JP7061461B2 - Water-soluble transmembrane protein and its preparation and usage - Google Patents

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 March 26, 2015, both of which claim priority. 35U. S. C. Under 119 (e), US Provisional Application No. 62 / 117,550 filed February 18, 2015, US Provisional Application No. 61 / 993,783 filed 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 for 35 U.S. S. C. Under 119 (e), US Provisional Application No. 62 / 117,550 filed February 18, 2015, US Provisional Application No. 61 / 993,783 filed 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 listings, 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 data bank. However, only 945 membrane protein structures with 28 unique structures containing 28 G protein-coupled receptors and no tetraspanin membrane proteins 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) are for water-soluble GPCRs in which certain hydrophobic amino acids located within the transmembrane domain have been replaced with polar amino acids. Describes the improved method. However, this method requires a large labor force. Further, although the modified transmembrane domain 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 a method for designing, selecting and / or producing a water-soluble membrane protein and peptide, a peptide designed, selected or produced from the peptide (and a transmembrane domain), a composition containing the peptide, and a method for using the same. In particular, the method comprises water-insoluble amino acids (leucine, isoleucine, valine and phenylalanine or simple letter codes L, I, V, F) and nonionic water-soluble amino acids (glutamine, threonine and tyrosine or simple letter code Q). , T, Y), the present invention relates to a method for designing a library of water-soluble membrane peptides such as GPCR variants and tetraspanin membrane proteins using the "QTY principle". In addition, two additional nonionic amino acids, asparagine (N) and serine (S), may be used for the substitution of 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 variants) or L, I or V (described as intrinsically disordered proteins). It should be understood that it is supposed to be possible. However, for the sake of brevity, the details of these other embodiments will be known to those of skill in the art by the teachings herein and will not be further elaborated 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 combines multiple 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). / S, Y) further includes polypeptides prepared by substitution. The variant can be characterized by a name (eg, CXCR4-QTY) in which the parent or intrinsic disordered 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), respectively. 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 tyrosine, respectively. A step of being replaced with (Y), 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) It comprises 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 running 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 certain embodiments, 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 step preformed by the data processing system. This 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 substituted to generate one member of the variant candidate library. And one or more different subsets of the plurality of hydrophobic amino acids are substituted 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 the α-helix secondary structure prediction result and the transmembrane domain 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 steps of performing the method by a data processor, which may further include memory attached thereto.

In certain embodiments, the method may further comprise 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 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). be. In certain embodiments, the method further comprises the step of creating a library of one of the candidate variants for all 1, 2, 3, 4, 5 or 6 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 variant 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 to 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 arrangement 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 threonine (or S or N). And / or the leucine residues of the α-helix domain of the protein are 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 phenylalanine is not substituted.

In certain embodiments, the combination mutant library comprises less than about 2 million members. In certain embodiments, the GPCR sequence comprises 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, a TMHMM 2.0 (transmembrane prediction using a hidden Markov model) 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, bacteria such as E. coli, yeast such as Saccharomyces cerevisiae or fission yeast, insect cells such as Sf9 cells, non-human mammalian cells or human cells). There may be.

In certain embodiments, the scripting procedure can include a VBA script. In certain embodiments, the scripting procedure can operate 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 receptors, thrombin receptors (proteoactive receptors (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 vasopresin 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 Bradykinin Receptor, Calcium Sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK1 _takikinin receptor, thyroid stimulating hormone (TSH) receptor, protease activation receptor, neuropeptide receptor, adenosine A2B receptor, P2Y purine receptor , 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) variant. For the purposes of the present invention, the term "natural" or "wild type" is intended to 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 cell-cell 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 Plasmodium malaria infection. The CD81 gene is localized in the tumor suppressor gene region and can be a candidate for mediating 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, cysteine-cysteine-glycine motif within a large extracellular loop.

