JP2004121261A - Method for enriching mutant protein having modified bonding property - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a phagemid expression vector containing a transcription control element operatively bonded to a gene fusion encoding a fusion protein. <P>SOLUTION: The phagemid expression vector comprises a gene fusion comprising the first gene encoding polypeptide and the second gene encoding at least one part of a phage coat protein, wherein (a) the vector contains no gene encoding a mature coat protein and (b) no perfect phage genome, (c) host cell transformation caused by the vector produces no phage particle in the absence of a helper phage, or (d) a DNA triplet codon encoding a termination codon capable of controlling mRNA is inserted between the fusion terminals of the first and the second genes or substituted instead of an amino acid-encoded triplet codon adjacent to the gene fusion connecting point. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to the production and systematic selection of new binding proteins with altered binding to target molecules. More specifically, the present invention relates to methods for producing exogenous polypeptides that mimic the binding activity of a natural binding partner.

Binding partners are substances that specifically bind to each other, usually through non-covalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.

In the past, binding partners have been identified by various methods, including collection from nature (eg, antibody-antigen and ligand-receptor pairing), and by accidental identification (eg, using random screening of candidate molecules). Drug development). In some cases, these two methods are combined. For example, variants of proteins or polypeptides (eg, polypeptide fragments) containing key functional residues involved in binding have been prepared. These polypeptide fragments are then derivatized by methods similar to traditional drug development. Examples of such derivatization would include methods such as cyclization to conformationally constrain the polypeptide fragment to obtain new candidate binding partners.

The problem with conventional methods is that natural ligands may not have properties suitable for all therapeutic applications. Furthermore, polypeptide ligands may not be available for some target substances. Also, methods for producing non-natural synthetic binding partners are often expensive and difficult, and usually require complex synthetic methods to obtain each candidate. The inability to characterize the resulting candidate structure so that rational drug design methods can be applied to further optimize the candidate molecule further hinders these methods. I have.

In an attempt to overcome these problems, Geysen ( Immun. Today 6: 364-369 (1985)) and Geysen et al . (Mol. Immun. 23: 709-715 (1986)) The use of polypeptide synthesis to provide a framework for the identification and production of A. was proposed. According to Geysen et al. (Ibid.), Short polypeptides, such as dipeptides, are first screened for the ability to bind to a target molecule. The most active dipeptide is then selected for the next test. The test consists of attaching another residue to the starting dipeptide (or by internally modifying the components of the first starting dipeptide) and then screening this set of candidates for the desired activity. This process is repeated until a binding partner with the desired properties is identified.

The method of Geysen et al is hampered by the inconvenience that the chemistry underlying the method, ie, peptide synthesis, produces molecules with unclear or varied secondary and tertiary structures. As the number of selection iterations increases, random interactions increase at an accelerated rate among the various substituents of the polypeptide, and a truly random population of interacting molecules with reproducible conformations is no longer obtained. For example, interactions between amino acid side chains, which are widely separated in sequence but adjacent in space, occur arbitrarily. In addition, sequences that do not promote a conformationally stable secondary structure may provide complex peptide-side chain interactions, which may interfere with side chain interactions of certain amino acids with the target molecule. Such complex interactions are facilitated by the flexibility of the polyamide backbone of the candidate polypeptide. Also, candidates can exist in a number of conformations, making it difficult to identify the conformer that interacts with or binds to the target with maximum affinity or specificity, and provides rational drug design. Make it difficult.

A final problem with Geysen's iterative polypeptide method is that there is currently no practical way to produce, screen and analyze highly diverse alternative peptides. Using 20 natural amino acids, the total number of all combinations of hexapeptides that must be synthesized is 64,000,000. Even if such a highly diversified peptide is produced, a mixture of such highly diversified peptides can be rapidly screened to select a peptide having a high affinity for a target molecule. There is no way. At present, each "attached" peptide must be recovered in large enough quantities to perform protein sequencing.

To overcome many of the problems inherent in Geysen's method, biological selection and screening were chosen as alternatives. Biological selection and screening is a powerful tool for exploring protein function and isolating mutant proteins with desired properties (Shortle, Protein Engineering, Oxender and Fox eds., ARLiss, Inc., NY pp. 103-108 (1988); and Bowie et al., Science 247: 1306-1310 (1990)]. However, the resulting selection or screening is only applicable to one or a few related proteins.

More recently, Smith and coworkers [Smith, Science 228: 1315-1317 (1985); and Parmley and Smith, Gene 73: 305-318 (1985)] reported that short gene fragments could be fused to fd phage ("fusion" Phage ") has shown that small protein fragments (10-50 amino acids) can be efficiently" displayed "on the surface of filamentous phage. The gene III minor coat protein (approximately 5 copies at one end of the virion) is important for proper phage assembly and infection by adherence to E. coli fimbria [Rasched et al., Microbiol. Rev. 50: 401. -427 (1986)]. Recently, "fusion phages" have been described as exogenous proteins (Devlin et al., Science 249: 404-406 (1990)) or antibodies (Scott et al., Science 249: 386-390 (1990); and Cwirla et al., Proc. Natl. Acad. USA 87: 6378-6382 (1990)] has been shown to be useful in displaying short mutant peptide sequences to identify peptides that can react with the peptide.

However, there are some important limitations when using such "fusion phages" to identify modified peptides or proteins with new or enhanced binding. First, the fusion phage is only displayed when displaying proteins with less than 100, preferably less than 50 amino acid residues, since large inserts probably disrupt gene III function, and thus disrupt phage assembly and infectivity. Has been shown to be useful [Parmley et al., Gene 73: 305-318 (1988)]. Second, conventional methods fail to select peptides from a library that have the greatest binding affinity for the target molecule. For example, after exhaustive selection of a random peptide library with an anti-β endorphin monoclonal antibody, Cwirla and coworkers found that high-affinity peptides fused to phage (Kd-0.4 μM) (Kd-10 μM) could not be separated. In addition, parental β-endorphin peptide sequences with extremely high affinity (Kd〜7 nM ) were not selected from the epitope library.

Ladner [WO 90/02802] discloses a method for selecting novel binding proteins displayed on the outer surface of virions and cells (heterologous proteins have up to 164 amino acid residues). Is allowed). This method contemplates isolating and amplifying the displayed protein to design a new group of binding proteins with the desired affinity for the target molecule. More specifically, Ladner displays a protein having an "initial protein binding domain" ranging from residue 46 (clambin) to residue 164 (T4 lysozyme) fused to the M13 gene III coat protein. "Fusion phage" are disclosed. Ladner teaches the use of these proteins "not too large" because it is relatively easy to arrange restriction sites in relatively small amino acid sequences, and a 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI) I prefer to use. Small fusion proteins, such as BPTI, are preferred when the target is a protein or macromolecule, while relatively large fusion proteins, such as T4 lysozyme, are preferred for small target molecules, such as steroids, where such large proteins are small. This is because it has crevices and grooves into which molecules can be fitted. This preferred protein, BPTI, is placed at the site of gene III disclosed by Smith et al . Or de la Cruz et al . [ J. Biol. Chem. 263: 4318-4322 (1988)], or at one of its ends, the second of gene III. It has been proposed to fuse with the synthetic copy so that "part of" the unmodified Gene III protein is present. Ladner does not address the problems of successfully selecting high affinity peptides from random peptide libraries, which hinders the prior art biological selection and screening methods.

Human growth hormone (hGH) is greatly involved in regulating normal human growth and development. This 22,000 dalton pituitary hormone exhibits a number of biological effects including, inter alia, linear growth (formation), lactation, macrophage activation, insulin-like and diabetic effects [Chawla, RK, Ann. Rev, Med. 34: 519 (1983); Edwards, CK et al., Science 239: 769 (1988); Thomer, MO et al., J. Clin. Invest. 81: 745 (1988)]. Growth hormone deficiency in children leads to dwarfism, which has been successfully treated by external administration of hGH for over a decade. hGH is a member of a group of homologous hormones including placental lactogen, prolactin, and other genetic and species variants or growth hormones [Nicoll, C.S. et al., Endocrine Reviews 7: 169 (1986)]. hGH is unique among these, exhibits broad species specificity, and has cloned somatic receptors [Leung, DW et al., Nature 330: 537 (1987)] or prolactin receptors [Boutin, JM et al., Ce 53 : 69 (1988)]. The cloned hGH gene is expressed in a secretory form in E. coli [Chang, CN et al., Gene 55: 189 (1987)], and its DNA and amino acid sequences have been reported [Goeddel et al., Nature 281: 544]. (1979); Gray et al., Gene 39: 247 (1985)]. The three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern of porcine growth hormone (pGH) has been reported with normal resolution and precision [Abdel-Meguid, S.S. et al., Proc. Natl. Acad. Sci. USA 84: 6434 (1987)]. Human growth hormone receptor and antibody epitopes have been identified by homolog-scanning mutagenesis [Cunningham et al., Science 243: 1330 (1989)]. The structure of a novel amino-terminal methionyl bovine growth hormone containing the spliced sequence of human growth hormone including histidine 18 and histidine 21 has been shown [US Pat. No. 4,880,910].

