific monoclonal antibody rhodopsin tag until homogeneous. The blue strain showed a single band on the SDS gel indicating its purity. As inferred from the size marker of the protein, it appears to be a pure homodimer (the natural membrane-bound CXCR4 crystal structure was a dimer). Western blots confirmed common CCR5e mutant monomers and homodimers by GPCR.

8. Secondary Structure Study of QTY CCR5e We obtained a water-soluble QTY mutant of GPCR CCR5e. Next, we performed secondary structure analysis using an Aviv model 410 circular dichroism device and confirmed that the GPCR QTY CCR5-e mutant had a typical α-helix structure. The present invention also conducted experiments at various temperatures to determine the Tm of the CCR5e mutant, that is, the thermal stability of the water-soluble CCR5e mutant. From these experiments, we determined that the Tm of the CCR5e mutant was about 46 ° C. This Tm is good for crystal screening experiments.

9. Ligand Binding Study of CXCR4 Using CCL12 (SDF1a) To ensure that the designed water-soluble QTY GPCRs still maintain their biological function, ie, recognize and bind their natural ligands. In addition, the present invention first studied water-soluble CXCR4 using its natural ligand CCL12 (also referred to as SDF1a) using ELISA measurements. The assay concentration is in the range of 50 nM to 10 μM. The measured Kd is about 80 nM. The Kd of the natural membrane-bound CXCR4 with SDF1a is about 100 nM. Therefore, the Kd of the water-soluble CXCR4 is within the permissible range. Further experiments or other measurements with the more sensitive SPR may be performed to produce more accurate Kd.

Although the present invention has been illustrated and described with reference to particularly preferred embodiments thereof, various changes in the form and details can be made without departing from the scope of the present invention included in the appended claims. Will be understood by those skilled in the art.

Claims (80)