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, where (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, respectively. Substituted with (N) or serine (S), (c) the isoleucine (I) and the valine (V) are independently substituted with treonine (T), asparagine (N) or serine (S), respectively. And (d) the phenylalanine is each substituted with tyrosine (Y), and then (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), 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 the CX3CR1 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-182. 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: 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 variant comprises SEQ ID NOs: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317. Contains 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) a step of culturing a bacterium in a growth medium under conditions suitable for protein production, and (b) a soluble fraction and an insoluble pellet fraction in which a lysate of the bacterium is divided into fractions. It comprises a step of producing a fraction and (c) a step of isolating the protein from a soluble fraction, wherein (1) the protein is a G protein conjugated receptor according to any one of claims 29-46. 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 controlled by 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 poly described herein is 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 administration of the water-soluble peptides of the invention are, 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 subject water-soluble peptide containing a modified α-helix domain. In certain embodiments, the cell is an animal cell (eg, human, non-human mammal, insect, bird, fish, reptile, amphibian or other cell), yeast or bacterial cell.

The 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 computer-executable instructions are stored, the computer-executable instruction causes the computer system to execute this method when executed by the computer system, and the computer-executable instruction is the computer-executable instruction. When performed 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 a memory connected 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-temporary 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 of skill 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 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 to scale, but 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 threonine and tyrosine are three water molecules, i.e. one hydrogen donor and two. Can be attached to 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 point. (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, TMHMM predictions of the CXCR4 variant (CXCR4-QTY) subjected to the QTY substitution method of the present invention no longer have 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 a schematic diagram of the flowchart which describes the processing process of the specific preferable embodiment of this invention. It is a schematic diagram 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 hydrophobic residues of seven transmembrane α-helices of natural proteins. 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 is capable of converting 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 invention also apply to other proteins that have a transmembrane (α-helix) region.

GPCRs typically have 7 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. The 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, respectively). It is divided into N-terminal and C-terminal loops, each of which has a free end). Thus, 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) is 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 domain result for the variant and confirm the water solubility of the variant, thereby selecting a water soluble variant of the membrane protein (eg, GPCR). Provides 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 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 are 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 are 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 are about 95%, 90%, 85%, 80% of all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. 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 Ls are substituted with Q, all I and V are substituted with T, and all Fs are substituted 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 variants are selected as members of the variant 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 variants are selected based on predetermined criteria such as a scoring system that takes into account α-helix secondary structure prediction results and / or transmembrane domain prediction results.

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 iteration storing in electronic memory or database. Create a mutant candidate library that can be used. Within the same library, all variants differ in substitution within the selected TM region (see above), but are otherwise the same in 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 acid L, I, V, F substitutions in 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, in a GPCR with 7 TM regions, if each of the ⁷ libraries has 8 members, the total number of combination mutants based on that library will be 87, or about 2.1 million. In certain embodiments, the combination mutant library comprises members of about 5 million, 4 million, 3 million, 2 million, 1 million or less than 500,000 species.

Thus, in a particular embodiment, step (2) replaces one subset of the plurality of hydrophobic amino acids within the same TM region of the protein (eg, GPCR) with one of the variant candidate libraries. Members of the species are made and one or more different subsets of the hydrophobic amino acids are substituted 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 domain prediction result. It is a combination that has been made.

Domains with different sequences will probably predict different water solubility and tendencies for α-helix formation, as will be appreciated by those of skill in the art. Assign a "score" to a particular predicted water-soluble or water-soluble range, a tendency or trend 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, for example, on a scale of 1-10 established characterized by an increase in the degree of water solubility. Alternatively, this score may be quantitative, such as describing the expected solubility in mg / mL units. Once scores have been evaluated for each domain, domain variants are easily compared (or ranked) by one or preferably both of those scores, both being water soluble and forming an alpha helix. You can select the domain variant 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 constrained computational models with their substitution combinations that are not effective in achieving a given biological function. It should also be noted that by doing so, 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 variant 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 variants produced by can be ranked more efficiently. Thus, a new variant candidate having a substitution pattern that closely matches the known successful variant substitution pattern is a substitution pattern or known that does not closely match the known successful variant substitution pattern. Can be ranked higher than other mutant candidates with a substitution pattern that closely matches the substitution pattern of the unsuccessful variant of.