Human growth hormone (hGH) causes a variety of physiological and metabolic effects in various animal models, including linear skeletal growth, lactation, macrophage activation, insulin-like and diabetic effects [RKChawla et al., Annu. .Med. 34: 519 (1983); OGPIsaksson et al., Annu. Rev. Physiol. 47: 483 (1985); CKEdwards et al., Science 239: 769 (1988); MOThorner and MLVance, J. Clin. Invest. 82: 745 (1988); JP Hughes and HGFriesen, Ann. Rev. Physiol. 47: 469 (1985)]. These biological effects are guided by the interaction between hGH and specific cellular receptors.

Accordingly, it is an object of the present invention to provide a rapid and efficient method for the systematic production of candidate binding agents.
It is another object of the present invention to produce conformationally stable candidate binding agents displayed on the surface of phagemid particles.
Another object of the present invention is to produce a candidate binding substance consisting of a fusion protein of a phage coat protein and a heterologous polypeptide [wherein the polypeptide is 100 amino acids or more in length and 1 subunit And displayed on a phagemid particle (the polypeptide is encoded in the phagemid genome)].
Yet another object of the invention is to provide or display all peptidyl moieties that may be involved in non-covalent binding interactions and to provide these moieties in a sterically restricted manner. It provides a method for producing and selecting a binding material with sufficient flexibility.

The purpose of the above is
(a) a replicable expression vector containing a first gene encoding a polypeptide and a second gene encoding at least a portion of a native or wild-type phage coat protein, wherein the first and the second The second gene is heterologous and the transcriptional regulatory element is operably linked to the first and second genes, thus resulting in a gene fusion encoding the fusion protein).
(b) mutating at one or more selected positions in the first gene of the vector to generate a family of related plasmids;
(c) transforming a suitable host cell with the plasmid;
(d) infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein;
(e) only a small amount of phagemid particles will deposit more than one copy of the fusion protein on the surface of the particles, under conditions suitable for the formation of recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host. Culturing the transformed and infected host cells, adjusting the conditions as indicated;
(f) contacting the phagemid particles with a target molecule to bind at least a portion of the phagemid particles to the target molecule; and (g) separating bound phagemid particles from unbound particles;
By providing a method for selecting a novel binding polypeptide comprising: Preferably, the method further comprises the step of transforming a suitable host cell with the recombinant phagemid particles that bind to the target molecule and repeating steps (d)-(g) one or more times.

Further, a method for selecting a novel binding protein consisting of more than one subunit is achieved by selecting a novel binding peptide as follows:
A replicable expression vector containing a transcription regulatory element operably linked to DNA encoding a desired protein comprising one or more subunits, wherein at least one of the subunits The DNA is fused to DNA encoding at least a portion of the phage coat protein).
Mutating the DNA encoding the desired protein at one or more selected positions to generate a family of related vectors;
Transforming a suitable host cell with the vector;
Infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein;
Only small amounts of phagemid particles display more than one copy of the fusion protein on the surface of the particles, under conditions suitable for the formation of recombinant phagemid particles containing at least part of the plasmid and capable of transforming the host. Culturing the transformed and infected host cells by adjusting the conditions as described above;
Contacting the phagemid particles with a target molecule to bind at least a portion of the phagemid particles to the target molecule; and separating bound phagemid particles from unbound particles;
Consisting of

In the method of the present invention, the plasmid is cultured under strict control of transcriptional regulatory elements such that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particles is less than about 1%. Preferably, the conditions are adjusted.

It is also preferred that the amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% of the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably, this amount is less than 20%.

Usually, in the methods of the invention, the expression vector will further contain a secretory signal sequence fused to the DNA encoding each subunit of the polypeptide, and the transcriptional regulatory element will be a promoter system. Preferred promoter systems are selected from LacZ, λ _PL , TAC, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.

Also, the first gene will usually encode a mammalian protein. Preferred proteins will be selected from: human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A chain, insulin B chain, proinsulin, relaxin A chain, relaxin B chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and leutinizing hormone (LH), glycoprotein hormone receptor, calcitonin, glucagon, factor VIII, antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hematopoietic growth hormone, tumor necrosis factors α and β, enkephalinase , Human serum albumi , Mueller inhibitors, mouse gonadotropin-related peptides, microbial proteins such as β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, hormone or growth factor receptor; integrin, thrombopoietin, protein A or D, rheumatoid factor A nerve growth factor such as NGF-β, platelet growth factor, transforming growth factor (TGF) such as TGF-α and TGF-β, insulin-like growth factors I and II, insulin-like growth factor binding protein, CD-4, DNase, latency-related peptide, erythropoietin, osteoinductive factors, interferons such as interferons α, β and γ, colony stimulating factors (CSF) such as M-CSF, GM-CSF and G-CSF, interleukin (IL), eg, IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay-accelerating factor, viral antigen, HIV envelope protein, eg, GP120, GP140, atrial natriuretic peptide A, B or C, immunoglobulins, as well as fragments of any of the above proteins.

Preferably, the first gene encodes a polypeptide of one or more subunits comprising about 100 or more amino acid residues, and comprises a plurality of rigid secondary proteins displaying a plurality of amino acids capable of interacting with a target. Will be folded to form a structure. Preferably, the first gene will be mutated at codons corresponding only to amino acids that can interact with the target, such that the integrity of the rigid secondary structure is preserved.

Usually, the method of the invention will use a helper phage selected from M13KO7, M13R408, M13-VCS and PhiX174. A preferred helper phage is M13KO7 and a preferred coat protein is the gene III coat protein of M13 phage. A preferred host is E. coli, which is a protease-deficient strain of E. coli. Novel hGH variants selected by the method of the present invention were detected. A phagemid expression vector was constructed containing a suppressible stop codon functionally located between this polypeptide and the nucleic acid encoding the phage coat protein.

The following discussion will be best understood by referring to FIG. In its simplest form, the method of the present invention provides a novel binding polypeptide (such as a protein ligand) having a desired, usually high affinity for a target molecule from a library of structurally related binding polypeptides. Consist of ways to choose. Libraries of structurally related polypeptides fused to phage coat proteins are prepared by mutagenesis, preferably containing a single copy of each related polypeptide containing the DNA encoding the polypeptide. Displayed on the surface of phagemid particles. These phagemid particles are then contacted with a target molecule and the particles with the highest affinity for the target are separated from the low affinity particles. The high affinity binder is then amplified by infection of the bacterial host and the competitive binding step is repeated. This process is repeated until the desired affinity polypeptide is obtained.

The novel binding polypeptides or ligands produced by the methods of the present invention are themselves useful as diagnostic or therapeutic agents (eg, agonists or antagonists) used in the treatment of biological organisms. In addition, rational drug design can be advanced using the structural analysis of the selected polypeptide.

結合 "Binding polypeptide" as used herein refers to any polypeptide that binds to a target molecule with selective affinity. Preferably, the polypeptide is a protein, most preferably a protein containing about 100 or more amino acid residues. Usually, the polypeptide will be a hormone or antibody or a fragment thereof.

As used herein, “high affinity” means an affinity constant (Kd) of <10 ⁻⁵ M, preferably <10 ⁻⁷ M under physiological conditions.

「" Target molecule "as used herein is any molecule for which it is desired to produce a ligand, and is not necessarily limited to proteins. However, the target is preferably a protein, most preferably the target is a receptor, such as a hormone receptor.

用 い る "Humanized antibody", as used herein, refers to an antibody in which the complementarity determining regions (CDRs) of a mouse or other non-human antibody are bound to the framework of a human antibody. The framework of a human antibody refers to all human antibodies except CDRs.

I. Selection of Polypeptide for Display on Phage Surface The first step in the method of the present invention is to select a polypeptide having a rigid secondary structure exposed on the surface of the polypeptide for display on the phage surface. That is.

用 い る "Polypeptide" as used herein refers to any molecule that can be expressed by a specific DNA sequence. A polypeptide of the present invention consists of more than one subunit, each subunit being encoded by a separate DNA sequence.