A computer-implemented method for performing the procedure for selecting water-soluble variants of G protein-coupled receptors (GPCRs).
The step of inputting the GPCR sequence for analysis and
A step of obtaining a variant of the GPCR in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helix segment (“TM region”) of the GPCR are substituted.
(A) The hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F).
(B) The leucine (L) is independently replaced with glutamine (Q), asparagine (N) or serine (S).
(C) The isoleucine (I) and the valine (V) are independently substituted with threonine (T), asparagine (N) or serine (S), and (d) the phenylalanine is tyrosine (Y), respectively. ), Followed by obtaining α-helix secondary structure results for the variant and confirming maintenance of the α-helix secondary structure within the variant.
A method comprising the step of obtaining a transmembrane region result for the mutant and confirming the water solubility of the mutant, thereby selecting the water-soluble variant of the GPCR. The method according to claim 1, wherein step (3) is performed before, simultaneously with, or after step (4). In step (2), one subset of the plurality of hydrophobic amino acids in the same TM region of the GPCR is replaced to prepare one member of the mutant candidate library, and the plurality of hydrophobicities are prepared. The method of claim 1 or 2, wherein one or more different subsets of amino acids are replaced to make additional members of the library. Claim that the combination score further comprises a step of ranking all members of the library based on the combination score, which is a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. The method according to 3. The method of claim 1, further comprising a step of ranking the variant using a ranking function. The method of claim 1, further comprising the step of performing the method using a data processor. The method of claim 6, further comprising a memory connected to the data processor. The method of claim 5, wherein the ranking function comprises a secondary structure component and a water soluble component. The method of claim 8, wherein the ranking function comprises a weighted value of the secondary structure component and / or the water soluble component. It further comprises the step of selecting the members of the N species having the highest combination score to form a first library of mutant candidates for the TM region, where N is a predetermined integer (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). The method of claim 4. 10. The method of claim 10, further comprising making one library of mutant candidates for all three other TM regions of the GPCR. The method according to claim 11, further comprising the step of substituting two or more TM regions of the GPCR with the corresponding TM regions in the mutant candidate library to prepare a combined mutant library. The method according to any one of claims 1 to 12, wherein substantially all (for example, all) of the leucine is replaced with glutamine. The method according to any one of claims 1 to 13, wherein substantially all (for example, all) of the isoleucine is replaced with threonine. The method of any one of claims 1-14, wherein substantially all (eg, all) of the valine is replaced with threonine. The method according to any one of claims 1 to 15, wherein substantially all (for example, all) of the phenylalanine is replaced with tyrosine. The method according to any one of claims 1 to 16, wherein one or more (eg, 1, 2 or 3) of the leucines are not substituted. The method according to any one of claims 1 to 17, wherein one or more (eg, 1, 2 or 3) of the isoleucines are not substituted. The method according to any one of claims 1 to 18, wherein one or more (eg, 1, 2 or 3) of the valine is not substituted. The method according to any one of claims 1 to 19, wherein one or more (eg, 1, 2 or 3) of the phenylalanine is not substituted. The method according to any one of claims 1 to 20, further comprising a step of producing / expressing the combination mutant. Claims 1-21 further include testing the combination variant for ligand binding (eg, by the yeast two-hybrid method), wherein those having substantially the same ligand binding as compared to the GPCR are selected. The method according to any one of the above. Claims 1 to 22, further comprising testing the combination variant for the biological function of the GPCR, wherein selecting one having substantially the same biological function as compared to the GPCR. The method according to any one item. The method of any one of claims 1-23, wherein the combination mutant library comprises less than about 2 million members. The method of any one of claims 1-24, wherein the sequence of the GPCR comprises information about the TM region of the GPCR. The method according to any one of claims 1 to 25, wherein the sequence of the GPCR is obtained from a protein structure database (eg, PDB, UniProt). The method according to any one of claims 1 to 26, wherein the TM region of the GPCR is predicted based on the sequence of the GPCR. 27. The method of claim 27, wherein the TM region of the GPCR is predicted using a TMHMM 2.0 (transmembrane prediction using a hidden Markov model) software module. 28. The method of claim 28, wherein the TMHMM 2.0 software module utilizes a dynamic baseline for peak search. The method according to any one of claims 1 to 29, further comprising the step of providing the polynucleotide sequence of each variant of the GPCR. The polynucleotide sequence is a codon optimized for expression in a host (eg, a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cell, a non-human mammalian cell or a human cell). The method according to claim 30. The method according to any one of claims 1 to 31, the present scripting procedure includes a VBA script. This scripting procedure can be operated by a Linux® system (eg, Ubuntu 12.04 LTS), a Unix® system, a Microsoft Windows operating system, an Android operating system or an Apple iOS operating system, claims 1 to 1. The method according to any one of 32. A water-soluble variant of a G protein-coupled receptor (GPCR) in which multiple hydrophobic amino acids have been substituted within the transmembrane (TM) domain α-helix segment (“TM region”) of GPCR.
(A) The hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F).
(B) The leucine (L) is independently replaced with glutamine (Q), asparagine (N) or serine (S).