0 to 1 that can be used to predict the tendency to form a transmembrane domain / protein when the TMHMM program is run as a stand-alone software module / package (eg, for Linux® systems). Generate a score. This score can be used as a quantitative prediction of water solubility in the method 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 expected α-helix secondary structure, and the expected 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 from 0 to 1 where 0 has no predicted TM region and 1 has the strongest tendency to form one or more TM regions. Can obtain transmembrane domain results. Thus, the two scores can be combined directly or with weights to represent an overall assessment of the secondary structure in which the combined score is maintained as well as the expected water solubility (measured by the tendency to form TM regions). .. For example, a combination score of 0 indicates that the variant does not have the predicted TM region but maintains the predicted α-helix secondary structure and is therefore the desired variant. 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 domain 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, α-helix secondary structure predictions weigh 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. The ranking function can be selected to include a weighting scheme that allocates the results and the rest to the transmembrane domain 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 with 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 candidate variants for 1, 2, 3, 4, 5, 6 or all remaining TM regions of the protein (eg, GPCR). Further includes 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 the TM regions of two or more (eg, all) of the protein (eg, GPCR) with the corresponding TM regions in the mutant candidate library to create a combined mutant library. Further includes steps to be performed. As used herein, the "corresponding TM region" refers to a TM region in a variant 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 combined variant.

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 phenylalanine is 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 an in vitro or in a biological system such as the yeast two-hybrid method), in this case with the ligand binding of the GPCR. 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 the delimited 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 Structure 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 organizations (PDBe, PDBj and RCSB). It is freely accessible. The PDB is overseen by the World Protein Structure Data Bank, or wwPDB. PDBs are 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 PDBs.

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 the transmembrane domain.

Another publicly available database capable of providing 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 genomic sequencing projects. It contains a lot of information about the biological function of proteins from the research literature. UniProt provides four core databases, namely UniProtKB (including subdivisions Swiss-Prot and TREMBL), UniPark, 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 annotations of the entry include a 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, spontaneous 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. Information may be obtained.

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 associated 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 facilitated based on known information. Not available on. In this embodiment, the invention is a method approved in the art such as the TMHMM 2.0 (Hidden Markov Model-based Penetration Prediction) program developed by the Center for Biological Sequence Analysis. Is used to calculate the TM area. 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 polynucleotide sequences can be readily 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 Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cells, 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 ), Collesistkinin (CCK ₁ ), V _1a Basopressin receptor , D5 dopamine receptor, fMLP formyl peptide receptor, GAL ₂ galanin receptor, EP ₃ prostanoid receptor, A ₁ adenosine receptor, α ₁ adrenergic receptor, BB ₂ bombesin receptor, B _{2 bradikinin} receptor Body, calcium sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK1 _takikinin receptor, thyroid stimulating hormone (TSH) receptor, protease activation receptor, neuropeptide receptor, 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 (eg, GPCR), if necessary, to identify the first transmembrane region of the membrane (transmembrane) 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 (penetration) 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 a first modified transmembrane mutant that is presumably water-soluble.
(6) Compare the respective structural and solubility scores of the first modified transmembrane variant that is presumably water soluble in the first library, preferably using the structural and solubility scores. The step of ranking the first modified transmembrane variant, which is presumably water soluble.
(7) Multiple presumably water-soluble first modified transmembrane variants (where the 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) Repeat steps (1)-(7) for the second, third, fourth, fifth, sixth, seventh or preferably all transmembrane regions of the protein (modified by this method). The total transmembrane domain created 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 any extracellular or intracellular domain of the protein. ,
(10) A step of producing a combined 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 are used to generate nucleic acid sequences for 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 presumably water-soluble transmembrane protein variants of up to ^Hn species. 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 variant protein (eg, GPCR) designed on the basis of the method of the invention. This aspect of the invention is, in part, by a water-soluble variant protein designed on the basis of the method of the invention (eg, GPCR), which is commonly used for expression in cell-free expression systems in vitro and for E. coli and the like. 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 the soluble fraction of the lysate of E. coli culture, as opposed to the insoluble aggregates or pellets in which most membrane proteins are typically present. 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 generate 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 invention will be further described below with reference to the general aspects of the invention.

Transmembrane Domain Prediction The particular method of the invention comprises the step of predicting the transmembrane domain 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 helices. It uses a hidden Markov model to predict transmembrane helices in proteins. The input protein sequence may be in FASTA format and its output can be presented as an html page containing images of predicted positions in 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 at the time of evaluation. It was determined to be the best program to perform a transmembrane prediction program.