As used herein, "rigid secondary structure", for example, alpha-helix, 3 ₁₀ helix, [pi helices, any showing a regular repeating structure found in such parallel and antiparallel β- sheet and reverse turns, Means a polypeptide segment. Also, certain "disordered" structures lacking recognizable geometric order form domains or "patches" of amino acid residues that allow them to interact with the target, and the overall shape of the structure in the structure Unless destroyed by amino acid substitutions, they are included in the definition of a rigid secondary structure. Certain types of disordered structures are thought to be combinations of reverse turns. The geometry of these rigid secondary structures is well defined by the φ and ψ twist angles around the α-carbon of the peptide “backbone”.

All that is required to expose the secondary structure to the polypeptide surface is to provide a domain or "patch" of amino acid residues that can be exposed to and bind to the target molecule. This is basically an amino acid residue that is replaced by mutagenesis, which results in a "library" of structurally related (mutant) binding polypeptides displayed on the surface of the phage. Obtained, from which a novel polypeptide ligand is selected. Generally, mutagenesis or substitution of amino acid residues towards the interior of the polypeptide is avoided, thereby preserving the overall structure of the rigid secondary structure. Some substitutions of amino acids in internal regions of the rigid secondary structure, especially with hydrophobic amino acid residues, may be tolerated because these conservative substitutions do not appear to distort the overall structure of the polypeptide. .

繰 Using repeated cycles of “polypeptide” selection, select higher affinity binding by phagemid selection of multiple amino acid changes selected by multiple selection cycles. Following the first round of phagemid selection (which involves the selection of amino acids or first regions in the ligand polypeptide), additional rounds of phagemid selection are performed on other regions or amino acids of the ligand polypeptide. This phagemid selection cycle is repeated until the desired affinity of the ligand polypeptide is achieved. To illustrate this method, phagemid selection of hGH in Example VIII was performed in cycles. In the first cycle, mutations were made to amino acids 172, 174, 176 and 178 of hGH and phagemids were selected. In the second cycle, amino acids 167, 171, 175 and 179 of hGH were phagemid selected. In the third cycle, amino acids 10, 14, 18 and 21 of hGH were phagemid selected. Optimal amino acid changes from the previous cycle can be introduced into the polypeptide before selection for the next cycle. For example, amino acid substitutions 174 (serine) and 176 (tyrosine) of hGH were introduced into hGH prior to phagemid selection of amino acids 167, 171, 175, and 179 of hGH.

From the foregoing, it will be appreciated that the amino acid residues forming the binding domain of the polypeptide may not be contiguously linked, but may be present on different subunits of the polypeptide. That is, the binding domain follows a specific secondary structure at the binding site, not the primary structure. Thus, mutations should be introduced into codons encoding amino acids within specific secondary structures at sites from the interior to the exterior of the polypeptide so that they have the potential to interact with the target. Normal. For illustration, the positions of residues in hGH that are known to strongly modulate binding to hGH-binding proteins are shown in FIG. 2 [Cunningham et al., Science 247: 1461-1465 (1990)]. That is, representative sites suitable for mutagenesis include residues 172, 174, 176 and 178 of helix 4 and residue 64 within the "disordered" secondary structure.

ポ リ It is not necessary that a polypeptide selected as a ligand for a target binds normally to the target. That is, for example, a glycoprotein hormone such as TSH can be selected as a ligand for the FSH receptor, and a library of mutant TSH molecules is used in the method of the present invention to obtain novel drug candidates.

That is, the present invention contemplates any polypeptide that binds to a target molecule and includes antibodies. Preferred polypeptides are those that have pharmaceutical use. More preferred polypeptides include: growth hormones (including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone); parathyroid hormone; thyroid stimulating hormone; thyroxine; A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; leutinizing hormone; glucagon; factor VIII; antibody; lung surfactant; plasminogen activator [eg urokinase or human tissue-type plasminogen. Activator (t-PA)]; bombesin; factor IX; thrombin; hematopoietic growth factor; tumor necrosis factors α and β; enkephalinase; serum albumin (eg, human serum albumin); Muller inhibitor; relaxin A chain; Relaxin B chain; prorelaxin; Sgonadotropin-related peptide; Microbial protein (eg, β-lactamase); Tissue factor protein; Inhibin; Activin; Vascular endothelial growth factor; Hormone or growth factor receptor; Integrin; Thrombopoietin; Protein A or D; Rheumatoid factor; Factors (eg, NGF-β); platelet-derived growth factors; fibroblast growth factors (eg, aFGF and bFGF); epidermal growth factor; transforming growth factors (TGF) (eg, TGF-α and TGF-β). Insulin-like growth factors I and II; insulin-like growth factor binding protein; CD-4; DNase; latency-related peptide; erythropoietin; osteoinductive factors; interferons (eg, interferon α, β and γ); CSF) For example, M-CSF, GM-CSF and G-CSF); interleukin (IL) (eg, IL-1, IL-2, IL-3, IL-4); superoxide dismutase; Natriuretic peptides A, B or C; viral antigens (eg, part of the HIV envelope); immunoglobulins; and fragments of any of the above polypeptides. In addition, one or more predetermined amino acid residues on the polypeptide can be substituted, inserted or deleted to obtain a product having, for example, improved biological properties. Also included are fragments of these polypeptides, particularly biologically active fragments. Further preferred polypeptides of the present invention are human growth hormone, atrial natriuretic peptides A, B and C, endotoxin, subtilisin, trypsin, and other serine proteases.

A more preferred polypeptide hormone is any amino acid sequence produced in a first cell, specifically for a receptor on the same cell type (autocrine hormone) or a second cell type (non-autocrine). A polypeptide hormone that can be defined as an amino acid sequence that binds and causes a characteristic physiological response in receptor-bearing cells. Such polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and pituitary gland polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle stimulating hormone, thyrotropin, villous gonadotropin, Corticotropin, α or β-melanocyte stimulating hormone, β-lipotropin, γ-lipotropin and endorphins; hypothalamic release inhibitory hormones such as corticotropin-releasing factor, growth hormone-releasing hormone, growth hormone-releasing factor; , B and C, and other polypeptide hormones.

II. Obtaining the First Gene (Gene 1) Encoding the Desired Polypeptide The gene encoding the desired polypeptide (ie, a polypeptide having a rigid secondary structure) can be obtained by methods known in the art. [See, generally, Sambrook et al., Molecular Biology: A laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)]. If the sequence of this gene is known, the DNA encoding this gene may be synthesized chemically [Merrfield, J. Am . Chem . Soc . 85: 2149 (1963)]. If the sequence of the gene is unknown, or if the gene has not been isolated before, a cDNA library (prepared from RNA from a suitable tissue in which the desired gene is expressed) Alternatively, it can be cloned from an appropriate genomic DNA library. The gene is then isolated using a suitable probe. Suitable probes for cDNA libraries include monoclonal or polyclonal antibodies (when a cDNA library is an expression library), oligonucleotides, and complementary or homologous cDNAs or fragments thereof. Probes that can be used to isolate the desired gene from a genomic DNA library include cDNAs or fragments thereof that encode the same or similar genes, homologous genomic DNA or DNA fragments, and oligonucleotides. It is. Screening of a cDNA or genomic library with the selected probe is performed using standard methods described in Chapters 10-12 of Sambrook et al. (Supra).

Another method for isolating the gene encoding the desired protein is to use the polymerase chain reaction (PCR) described in section 14 of Sambrook et al. (Supra). This method requires the use of oligonucleotides that hybridize to the desired gene. That is, in order to obtain an oligonucleotide, at least a portion of the DNA sequence of this gene must be known.
After isolating the gene, it can be inserted into an appropriate vector for amplification, preferably a plasmid, as generally described by Sambrook et al. (Supra).

III. Construction of Replicable Expression Vectors Although several types of vectors are available and can be used in the practice of the present invention, plasmid vectors are the preferred vectors for use in the present invention. This is because these vectors can be constructed relatively easily and can be easily amplified. In general, plasmid vectors contain various components, including promoters, signal sequences, phenotypic selection genes, origins of replication, and other necessary components, as known to those skilled in the art.

The most commonly used promoters in prokaryotic vectors, lacZ promoter system, the alkaline phosphatase phoA promoter, the bacteriophage .lambda.P _L promoter (a temperature sensitive promoter), tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), Includes tryptophan promoter, and bacteriophage T7 promoter. See Section 17 of Sambrook et al. (Supra) for a general description of promoters. Although there are most commonly used promoters, other suitable microbial promoters can be used as well.