(C) The isoleucine (I) and the valine (V) are independently substituted with threonine (T), asparagine (N) or serine (S), and (d) the phenylalanine is tyrosine (Y), respectively. A water-soluble variant characterized in that the secondary structure of the α-helix is maintained by all seven TM regions of the variant thereafter and the expected transmembrane region is absent. 34. Claim 34, which comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64. Water-soluble mutant of. 35. The water-soluble variant of claim 35, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57. The water-soluble variant according to claim 35 or 36, which binds to a CXCR4 ligand. 34. The water-soluble according to claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114 and 116-123. Sex mutant. 38. The water-soluble variant of claim 38, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124. The water-soluble variant of claim 38 or 40 that binds to the CX3CR1 ligand. 34. The water-soluble claim 34, which comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-182. Sex mutant. The water-soluble variant of claim 41, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183. The water-soluble variant of claim 41 or 42 that binds to a CCR3 ligand. 34. The water-soluble according to claim 34, which comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234. Sex mutant. The water-soluble variant of claim 44, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235. The water-soluble variant of claim 44 or 45 that binds to a CCR5 ligand. 34. The water-soluble claim 34, which comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290. Sex mutant. The water-soluble variant of claim 47, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. The water-soluble variant of claim 47 or 48 that binds to a CXCR3 ligand. Any one of SEQ ID NOs: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323 or 325. 34. The water-soluble variant of claim 34, comprising one or more transmembrane domains described in. The water-soluble mutant according to claim 50, wherein the water-soluble mutant is water-soluble and binds to a ligand of a homologous natural transmembrane protein. (A) A step of culturing bacteria in a growth medium under conditions suitable for protein production, and
(B) A step of dividing the lysate of the bacterium into fractions to generate a soluble fraction and an insoluble pellet fraction, and
(C) Including the step of isolating the protein from the soluble fraction.
(1) The protein is a variant of the G protein-coupled receptor (GPCR) according to any one of claims 29 to 46.
(2) A protein in a bacterium (eg, E. coli) characterized in that the yield of the protein is at least 20 mg / L (eg, 30 mg / L, 40 mg / L, 50 mg / L or more) of the growth medium. How to produce. 47. The method of claim 47, wherein the bacterium is Escherichia coli BL21 and the growth medium is an LB medium. The method of claim 47 or 48, wherein the protein is encoded by a plasmid within the bacterium. The method of any one of claims 47-49, wherein expression of the protein is under the control of an inducible promoter. The method of claim 50, wherein the inducible promoter is inducible by IPTG. The method according to any one of claims 47 to 51, wherein the lysate is produced by sonication. The method according to any one of claims 47 to 52, wherein the lysate is centrifuged at 14,500 xg or more to produce the soluble fraction. A non-transitory computer-readable medium in which a series of instructions for performing the method according to any one of claims 1 to 33 is stored. A data processing system that operates to select water-soluble variants of membrane proteins, includes a data processor that operates to perform amino acid substitutions, and by a ranking function that includes secondary and water-soluble components. A system for ranking protein variants. The system of claim 60, further comprising a library of membrane proteins for processing by the system. 60. The system of claim 60, further comprising a memory connected to the data processor that stores coded instructions for executing a replacement processor. The system of claim 60, which operates on steps (a), (b), (c) and (d) of the method of claim 1. The system of claim 60, further comprising a ranking function that is a weighted combination based on the secondary structure component. The system of claim 60, which communicates with an external program over a network. The system of claim 60, further comprising a database for storing water-soluble variants. 60. The system of claim 60, further comprising instructions for performing dynamic baseline processing. 60. The system of claim 60, further comprising an interface for selecting method parameters. The system of claim 60, further comprising the step of inputting the sequence of claim 35-50. A computer implementation method for performing procedures for selecting water-soluble variants,
The process of processing data to identify membrane protein sequences for analysis,
A step of obtaining a variant of the membrane protein in which a plurality of hydrophobic amino acids of the transmembrane (TM) domain α-helix segment (“TM region”) of the membrane protein is substituted is included.
The data processor
Determining the results of the α-helix secondary structure of the mutant to confirm the maintenance of the α-helix secondary structure in the mutant.
A method comprising determining the transmembrane region result of the mutant, confirming the water solubility of the mutant, and selecting a water-soluble variant of the membrane protein. The replacement is
(A) Select the hydrophobic amino acid from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F).
(B) Replacing leucine (L) independently with glutamine (Q), asparagine (N) or serine (S),
(C) Isoleucine (I) and valine (V) are independently replaced with threonine (T), asparagine (N) or serine (S), and (d) the phenylalanine is replaced with tyrosine (Y), respectively. The method of claim 70, comprising substituting. Substituting one subset of the plurality of hydrophobic amino acids in one same TM region of the GPCR to make one member of the mutant candidate library, and one or more different subsets of the plurality of hydrophobic amino acids. 71. The method of claim 71, wherein a further member of the library is made by substituting. Claim that the combination score further comprises a step of ranking all members of the library based on the combination score, which is a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. 70. 70. The method of claim 70, further comprising a step of ranking the variant using a ranking function. The method of claim 70, further comprising a memory connected to the data processor. The method of claim 74, wherein the ranking function comprises a secondary structure component and a water soluble component. The method of claim 76, wherein the ranking function comprises a weighted value of the secondary structure component and / or the water soluble component. Further comprising selecting the N species member having the highest combination score to form a first library of mutant candidates for the TM region, where N is a predetermined integer (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). The method of claim 73. 28. The method of claim 78, further comprising making one library of mutant candidates for all three other TM regions of GPCRs: 1, 2, 3, 4, 5 or 6. The method of claim 79, further comprising the step of substituting the two or more TM regions of the GPCR with the corresponding TM regions in the mutant candidate library to prepare a combined mutant library.

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