The programs compared in that 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 / Program (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. 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) predict the transmembrane region 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 (non-homologous membrane proteins) using a special score matrix previously obtained. The method provides a highly accurate hydrophobic profile of the query from which the location of the transmembrane segment candidate can be obtained. The novelty of the DAS-TMfilter algorithm is a second prediction cycle for predicting TM segments in an array in the TM library. To use the DAS server, the user can use www. sbc. su. The protein sequence is input at 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 G.E 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 G.E 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 a protein using the 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 topology. 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 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 in one place 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). Have been described. For PoyPhobis, 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 See Acids Res. 35: W429-32, 2007 (all cited technical content is incorporated by reference).

SOSUI is intended to distinguish between membrane proteins and soluble proteins, as well as the prediction of transmembrane helices. SOSUI predicts the transmembrane domain using Hydrophobicity Analysis for Topology and Probe Helix Method for Tertial 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 orientations in query sequences. Specifically, the TM Pred algorithm is based on 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 makes standard hydrophobic analysis accurate by detecting end (“end”, start and end) candidates for the transmembrane domain. This allows us to discard highly hydrophobic regions that are not separated by well-defined start and end configurations and identify putative transmembrane segments that are not distinguishable 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 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, this 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, the neural network incorrectly predicts 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 (embedded by reference).

Predicting the α-helix secondary structure of a protein The specific method of the present invention comprises 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 dominant states: helix, sheet or random coil. 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 to form secondary structural 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, at a location (and close to it, 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 in itself may indicate 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 alpha-helical structure rather than a random coil. Thus, in the method of the 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 popular publicly available predictive web servers.

According to these tests, the most accurate methods at this time are 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 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 structure to amino acids in a protein, taking into account the atomic resolution coordinates of the protein. DSSP begins by simply identifying intraskeletal hydrogen bonds in proteins 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 symbols G, H and I, and their residues are recognized by having a repetitive 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 other rules do not 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).

PSIDRED (secondary structure prediction by PSI-BLAST) is a technique used to investigate protein structure. It uses a neural network or machine learning method in its algorithm. It is a server-side program featuring a website that serves 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 / psipred. The idea of this method is a machine learning method that predicts the secondary structure of a new amino acid sequence using information on evolutionarily related proteins. 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: array profile creation, first secondary structure prediction and predicted structure filtering. PSIDRED 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. This network has a single hidden layer consisting of 75 units and 3 output nodes (1 for each secondary structural element ie helix, sheet, coil). A second neural network is used to filter the expected 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 structural element ie helix, sheet, coil). The three final output nodes send the score of each secondary 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 is performed according to the "QTY code" of the present invention without limiting the substitution in.

In that particular embodiment, the method initiates 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 a TMHMM program (240). If this prediction does not yield any TM region in 242, 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 is terminated (260).

On the other hand, if one or more suitable programs in 242 predict one or more TM regions, then 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 candidate water-soluble variant 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. The TM region can then be predicted (220), confirmed (222), and generate mutant sequences (224). Optionally, if this result indicates that one or more of the one or more α-helix secondary structures in the original protein have been disrupted at 214, 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 variant has higher water solubility than the original protein but maintains 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 variants obtained by further steps 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 domain 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 searching for additional data (eg, sequence data) about the protein selected for analysis.