好 ま し い Preferred promoters for the practice of the present invention are those that can be strictly regulated to control the expression of the fusion gene. It is believed that a problem that remained unrecognized in the prior art was that the display of multiple copies of the fusion protein on the surface of the phagemid particles led to multiple point binding of the phagemid to the target. This effect, called "chelation", selects multiple erroneous "high-affinity" polypeptides when multiple copies of the fusion protein are displayed on phagemid particles in close proximity to each other and the target is "chelated" It is thought to give results. When multiple point binding occurs, the effective or apparent Kd will be as high as the product of the individual Kd for each copy of the indicated fusion protein. This effect may be the reason Cwirla and coworkers (above) were unable to separate medium-affinity peptides from relatively high-affinity peptides.

By tightly regulating the expression of the fusion protein so that only small amounts (ie, less than about 1%) of the phagemid particles contain multiple copies of the fusion protein, the "chelation" is overcome and the high affinity It has been discovered that proper selection of the polypeptide is possible. That is, depending on the promoter, the culture conditions of the host are adjusted to maximize the number of phagemid particles containing a single copy of the fusion protein and minimize the number of phagemid particles containing multiple copies of the fusion protein.

好 ま し い Preferred promoters used in the practice of the present invention are the lacZ promoter and the phoA promoter. This lacZ promoter is regulated by the lac repressor protein laci, and thus transcription of the fusion gene can be controlled by manipulating the amount of the lac repressor protein. By way of illustration, the lacZ promoter-containing phagemid is grown in a cell line containing a copy of the lac repressor gene, a repressor of the lacZ promoter. Examples of cell lines containing the laci gene include JM101 and XL1-Blue. Alternatively, host cells can be co-transfected with a plasmid containing both the repressor laci and the lacZ promoter. Sometimes both of the above methods are used simultaneously. That is, phagemid particles containing the lacZ promoter are grown in a cell line containing the laci gene, and the cell line is co-transfected with a plasmid containing both the lacZ and laci genes. Usually, when it is desired to express the gene in the above transfected host, an inducer such as isopropylthiogalactoside (IPTG) is added. However, in the present invention, this step is omitted, and (a) the expression of the gene III fusion protein is minimized to minimize the copy number (ie, the number of gene III fusions relative to the number of phagemids); ) Prevents poor or improper packaging of phagemid caused by inducers such as IPTG even at low concentrations. Usually, when no inducer is added, the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins / number of phagemid particles). The most preferred promoter used in the practice of the present invention is phoA. It is thought that this promoter is regulated by the amount of inorganic phosphate in the cell, which phosphate down regulates the activity of the promoter. That is, the activity of the promoter can be increased by reducing the phosphate of the cell. The desired result is achieved by growing the cells in a phosphate-rich medium, such as 2YT or LB, and controlling the expression of the gene III fusion.

Another useful component of the vectors used in practicing the present invention is a signal sequence. Usually, this sequence is located immediately 5 'to the gene encoding the fusion protein, and thus is transcribed at the amino terminus of the fusion protein. However, in certain cases, this signal sequence has been shown to be at another 5 'position in the gene encoding the protein to be secreted. This sequence targets the protein to which it binds across the inner membrane of the bacterial cell. DNA encoding the signal sequence can be obtained as a restriction endonuclease fragment from any gene encoding a protein having the signal sequence. Suitable prokaryotic signal sequences can be obtained, for example, from genes encoding LamB or OmpF [Wong et al., Gene 68: 193 (1983)], MalE, PhoA and other genes. A preferred prokaryotic signal sequence for the practice of the present invention is the Escherichia coli thermostable enterotoxin II (STII) signal sequence described by Chang et al. [ Gene 55: 189 (1987)].

別 Another useful component of the vector used in the practice of the present invention is a phenotypic selection gene. A common phenotypic selection gene is a gene that encodes a protein that confers antibiotic resistance on a host cell. By way of illustration, the ampicillin resistance gene (amp) and the tetracycline resistance gene (tet) are easy to use for this purpose.

Construction of a suitable vector containing the above components and a gene encoding the desired polypeptide (gene 1) is performed using standard recombinant DNA techniques described by Sambrook et al. (Supra). The isolated DNA fragments that are ligated to form the vector are cut, processed, and ligated together in a particular order and orientation to create the desired vector.

(4) Cleavage the DNA using an appropriate restriction enzyme or enzymes in an appropriate buffer. Typically, about 0.2-1 μg of plasmid or DNA fragment is used with about 1-2 units of the appropriate restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the restriction enzyme manufacturer. Usually, an incubation time of about 1 or 2 hours at 37 ° C. is appropriate, but some enzymes require higher temperatures. After incubation, enzymes and other impurities are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and DNA is recovered from the aqueous fraction by precipitation with ethanol.

末端 In order to ligate the DNA fragments together to obtain a functional vector, the ends of these DNA fragments must be compatible with each other. In some cases, these ends are compatible after endonuclease digestion. However, in order to make them compatible for ligation, it may be necessary first to convert sticky ends, usually produced by endonuclease digestion, to blunt ends. To blunt the ends, the DNA is digested at least 15 ° C. with 15 units of Klenow fragment of DNA polymerase I (Klenow) in a suitable buffer in the presence of four deoxynucleotide triphosphates. Process for a minute. Next, the DNA is purified by phenol-chloroform extraction and ethanol precipitation.

切断 Using DNA gel electrophoresis, the cut DNA fragments can be size-separated and selected. DNA can be electrophoresed on either agarose or polyacrylamide matrices. The choice of matrix will depend on the size of the DNA fragments to be separated. After electrophoresis, by electroelution, as described in sections 6.30-6.33 of Sambrook et al., Supra, or by melting agarose and extracting DNA therefrom when low melting agarose is used as the matrix. Extract the DNA from the matrix.

ＤＮＡ Put approximately the same amount of DNA fragments to be ligated together (pre-digested with appropriate restriction enzymes so that the ends of each fragment to be ligated are compatible). This solution will also contain ATP, ligase buffer and ligase, eg, about 10 units of T4 DNA ligase per 0.5 μg of DNA. When ligating a DNA fragment into a vector, the vector is first linearized by cutting it with the appropriate restriction endonuclease (s). The linearized vector is then treated with alkaline phosphatase or bovine intestinal phosphatase. This phosphatase treatment prevents self-ligation of the vector during the ligation step.

After ligation, the vector into which the foreign gene has been inserted is introduced into a suitable host cell. Prokaryotes are preferred host cells for the present invention. Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325), E. coli X1776 (ATCC No. 31,537), E. coli XL-1 Blue (stratagene). , And E. coli B, but many other strains of E. coli (eg, HB101, NM522, NM538, NM539), and many other prokaryotic genera and species can be used as well. In addition to the E. coli strains listed above, Bacillus (eg, Bacillus Subtilis ), other enteric bacteria (eg, Salmonella typhimurium or Serratia marcesans ), and various Pseudomonas species can all be used as hosts.

Transformation of prokaryotes is readily accomplished using the calcium chloride method described in Section 1.82 of Sambrook et al. (Supra). Alternatively, these cells can be transformed using electroporation [Neumann et al., EMBO J. 1: 841 (1982)]. Transformed cells are usually selected by growing on antibiotics such as tetracycline (tet) or ampicillin (amp). Resistant to substances).

After selection of the transformed cells, the cells are grown in culture and the plasmid DNA (or other vector into which the foreign gene has been inserted) is then isolated. Plasmid DNA can be isolated using methods known in the art. Two suitable methods are small-scale DNA preparation and large-scale DNA preparation described in sections 1.25 to 1.33 of Sambrook et al. (Supra). The isolated DNA can be purified by methods known in the art, such as those described in Section 1.40 of Sambrook et al. (Supra). The purified plasmid DNA is then analyzed by restriction mapping and / or DNA sequencing. Usually, DNA sequencing is performed by either the method of Messing et al. [ Nucleic Acids Res. 9: 309 (1981)] or the method of Maxam et al . [ Meth. Enzymol. 65: 499 (1980)].

IV. Gene Fusion The present invention contemplates fusing a gene encoding a desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is produced during transcription. . Generally, gene 2 is a phage coat protein gene, preferably a gene III coat protein of phage M13 or a fragment thereof. Fusion of genes 1 and 2 can be achieved by inserting gene 2 at a particular site on a plasmid containing gene 1, or by inserting gene 1 at a particular site on a plasmid containing gene 2. it can.