3: In step 3, the user can input, upload or acquire data identifying the transmembrane domain. 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 domain 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 domain is generally characterized by an α-helix conformation. For example, using a software module / package named TMHMM 2.0 (transmembrane prediction using a hidden Markov model) developed by the Biological Sequence Center (www.cbs.dtu.dk/services/TMHMM). Helix predictions can be made. 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 initial 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 domain. 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 domains were initially identified. However, setting the TMHMM 2.0 baseline value to 0.07 identified a total of seven correct transmembrane domains. 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 into a total of 15 fragments (ie, 7) according to the TM region information. Divide 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 domain (typically not required, but most proximal to the N-terminus of the protein) is initially selected for mutation. Then some or all of the hydrophobic amino acids (L, I, V and F) in the first transmembrane domain are combined 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 substituted 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 domain hydrophobic. As an addition or alternative, it may be desirable to choose to replace all of the leucine (L) in the transmembrane domain. As an addition or alternative, it may be desirable to selectively replace all isoleucine (I) in the transmembrane domain. As an addition or alternative, it may be desirable to select and replace all valine (V) in the transmembrane domain. As an addition or alternative, it may be desirable to selectively replace all phenylalanine (F) in the transmembrane domain. In addition or as an alternative, it may be advantageous to retain one or more phenylalanines within the transmembrane domain. In addition or as an alternative, it may be advantageous to retain one or more valines in the transmembrane domain. In addition or as an alternative, it may be advantageous to retain one or more leucines in the transmembrane domain. In addition or as an alternative, it may be advantageous to retain one or more isoleucines in the transmembrane domain. 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 domain characterized by three or more contiguous hydrophobic amino acids.

7: In step 7, the transmembrane domain 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 a QTY substitution corresponds to the original protein. It exchanges with the TM region to generate a transmembrane variant or "estimated variant" (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 domain prediction method (84) described herein (eg, predicted loss of TM region). The variant is also evaluated for a score of its sequence's tendency to form an α-helix (82). The mutant is also used in the water solubility prediction method described herein. For example, the variant 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, and the low tendency to form TM regions or the like thereof. 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 in 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 may be impaired, then either highly water soluble or 0, 1, 2 or 3 hydrophobic amino acids (eg, higher weight for water solubility prediction results). Characteristic transmembrane variants can be selected. Alternatively or additionally, advanced α-helix 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 combination score above, 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 therefore 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 (and higher) transmembrane domains 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 or one 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, all hydrophobic amino acids are replaced with hydrophilic amino acids, and thus the domain / TM region, which is characterized by maximizing its water solubility score, and the three. It can be combined with a second domain / TM region that holds 4 or 5 hydrophobic amino acids. As is known in the art, such selected putative variants are "recombined" with extracellular and intracellular domains to provide the first combinatorial library of putatively water-soluble protein variants. Can be made.

In certain embodiments, all or fragments of a presumably 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 result in the expression of 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 capable of producing water-soluble protein variants that have three-dimensional structure and retain ligand binding function (including binding affinity) or other functions. The software may include a learning module that uses 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 study 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 in the region. Random combinations of various fragments form about 2 ^million (87) variants of the full-length GPCR gene. The expected number of variants 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 variants. Can be characterized by the estimated number of variants in each transmembrane domain available to produce.

Once the first combinatorial library or group 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. It is preferred that these nucleic acid molecules be designed to perform 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. See com / resources / gentlesigner. 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 invention includes a transmembrane domain variant obtained or obtained from the methods described herein and a nucleic acid molecule encoding it.

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 with Q, T, T or Y, respectively. We also envision a water-soluble GPCR variant (“sGPCR”) characterized by multiple transmembrane domains independently characterized by at least about 90% hydrophobic amino acid residues (L, I, V and F). .. The sGPCRs of the 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. Further comprising 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 research 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, a decrease in 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 is 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. (2001) Nature 410: 50-56) found that CXCR4 expression levels were higher in primary tumors relative to normal mammary glands or epithelial cells. I found it. In addition, CXCR4 antibody treatment was found to inhibit metastasis to regional lymph nodes as compared to control isotypes, all of which had metastasized to lymph nodes and lungs (Muller et al. (2001)). Therefore, decoy treatment models are 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, allergies, asthma, AIDS-related encephalitis, AIDS-related plaque rash, AIDS-related interstitial pneumonia, AIDS-related bowel disease, AIDS-related. It is selected from the group consisting of perigate pneumonia and AIDS-related glomerulonephritis.

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

The present invention also includes pharmaceutical compositions containing said water-soluble polypeptides and pharmaceutically acceptable carriers or diluents.

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 contain 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, intrathecal 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. The parenteral preparation may also contain antibacterial agents such as benzyl alcohol or methylparaben, such as 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 infiltrates, 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 a depot injection or implant 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. Examples of the transdermal preparation include patches, ointments, creams, gels, and ointments. 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, alleviating or ameliorating the symptoms, or preventing or preventing further progression of the disease, illness or disorder. Including inhibiting. 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 other things, 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 equipment 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 may be 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-tiered system, which includes components distributed among one or more server systems performing various functions. Thus, embodiments are not limited to execution on any particular system or group of systems. Further, embodiments, functions and methods may be implemented in software, hardware or firmware or any combination thereof. Accordingly, embodiments, 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.