O Insertion of the gene into the plasmid requires that the plasmid be cut at the exact location where the gene is to be inserted. That is, there must be a restriction endonuclease site at this position (preferably the only site so that the plasmid is cut at only one site during restriction endonuclease digestion). The plasmid is digested, phosphatase treated and purified as described above. The gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be achieved when the ends of the plasmid match the ends of the gene to be inserted. When cutting the plasmid with restriction enzymes and isolating the gene to be inserted (creating blunt ends or compatible cohesive ends), the bacteriostat is used as described in Section 1.68 of Sambrook et al. (Supra). The DNA can be directly ligated together using a ligase such as phage T4 DNA ligase and incubating the mixture at 16 ° C. for 1-4 hours in the presence of ATP and ligase buffer. If the ends are not compatible, they are first ligated to the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase (these four deoxyribonucleotides are used to fill the protruding single-stranded ends of the digested DNA). (Requires triphosphoric acid). Alternatively, these ends can be blunted using nucleases such as nuclease S1 or mung bean nuclease, which function by retracting the protruding single strand of DNA. The DNA is then ligated using the ligase described above. In some cases, the reading frame of the coding region changes, so that the end of the gene to be inserted may not be smoothed. Oligonucleotide linkers may be used to overcome this problem. This linker serves as a bridge connecting the plasmid and the gene to be inserted. These linkers can be prepared by synthesis as double-stranded or single-stranded DNA using conventional methods. These linkers have one end that matches the end of the gene to be inserted and are first linked to this gene using the ligation method described above. The other end of the linker is designed to be compatible with the plasmid for ligation. When designing the linker, care must be taken not to destroy the reading frame of the gene to be inserted or of the gene contained on the plasmid. In some cases, it may be necessary to design the linker so that they encode some of the amino acids, or one or more amino acids.

A DNA encoding a stop codon can be inserted between gene 1 and gene 2. Such stop codons are UAG (Amber), UAA (Ocher) and UGA (Opel) [Davis et al., Microbiology, Harper & Row, New York, pp. 237, 245-47 and 274 (1980)]. A stop codon expressed in a wild-type host cell results in the synthesis of the gene 1 protein product without the gene 2 protein attached. However, growth in suppressor host cells will result in the synthesis of detectable amounts of the fusion protein. Such suppressor host cells contain a tRNA that has been modified to insert an amino acid at the stop codon position of the mRNA, thus producing a detectable amount of the fusion protein. Such suppressor host cells are well known and disclosed, for example, as the suppressor strain of E. coli [Bullock et al., Bio Techniques 5: 376-379 (1987)]. Such stop codons can be placed in the mRNA encoding the fusion polypeptide using any accepted method.

The repressible codon can be inserted between a first gene encoding a polypeptide and a second gene encoding at least a portion of a phage coat protein. Alternatively, this suppressible stop codon can be inserted adjacent to the fusion site by substitution of the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein. Propagation of the phagemid containing this repressible codon in a suppressor host cell results in the detectable production of a fusion polypeptide containing the polypeptide and the coat protein. When the phagemid is grown in non-suppressor host cells, termination at the inserted repressible triplet encoding UAG, UAA or UGA results in substantially unfused polypeptide to the phage coat protein. Synthesized. In non-suppressor cells, the polypeptide is synthesized and secreted from the host cell due to the absence of the fused phage coat protein, which otherwise anchors the polypeptide to the host cell.

V. Alteration (mutation) of gene 1 at selected position
Gene 1 encoding the desired polypeptide can be modified at one or more selected codons. An alteration is defined as a substitution, deletion or insertion of one or more codons in the gene encoding the polypeptide. This modification results in a change in the amino acid sequence of the polypeptide as compared to the unmodified or native sequence of the same polypeptide. Preferably, the alteration is by replacing at least one amino acid with any other amino acid in one or more regions of the molecule. This modification can be made by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated mutagenesis and cassette mutagenesis.

A. Oligonucleotide-mediated mutagenesis Oligonucleotide-mediated mutagenesis is a preferred method for preparing gene 1, substitution, deletion and insertion variants. This method is well known in the art as described by Zoller et al. [ Nucleic Acids Res. 10: 6487-6504 (1987)]. Briefly, gene 1 is modified by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template of a plasmid containing gene 1 of the unmodified or native DNA sequence. . After hybridization, the DNA polymerase is used to synthesize the complete second complement of this template, into which the oligonucleotide primers have been introduced, encoding the selected modification in gene 1.

Usually, oligonucleotides of at least 25 nucleotides in length are used. Optimal oligonucleotides will have 12-15 nucleotides that are perfectly complementary to the template on each side of the nucleotide (s) encoding the mutation. This ensures that the oligonucleotide properly hybridizes to the single-stranded DNA template molecule. This oligonucleotide is readily synthesized using methods known in the art, for example, as described by Crea et al. [ Proc. Natl. Acad. Sci. USA 75: 5765 (1978)].

This DNA template is described in Veira et al . [ Meth. Enzymol . 153: 3 (1987)] or a vector derived from a bacteriophage M13 vector (M13mp18 and M13mp19 vectors available from commercial sources are suitable) . It can be obtained only by a vector containing such a single-stranded phage origin of replication. That is, the DNA to be mutated must be inserted into one of these vectors to create a single-stranded template. The preparation of single-stranded templates is described in sections 4.21 to 4.41 of Sambrook et al. (Supra).

オ リ ゴ To modify the native DNA sequence, the oligonucleotide is hybridized with a single-stranded template under appropriate hybridization conditions. Next, a DNA polymerase, usually Klenow fragment of DNA polymerase I, is added, and the complementary strand of the template is synthesized using this oligonucleotide as a primer for synthesis. Thus, a heteroduplex molecule in which one strand of DNA encodes a mutated form of gene 1 and the other strand (the first template) encodes the native, unmodified sequence of gene 1. Is formed. The heteroduplex molecule is then introduced into a suitable host cell, usually a prokaryote such as E. coli JM101. After growing the cells, they are plated on agarose plates and screened using oligonucleotide primers radiolabeled with 32-phosphate to identify bacterial colonies containing the mutated DNA.

方法 The method just described can be modified such that a heteroduplex molecule is created in which both strands of the plasmid contain the mutation (s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP) and deoxyribothymidine (dTTP), is available from a modified thio-deoxyribocytosine (Amersham) called dCTP- (aS). ) And mix. This mixture is added to the template-oligonucleotide complex. Addition of a DNA polymerase to this mixture creates a DNA strand identical to the template except for the mutated base. In addition, this novel DNA strand contains dCTP- (aS) instead of dCTP, which serves to protect this strand from restriction endonuclease digestion. After nicking the heteroduplex template strand with an appropriate restriction enzyme, digesting this template strand with ExoIII nuclease or another appropriate nuclease beyond the region containing the site (s) to be mutated. Can be. The reaction is then stopped to leave molecules that are only partially single-stranded. A complete DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP and DNA ligase. This homoduplex molecule can then be introduced into a suitable host cell such as E. coli JM101 as described above.

Mutants having more than one amino acid to be substituted can be created by any of several methods. When these amino acids are in close proximity to each other in a polypeptide chain, they can be mutated simultaneously with one oligonucleotide encoding all of the desired amino acid substitutions. However, when amino acids are present at some distance from each other (separated by about 10 or more amino acids), creating a single oligonucleotide encoding all of the desired changes is a comparative Difficult. Alternatively, one of two different methods can be used.

In the first method, an independent oligonucleotide is created for each amino acid to be substituted. Then, if the oligonucleotides are simultaneously annealed to the single-stranded template DNA, the second DNA strand synthesized from this template will encode all of the desired amino acid substitutions. This first round is the same as described for the single mutation. That is, using the wild-type DNA as a template, an oligonucleotide encoding the first desired amino acid substitution (s) is annealed to the template, and then a heteroduplex DNA molecule is created. The second round of mutagenesis uses the mutated DNA obtained from the first round of mutagenesis as a template. That is, the template already contains one or more mutations. An oligonucleotide encoding another desired amino acid substitution (s) is then annealed to this template, and the resulting DNA strand encodes mutations from both the first and second rounds of mutagenesis. are doing. The obtained DNA can be used as a template or the like in the third round of mutagenesis.

B. Cassette Mutagenesis This method is also a preferred method for preparing substitution, deletion and insertion mutants of Gene 1. This method is based on the method described by Wells et al. [ Gene 34: 315 (1985)]. The starting material is a plasmid (or other vector) containing gene 1, the gene to be mutated. The codon in gene 1 to be mutated has been determined. There must be only one restriction endonuclease site on each side of the determined mutation site (s). If such restriction sites are not present, they can be created using the oligonucleotide-mediated mutagenesis method described above and introduced into gene 1 at the appropriate location. After introducing restriction sites into the plasmid, the plasmid is cut at these sites to linearize it. Double-stranded oligonucleotides encoding the DNA sequence between these restriction sites and containing the desired mutation are synthesized by conventional methods. The two chains are synthesized separately and then hybridized together in a conventional manner. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends that match both ends of the linearized plasmid, so that it can be directly ligated to the plasmid. This plasmid will then contain the mutated DNA sequence of gene 1.