Referring to FIG. 10, a block diagram of a distributed computer system 300 in which various aspects and functions are implemented is shown. As shown in the figure, the distributed computer system 300 includes one or more computer systems for exchanging 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 distributed computer system 300 shows three networked computer systems, but the distributed computer system 300 is not so limited and any number 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, Italian, 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 coded to be executed 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 individualized 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 techniques 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, the processor 310 or some other controller allows faster access to information from the non-volatile recording medium by the 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 related to the data storage element 318 after the processing is completed. Copy to. 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 the execution on the computer system 302 shown in FIG. The various aspects and functions can be performed on one or more computers with different architectures or components than 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 disclosed herein. You may be. Another example, on the other hand, 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 their own hardware and operating system. It 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 control device, such as processor 310, runs an operating system. Examples of specific operating systems that can be run are from 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 iOS 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.

Together, the processor 310 and the operating system define a computer platform in 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 embodiment 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 logical 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 the aspect of a graphical user interface 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 components configured to perform the functions described herein (eg, dedicated hardware, executable code, data structures). Or an object).

In some examples, the components disclosed herein may read parameters that affect the functionality performed by such components. 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 drives). The parameters are also logically stored in their own data structure (such as a database or file defined by a userspace application) or a commonly shared data structure (such as an application registry defined by an operating system). May be good. Also, some examples provide both a system and a user interface that causes an external entity to modify parameters and thereby configure the behavior of such components.

The software for performing the calculation method as a whole is shown in FIG. 11A, where the user selects the operating parameters to perform the procedure on the computer, as described earlier herein (as described herein). 402), where one or more sequences are entered (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 accordingly (450).

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 modifications to the disclosed embodiments are obvious 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 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 a TM1 sequence. 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: 2. As an addition or alternative, the protein containing TM1 herein is 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 intrinsically disordered 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 recombinantly expressed, each containing one transmembrane domain variant in 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 in living yeast cells. Ligand binding was detected by gene activation with a yeast two-hybrid system, and then samples were sequenced. Nineteen CXCR4 mutants were sequenced. The results are 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 subjected 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) below. 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 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 (ununderlined sequences) of SEQ ID NO: 66. 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: 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 intrinsically disordered 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 recombinantly 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 to create a library containing multiple proteins.

The library thus prepared was then assayed in an aqueous medium for binding to the CX3CR1 homologous ligand (CXCL1) as described in Example 1. Ligand binding was detected and then the sample was sequenced. Seven variants were sequenced. The results are 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) below. 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 intrinsically disordered 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 recombinantly expressed, each containing one transmembrane domain variant in 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 variants were sequenced. The results are 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) below. 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: 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. 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: 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 intrinsically disordered 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:
LQNQAISDQFFQTVPFWAHY (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)

EKFRNYLLVFFQKHIAKRFCKCCSIFQEAPERASSVYTRSTGEQEISVGL (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 recombinantly expressed, each containing one transmembrane domain variant in each variant list between the intracellular and extracellular domains of SEQ ID NO: 186, 195, 204, 207. , 209, 218, 226 and 235 to create a library containing multiple proteins.

The library so 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 variant was sequenced. The results are 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 to align 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 intrinsically disordered protein sequence of CXCR3 was applied 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 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 systems, in this case yeast, as is known in the art. Multiples with intracellular and extracellular loops, each of which is recombinantly expressed with this coding sequence, each containing one transmembrane domain variant within each variant list between its intracellular and extracellular domains. A library containing the protein of 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 a transmembrane domain, 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 described protein or the native V, L, I, and F amino acids described 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.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 described protein or the native V, L, I, and F amino acids described 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.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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) is obtained.

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, 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 described protein or the native V, L, I, and F amino acids described 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.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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) is obtained.

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, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more. Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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) is obtained.

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, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 described protein or the native V, L, I, and F amino acids described 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.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 a transmembrane domain, 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 the protein depicted or one, two, three or one of the native L, I, V and F amino acids described in the wild-type sequence. Includes one or more additional transmembrane regions (underlined sequences) within a homologous sequence that optionally retains four or more. Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 complex involved in signal transduction. 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 domain exemplifies the modified domain of the invention, including (SEQ ID NO: 326, 327, 328, 329, 330, 331, 332, 333, respectively):

Thus, for example, the 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 described protein or the native V, L, I, and F amino acids described 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.

Further transmembrane domain variants can be selected as described in Example 1 by subjecting the wild-type sequence to the methods 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 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 14,637 × g (12,000 mp) into two strokes. Divided into minutes. We then detected the location of the CXCR4-QTY protein using Western blot analysis of specific anti-rho-tagged monoclonal antibodies. The present invention 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 Then, the present inventions 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 tagged monoclonal antibody beads were not used. Therefore, not enough beads were added during purification, the protein was in the effluent lane, and was further washed away, resulting in no significant amount of CXCR4-QTY protein binding to the beads. Despite the significant loss, we can still obtain about 6 mg for 150 ml of culture, as can be seen from lanes 8-10 (eluent 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%. The present invention performed secondary structure measurements using circular dichroism (CD). We 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 observed that the purified CXCR4-QTY protein was relatively stable up to 55 ° C., the protein was only partially denatured gradually and the CD signal reduction was about 15%. At 55 ° C to 65 ° C, the denaturation increased towards 65 ° C, denaturation transitions 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 Method of Use)”. 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 E. 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 without 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 detergents, the cell-free system produced soluble protein.

The present invention cloned QTY variants 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 E. coli of a water-soluble GPCR CCR5e mutant. The CCR5e variant had 58 amino acid changes (about 18% change). Aqueous GPCR CCR5e variants were purified to homogenization using a specific monoclonal antibody rhodopsin tag. 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 blotting confirmed monomers and homodimers of common CCR5e variants in GPCRs.

8. Secondary structure study of QTY CCR5e We obtained a water-soluble QTY mutant of GPCR CCR5e. The present invention then 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 Studies 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, we first studied water-soluble CXCR4 with its natural ligand CCL12 (also referred to as SDF1a) using ELISA measurements. Assay concentrations range from 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 an acceptable 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, it is possible to make various changes in the form and details without departing from the scope of the invention covered by the appended claims. Will be understood by those skilled in the art.

Claims (10)