VI. Preparation of DNA Encoding the Desired Protein In another aspect, the present invention contemplates the production of variants of the desired protein containing one or more subunits. Usually, each subunit is encoded by another gene. Each gene encoding each subunit can be obtained by methods known in the art (see, eg, Section II). In some cases, it may be necessary to obtain genes encoding different subunits using independent methods selected from any of the methods described in Section II.

When constructing a replicable expression vector in which the desired protein contains more than one subunit, all subunits are replaced by the same promoter, usually located 5 'to the DNA encoding the subunit. Alternatively, each subunit may be regulated by an independent promoter appropriately oriented in the vector, each promoter being operably linked to the DNA to be regulated. The selection of the promoter is performed as described in section III above.

Reference should be made to FIG. 10 when constructing a replicable expression vector containing DNA encoding a desired protein having multiple subunits, where the vector is illustrated for illustration. DNA encoding each subunit of the antibody fragment is shown. This figure shows that one of the subunits of the desired protein is usually fused to a phage coat protein such as M13 gene III. Usually, this gene fusion will contain its own signal sequence. It can be seen that another gene encodes another subunit or group of subunits, and each subunit usually has its own signal sequence. FIG. 10 also shows that a single promoter can regulate the expression of both subunits. Alternatively, each subunit may be independently regulated by a different promoter. The desired protein subunit-phage coat protein fusion construct can be prepared as described in Section IV above.

When constructing a group of variants of the desired multi-subunit protein, the DNA encoding each subunit in the vector may be mutated at one or more positions in each subunit. When multiple subunit antibody variants are constructed, preferred mutagenesis sites encode amino acid residues located in the complementarity determining regions (CDRs) of either the light, heavy or both chains. Matches the codon that is. Usually, this CDR is called a hypervariable region. The method of mutating the DNA encoding each subunit of the desired protein is performed substantially as described in Section V above.

VII. Preparation of Target Molecules and Target Proteins, such as Receptors for Binding to Phagemids, can be isolated from natural sources or prepared by recombinant methods by methods known in the art. To illustrate, glycoprotein hormone receptors McFarland et al [Science 245: 494-499 (1989)] is can be prepared by methods described, non-glycosylated, which was expressed in E. coli Fuh et al [J . Biol. Chem. 265: 3111-3115 (1990)]. Other receptors can be prepared by a conventional method.

Purify the target protein with a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate, polyacrylic and polymethacrylic acid copolymers, nylon, neutral and ionic carriers. Can be combined. Binding of the target protein to the matrix can be performed by methods described in the literature [ Methods in Enzymology 44 (1976)] or by other means known in the art.

After binding the target protein to the matrix, the immobilized target is contacted with a library of phagemid particles under conditions suitable for binding at least a portion of the phagemid particles to the immobilized target. Usually, conditions including pH, ionic strength, temperature, etc. will be similar to physiological conditions.

結合 Separate bound phagemid particles with high affinity for the immobilized target (“binders”) from low affinity particles (and thus particles that do not bind to the target) by washing The conjugate can be dissociated from the immobilized target by various methods. These methods include competitive dissociation using changes in wild-type ligand, pH and / or ionic strength, and methods known in the art.

感染 Infect suitable host cells with the conjugate and helper phage, and culture the host cells under conditions appropriate for phagemid particle amplification. The phagemid particles are then collected and the selection step is repeated one or more times until a conjugate having the desired affinity for the target molecule is selected.

に よ り Optionally, a library of phagemid particles can be contacted sequentially with more than one immobilized target to improve selectivity for a particular target. For example, ligands such as hGH often have more than one natural receptor. In the case of this hGH, both the growth hormone receptor and the prolactin receptor bind to the hGH ligand. It would be desirable to improve the selectivity of hGH over the growth hormone receptor over that of the prolactin receptor. This involves first contacting the phagemid particle library with the immobilized prolactin receptor, eluting it with low affinity (ie, lower than wild-type hGH) for the prolactin receptor, and then binding this low affinity prolactin "binding" This can be accomplished by contacting the "body" or unconjugated with the immobilized growth hormone receptor and selecting a high affinity growth hormone receptor conjugate. In this case, mutants of hGH that have low affinity for the prolactin receptor have therapeutic use even when the affinity for the growth hormone receptor is slightly lower than that of wild-type hGH. Will be. This same method can be used to improve the selectivity of a particular hormone or protein for its primary functional receptor over its clearance receptor.

別 In another aspect of the present invention, an improved substrate amino acid sequence can be obtained. These will be useful in creating better “cleavage sites” for protein linkers or for better protease substrates / inhibitors. In this embodiment, an immobilizable molecule (eg, an hGH-receptor, biotin-avidin, or a molecule capable of covalently binding to a matrix) is fused to gene III via a linker. The linker is preferably 3-10 amino acids in length and acts as a substrate for a protease. Phagemids are constructed as described above (where the DNA encoding the linker region is randomly mutated to prepare a random library of phagemid particles with different amino acid sequences at the binding sites). This library of phagemid particles is then immobilized on a matrix and exposed to the desired protease. Phagemid particles having a preferred or better substrate amino acid sequence in the liner region of the desired protease will be eluted, and an enriched pool of phagemid particles encoding the preferred linker will be obtained first. These phagemid particles are then repeated several more times to obtain an enriched pool of particles encoding consensus sequence (s) (see Examples XIII and XIV).

VIII. Growth Hormone Variants and Methods of Use The cloned gene of hGH is expressed in a secreted form in Eschericha cola [Chang, CN et al., Gene 55: 189 (1987)], and its DNA and amino acid sequences have been reported [Goeddel et al., Nature 281: 544 (1979); Gray et al., Gene 39: 247 (1985)]. The present invention describes novel hGH variants obtained using the phagemid selection method. Human growth hormone variants containing substitutions at positions 10, 14, 18, 21, 167, 171, 172, 174, 175, 176, 178 and 179 have been described. Mutants with relatively high binding affinity are described in Tables VII, XIII and XIV. Amino acid nomenclature for describing these variants is provided below. Growth hormone variants may be administered and formulated in the same way as regular growth hormone. The growth hormone variants of the present invention can be expressed in any recombinant system capable of expressing native or met hGH.

Formulations of hGH for therapeutic administration include carriers, excipients or stabilizers that optionally allow the desired degree of purification of the hGH to be physiologically acceptable [ Remington's Pharmaceutical Sciences , 16th ed., Osol, A. Ed. (1980). )] Is prepared for storage in the form of a lyophilized cake or aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, for example, phosphate, citrate, and other organic acids. Buffer); antioxidants (including ascorbic acid); low molecular weight (less than about 10 residues) polypeptides; proteins (eg, serum albumin, gelatin, or immunoglobulin); hydrophilic polymers (eg, polyvinylpyrrolidone) Amino acids (eg, glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (eg, EDTA); divalent metal ions (Eg, zinc, cobalt or copper); sugar alcohols (eg, mannitol or Rubitoru); counter ion forming the salt (e.g., sodium); and / or nonionic surface active agents [e.g., Tween, Pluronics or polyethylene glycol (PEG)] are included. The formulation of the present invention may further contain a pharmaceutically acceptable buffer, amino acid, bulking agent, and / or nonionic surfactant. These include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like. Examples of these would include trimethylamine salt ("Tris buffer"), and disodium edetate. The phagemid of the present invention can be used to produce large amounts of hGH mutants without phage proteins. To express hGH variants that do not contain the gene III portion of the fusion, pS0643 and derivatives can simply be grown in non-suppressor strains such as 16C9. In this case, the amber codon (TAG) leads to the termination of translation and free hormone is obtained without the need for independent DNA construction. The hGH variant is secreted from the host and can be isolated from the culture medium.

Replacing one or more of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 with any amino acid other than the amino acid found at that position in native hGH shown herein; Can be replaced by That is, 1, 2, 3, 4, 5, 6, 7, or all of the amino acids F10, M14, H18, H21, R167, D171, T175, and I179 shown here are all 20 amino acids listed below. Can be substituted with any of the other 19 amino acids. In a preferred embodiment, all eight amino acids listed here are replaced with another amino acid. The most preferred eight amino acids to be substituted are shown in Table XIV in Example XII.
Amino acid nomenclature
Ala (A)
Arg (R)
Asn (N)
Asp (D)
Cys (C)
Gln (Q)
Glu (E)
Gly (G)
His (H)
Ile (I)
Leu (L)
Lys (K)
Met (M)
Phe (F)
Pro (P)
Ser (S)
Thr (T)
Trp (W)
Tyr (Y)
Val (V)
The nomenclature for single letter hGH variants is to list the hGH amino acid to be deleted (eg, glutamate 179) first, followed by the amino acid to be inserted (eg, serine), resulting in (E1795S).

Without further elaboration, it is believed that one skilled in the art can, using the preceding description and illustrative examples, fully implement and utilize the present invention. That is, the following specific examples illustrate preferred embodiments of the present invention and should not be considered in any way as limiting the remainder of the disclosure.

Preparation Plasmid phGH-M13gIII Construction and hGH- phagemid particles of Example 1 plasmid (FIG. 1), M13KO7 ⁷ and hGH production plasmid pBO473 [Cunningham, BC, et al., Science 243: 1330-1336 (1989)] was constructed from. Synthetic oligonucleotide:
5'-AGC-TGT-GGC-TTC- GGG-CCC -TTA-GCA-TTT-AAT-GCG-GTA-3 '
Was used to introduce a unique ApaI restriction site (underlined) after the last Phe191 codon of hGH in pBO473. Oligonucleotides:
5'-TTC-ACA-AAC-GAA- GGG-CCC - CTA -ATT-AAA-GCC-AGA-3 '
Was used to introduce a unique ApaI restriction site (first underline), and a Glu197-amber stop codon (second underline) in M13KO7 gene III. Oligonucleotides:
5'-CAA-TAA-TAA-CGG- GCT-AGC -CAA-AAG-AAC-TGG-3 '
Introduces a unique NheI site (underlined) after the 3 'end of the gene III coding sequence. The resulting 650 base pair (bp) ApaI-NheI fragment from the double mutated M13KO7 gene III was cloned into the large ApaI-NheI fragment of pBO473 to create plasmid pSO132. As a result, the carboxy terminus of hGH (Phe191) was fused to the Pro198 residue of the gene III protein while inserting the glycine residue encoded from the ApaI site. It was under the control of the sequence [Chang, CN et al., Gene 55: 189-196 (1987)]. The phoA promoter was replaced with a lac promoter and operator for inducible expression of the fusion protein in rich media. A 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and Cap binding site was ligated to the oligonucleotide:
5'-CACGACA GAATTC CCGACTGGAAA-3 'and 5'-CTGTT TCTAGA GTGAAATTGTTA-3'
[These border the desired lac sequence and introduce EcoRI and XbaI restriction sites (underlined)]
Was prepared by PCR of plasmid pUC119 using This lac fragment was gel purified and ligated to the large EcoRI-XbaI fragment of pSO132 to create plasmid phGH-M13gIII. The sequences of all processed DNA junctions were confirmed by dideoxy sequencing (Sanger, F. et al., Proc . Natl . Acad . Sci . USA 74: 5463-5467 (1977)). The R64A mutant hGH phagemid was constructed as follows. That is, the NsiI-BglII mutant fragment of hGH [Cunningham et al. (Supra)] encoding the Ala substitution of Arg64 (R64A) [Cunningham, BC, Wells, JA, Science 244: 1081-1085 (1989)] was used. It was cloned between the corresponding restriction sites in the phGH-M13gIII plasmid (FIG. 1) to replace the wild-type hGH sequence. The R64A hGH phagemid particles were grown and titrated as described below for wild-type hGH-phagemid.

Plasmids were introduced into a male strain of E. coli (JM101) and selected on carbenicillin plates. Single transformants were grown for 4 hours at 37 ° C. in 2YT medium (2 ml) and infected with M13KO7 helper phage (50 μl). The infected culture was diluted in 2YT (30 ml), cultured overnight, and phagemid particles were collected by precipitation with polyethylene glycol [Vierra, J., Messing, J., Methods in Enzymology 153: 3-11 (1987). ]. Usually, the titer of the phagemid particles was in the range of 2~5x10 ¹¹ cfu / ml. The particles were purified to homogeneity by CsCl density centrifugation [Day, LA, J. Mol . Biol. 39: 265-277 (1969)] to remove any fusion proteins not bound to virions.

Example II Immunochemical analysis of hGH on fusion phage Rabbit polyclonal antibody to hGH was purified using protein A and at a concentration of 2 μg / ml in 50 mM sodium carbonate buffer (pH 10) at 4 ° C. for 16-20 hours. , Microtitration plates (Nunc). After washing in PBS containing 0.05% Tween 20, the hGH or hGH-phagemid particles were treated with buffer A [50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg / ml bovine serum albumin, and 0.5 mg / ml bovine serum albumin. In 0.05% Tween 20] to 2.0-0.002 nM . After 2 hours at room temperature (rt), the plate was washed extensively and the indicated Mab [Cunningham et al. (Supra)] was added at 1 μg / ml in buffer A for 2 hours at rt. After washing, horseradish peroxidase-conjugated goat anti-mouse IgG antibody was allowed to bind for 1 hour at rt. After the last wash, peroxidase activity was assayed using the substrate o-phenylenediamine.

Example III Binding and Enrichment of hGH Binding Protein to Polyacrylamide Beads Oxirane polyacrylamide beads (Sigma) were added to the extra cysteine residue introduced by site-directed mutagenesis at position 237 (affects the binding of hGH Conjugated with the purified extracellular domain of the hGH receptor (hGHbp) containing [ J. Wells: unpublished] [Fuh, G. et al., J. Biol. Chem. 265: 3111-3115 (1990)] . It has become. The hGHbp was conjugated to 1.7 pmol hGHbp / mg dry oxirane beads as determined by the binding of [ ¹²⁵ I] hGH to the resin, as recommended by the supplier. Then, all unreacted oxirane groups were blocked with BSA and Tris. As a control for non-specific binding of phagemid particles, BSA was similarly bound to the beads. Buffer for adsorption and washing contained 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg / ml BSA and 0.02% Tween 20. The elution buffer contained 200 nM hGH or 0.2 M glycine (pH 2.1) in addition to the wash buffer. The parental phage M13KO7 is mixed with the hGH phagemid particles in a ratio of approximately 3000: 1 (initial mixture) and at room temperature in a 50 μl volume with either absorbent (5 μl each; 0.2 mg acrylamide beads) for 8-12 hours. Mixed. The beads were pelleted by centrifugation and the supernatant was carefully removed. The beads were resuspended in wash buffer (200 μl) and mixed for 4 hours at room temperature (wash 1). After the second wash (wash 2), the beads were eluted twice with 200 nM hGH each for 6-10 hours (eluate 1, eluate 2). The final elution was performed for 4 hours using glycine buffer (pH 2.1) to remove the remaining hGH phagemid particles (eluate 3). Each fraction was appropriately diluted in 2YT medium, mixed with fresh JM101, incubated at 37 ° C. for 5 minutes, and plated on LB or LB carbenicillin plates using 2YT soft agar (3 ml).

Example IV Construction of hGH-phagemid particles with a mixture of gene III products The gene III protein consists of 410 residues and is divided into two domains separated by a variable linker sequence [Armstrong, J. et al., FEBS Lett. 135 : 167-172 (1981)]. The amino-terminal domain is required for attachment to E. coli fimbria, while the carboxy-terminal domain is incorporated into the phage coat and is required for proper phage assembly [Crissman, JW, Smith, GP , Virology 132: 445-455 (1984)]. The signal sequence and amino terminal domain of gene III were replaced with the stII signal and the entire hGH gene [Chang et al., Supra] by fusion to residue 198 in the carboxy terminal domain of gene III (FIG. 1). This hGH-gene III fusion was placed under the control of the lac promoter / operator in a plasmid (phGH-M13gIII; FIG. 1) containing the β-lactamase gene of pBR322 and the ColE1 origin of replication and the intergenic region of phage f1. This vector can be easily maintained as a small plasmid vector by selection with carbenicillin, thereby avoiding reliance on a functional gene III fusion for propagation. This plasmid can also be efficiently packaged into virions (ie, phagemid particles) by infection with helper phage such as M13KO7 [Viera et al. (Supra)], thereby avoiding the problem of phage assembly. . The infectivity titer of phagemid based on transduction for carbenicillin resistance in this system varied from 2 to 5 × 10 ¹¹ colony forming units (cfu) / ml. The titer of the M13KO7 helper phage in these phagemid storage material is 10 ¹⁰ plaque forming units (pfu) / ml.