A computer-implemented method for performing the procedure of selecting a water-soluble variant of a G protein-coupled receptor (GPCR) that has binding properties to a ligand .
(1) Accepting the GPCR sequence for analysis,
(2) By substituting substantially all hydrophobic amino acids in the transmembrane (TM ) region of the GPCR according to the procedures (a) to (d) below, an amino acid sequence of the mutant of the GPCR is prepared. ,
(A) The hydrophobic amino acids are leucine (L), isoleucine (I), valine (V) and phenylalanine (F ) .
(B) Substantially all but one, two or three of the leucine (L) are independently replaced with glutamine (Q), asparagine (N) or serine (S).
(C) Except for 1, 2 or 3 of the isoleucine (I) and substantially all except 1, 2 or 3 of the valine (V) independently, threonine (T) and asparagine. Substituted with (N) or serine (S) and (d) substantially all but one, two or three of the phenylalanine with tyrosine (Y).
After that, based on the amino acid sequence of the variant of the GPCR prepared in (3) step (2), the α-helix secondary structure of the variant of the GPCR was determined, and the determined α-helix secondary structure was determined. From the structural results, it was confirmed that the α-helix secondary structure was maintained in the variant of the GPCR, and step (3) was performed before step (4) or in combination with step (4). You may go after (4),
(4) In order to confirm the water solubility of the variant of the GPCR, the TM region result in the amino acid sequence of the variant of the GPCR is determined, and the TM region result is that the TM region does not exist. Alternatively, when it is predicted that the tendency to form a TM region is low, the water solubility of the mutant of the GPCR is confirmed based on the amino acid sequence of the mutant of the GPCR.
( 5) The amino acid sequence of the variant of the GPCR, which maintains the α-helix secondary structure according to step (3) and is confirmed to have water solubility in step (4), is the water-soluble variant of the GPCR. Select as the amino acid sequence of the body ,
Method . One or two of the leucines have not been replaced and
Optionally, one or two of the isoleucines have not been substituted.
Optionally, one or two of the valine has not been replaced and
Optionally, one or two of the phenylalanines have not been substituted,
The method according to claim 1 . (A) In step (2) of the method, all (100%) of the leucine in the TM region is replaced with glutamine, and all (100%) of the isoleucine in the TM region is replaced with threonine, and the TM region. All (100%) of the valine is replaced with threonine and all (100%) of the phenylalanine in the TM region is replaced with tyrosine or (b) all of the leucine is replaced with glutamine. All the isoleucines are replaced with threonine, all the valine is replaced with threonine, and all the phenylalanine is replaced with tyrosine.
The method according to claim 1. The GPCR sequence contains information about the TM region of the GPCR.
Optionally, the GPCR sequence is obtained from a protein structure database .
Optionally, use a TMHMM 2.0 (Transmembrane Prediction Using Hidden Markov Model) software module that optionally utilizes a dynamic baseline for the TM region of the GPCR based on the sequence of the GPCR. Predict
The method according to any one of claims 1 to 3 . The method according to any one of claims 1 to 4 , further comprising the step of providing the polynucleotide sequence of each variant of the GPCR. The method of claim 5 , wherein the polynucleotide sequence is a codon optimized for expression in the host . The method is a scripting procedure that includes a VBA script.
Optionally, the scripting procedure can be operated by a Linux® system, a Unix® system, a Microsoft Windows operating system, an Android operating system or an Apple iOS operating system.
The method according to any one of claims 1 to 6 . A non-temporary computer-readable medium in which a series of instructions for performing the method according to any one of claims 1 to 7 is stored. A computer-implemented method for performing procedures for selecting water-soluble variants of membrane proteins that have binding properties to a ligand .
(1) The data processor is activated to identify the sequence of the membrane protein for analysis.
(2) Substituting substantially all hydrophobic amino acids in the transmembrane (TM ) region of the membrane protein according to the procedures (a) to (d) below can be used to obtain the amino acid sequence of the variant of the membrane protein. make,
(A) The hydrophobic amino acids are leucine (L), isoleucine (I), valine (V) and phenylalanine (F ) .
(B) Substantially all but one, two or three of the leucine (L) are independently replaced with glutamine (Q), asparagine (N) or serine (S).
(C) Except for 1, 2 or 3 of the isoleucine (I) and substantially all except 1, 2 or 3 of the valine (V) independently, threonine (T) and asparagine. Substituted with (N) or serine (S) and (d) substantially all but one, two or three of the phenylalanine with tyrosine (Y).
(3) Based on the amino acid sequence of the variant of the membrane protein prepared in step (2), the α-helix secondary structure of the membrane protein in the variant is determined, and the determined α-helix secondary structure is determined. From the structural results, it was confirmed that the α-helix secondary structure was maintained in the variant of the membrane protein, and step (3) was performed before step (4) or in combination with step (4). It may be done after step (4),
(4) In order to confirm the water solubility of the variant of the membrane protein, the TM region result in the amino acid sequence of the variant of the membrane protein is determined, and the TM region result has the TM region. When it is predicted that the membrane protein does not form or the tendency to form the TM region is low, the water solubility of the variant of the membrane protein is confirmed based on the amino acid sequence of the variant of the membrane protein, and (5). ) The amino acid sequence of the variant of the membrane protein, which maintains the α-helix secondary structure according to step (3) and is confirmed to have water solubility in step (4), is the water-soluble variant of the membrane protein. Select as the body's amino acid sequence ,
Method . (A) In step (2) of the method, all (100%) of the leucine in the TM region is replaced with glutamine, and all (100%) of the isoleucine in the TM region is replaced with threonine, and the TM region. All (100%) of the valine is replaced with threonine and all (100%) of the phenylalanine in the TM region is replaced with tyrosine or (b) all of the leucine is replaced with glutamine. All the isoleucines are replaced with threonine, all the valine is replaced with threonine, and all the phenylalanine is replaced with tyrosine.
The method according to claim 9 .

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