This system was used to confirm a previous study [Parmley, Smith (supra)] that homogenous expression of large proteins fused to gene III was detrimental to phage production (data not shown). For example, induction of the lac promoter in phGH-M13gIII by addition of IPTG resulted in low phagemid titers. Furthermore, phagemid particles obtained by co-infection with M13KO7 containing an amber mutation in gene III gave very low phagemid titers (<10 ¹⁰ cfu / ml). We thought that multiple copies of the gene III fusion bound to the phagemid surface could lead to multiple point binding ("chelation") of the fused phage to the immobilized target protein. Therefore, in order to control the number of copies of the fusion protein, by culturing the plasmid in E. coli JM101 containing the lac repressor protein, constitutively high levels (lacl ^Q), the transcription of hGH- gene III fusions Restricted. E. coli JM101 cultures containing phGH-M13gIII were grown maximally and infected with M13KO7 in the absence of the lac operon inducer (IPTG) (however, this system was flexible and had other gene III Can be balanced with co-expression of the fusion protein). From the ratio of hGH per virion (based on hGH immunoreactive material in phagemid purified on a CsCl gradient), we found that about 10% of phagemid particles contained one copy of the hGH gene III fusion protein. It is estimated. Thus, the titer of the fusion phage displaying the hGH gene III fusion is about 2-5 × ^{10 10} / ml. This number is considerably greater than the titer of E. coli cultures led them ^{(~10 8 ~10 9 / ml)} . That is, on average, all E. coli cells produce 10-100 copies of phage decorated with the hGH gene III fusion protein.

Example V Structural Integrity of hGH-Gene III Fusions Immunoblot analysis of hGH-gene III phagemids (FIG. 2) shows that hGH cross-reactants co-migrate with phagemid particles in agarose gels. This indicates that hGH is tightly bound to the phagemid particles. The hGH-gene III fusion protein from this phagemid particle migrated as a single immunostained band, indicating little degradation of hGH when hGH bound to gene III. The wild-type gene III protein is clearly present, as about 25% of the phagemid particles are infectious. This is similar to the concentration assessed by UV absorption and the specific infectivity assessment performed on similarly purified wild-type M13 phage (by CsCl density gradient method) [Smith, GP (supra) and Parmley, Smith ( the above)〕. That is, both the wild-type gene III and the hGH-gene III fusion protein are displayed in the phage pool.

It was important to confirm that the indicated tertiary structure of hGH was maintained in order to obtain confidence that the results of the binding selection were converted to the native protein. The monoclonal antibody against hGH (Mab) was used to assess the structural integrity of the indicated hGH gene III fusion protein (Table I).
Table I. Binding of eight different monoclonal antibodies (Mab) to hGH and hGH phagemid particles *
IC ₅₀ (nM)
Mab hGH hGH-phagemid 1 0.4 0.4
2 0.04 0.04
3 0.2 0.2
4 0.1 0.1
5 0.2> 0.2
6 0.07 0.2
7 0.1 0.1
8 0.1 0.1
* Values in the table are hGH giving half-maximal binding to a particular Mab
Alternatively, it represents the concentration (nM) of hGH-phagemid particles. The standard error of these measurements is usually ± 30% or less of the indicated value. See Raw Materials and Methods for details.

The epitopes on hGH for these Mabs have been mapped [Cunningham et al., Supra], and binding to seven of the eight Mabs requires that hGH be properly folded. The IC ₅₀ values of all Mab were equivalent to wild-type hGH except for Mab 5 and 6. Both Mab 5 and 6 are known to have binding determinants near the carboxy terminus of hGH that are blocked in the gene III fusion protein. The relative IC ₅₀ values of Mab1 reacting with both native and denatured hGH remain unchanged compared to the conformationally sensitive Mab 2-5, 7 and 8. Thus, Mab1 serves as a good internal control of any errors in adapting the concentration of the hGH standard to the concentration of the hGH-gene III fusion.

Example VI Enrichment of Binding to Receptor Affinity Beads Previous investigators [Parmley, Smith (supra); Smith (supra); Cwirla et al. (Supra); and Devlin et al. (Supra)] were streptavidin-coated. Phages were fractionated by sorting on polystyrene petri dishes or microtiter plates. However, chromatographic systems allow for more efficient fractionation of phagemid particles displaying muteins with different binding affinities. We selected non-porous oxirane beads (Sigma) to avoid the capture of phagemid particles in the chromatography resin. Furthermore, these beads have a small particle size (1 μm), maximizing the ratio of surface area to volume. The extracellular domain of the hGH receptor containing free cysteino residues (hGHbp) [Fuh et al. (Supra)] binds efficiently to these beads, and the phagemid particles bind to beads bound only to bovine serum albumin. It showed very low non-specific binding (Table II).

Table II . By specific binding of hormone phage to hGHbp coated beads
Enrichment of hGH-phage over the obtained M13KO7 phage * 1
Sample Absorbing substance * 3 Total pfu Total cfu ratio (cfu / pfu) Enrichment * 4
First mixture * 2 8.3x10 ¹¹ 2.9x10 ⁸ 3.5x10 ^-4 (1)
Supernatant BSA 7.4x10 ¹¹ 2.8x10 ⁸ 3.8x10 ^-4 1.1
hGHbp 7.6x10 ¹¹ 3.3x10 ⁸ 4.3x10 ^-4 1.2
Cleaning solution 1 BSA 1.1x10 ¹⁰ 6.0x10 ⁶ 5.5x10 ^-4 1.6
hGHbp 1.9x10 ¹⁰ 1.7x10 ⁷ 8.9x10 ^-4 2.5
Cleaning solution 2 BSA 5.9x10 ⁷ 2.8x10 ⁴ 4.7x10 ^-4 1.3
hGHbp 4.9x10 ⁷ 2.7x10 ⁶ 5.5x10 ^-2 1.6x10 ²
Eluent 1 (hGH) BSA 1.1 × 10 ⁶ 1.9 × 10 ³ 1.7 × 10 ^-3 4.9
hGHbp 1.2x10 ⁶ 2.1x10 ⁶ 1.8 5.1x10 ³
Eluate 2 (hGH) BSA 5.9 × 10 ⁵ 1.2 × 10 ³ 2.0 × 10 ^-3 5.7
hGHbp 5.5x10 ⁵ 1.3x10 ⁶ 2.4 6.9x10 ³
Eluate 3 (pH 2.1) BSA 4.6 × 10 ⁵ 2.0 × 10 ³ 4.3 × 10 ^-3 12.3
hGHbp 3.8x10 ⁵ 4.0x10 ⁶ 10.5 3.0x10 ⁴
* 1 The titer of M13KO7 and hGH-phagemid particles in each fraction was measured by multiplying the value of plaque forming unit (pfu) or carbenicillin resistant colony forming unit (cfu) by a dilution factor, respectively. See Example IV for details.
* 2 The ratio of M13KO7 to hGH-phagemid particles was adjusted to 3000: 1 in the initial mixture.
* 3 The absorbent was conjugated to BSA or hGHbp.
* 4 Enrichment was calculated by dividing the cfu / pfu ratio after each step by the cfu / pfu ratio of the initial mixture.

In a representative enrichment experiment (Table II), one part of hGH phagemid was mixed with> 3,000 parts of M13KO7 phage. After one cycle of binding and elution, 10 ⁶ phages were recovered and the ratio of phagemid to M13KO7 phage was 2: 1. That is,> 5000-fold enrichment was obtained by one binding selection step. Additional elution by free hGH or acid treatment to remove residual phagemids resulted in higher enrichment. This enrichment was comparable to the enrichment obtained by Smith and coworkers using batch elution from coated polystyrene plates [Smith, GP (supra) and Parmley, Smith (supra)]. However, even smaller volumes are used for beads (200 μl vs 6 ml). There was little enrichment of hGH phagemid over M13KO7 when using beads bound only to BSA. The slight enrichment observed for the control beads (-10 fold relative to pH 2.1 elution; Table 2) is due to trace impurities of bovine growth hormone binding protein present in BSA binding to the beads. Would be something. Nevertheless, these data indicate that hGH phage enrichment is dependent on the presence of hGHbp on the beads, suggesting that binding occurs by a specific interaction between hGH and hGHbp.

We evaluated the enrichment of wild-type hGH over relatively weak binding variants of hGH on the fusion phagemid to further investigate the specificity of the enrichment and to determine the decrease in binding affinity for purified hormone after fusion phagemid selection. Associated with the enrichment factor. A fusion phagemid was constructed using the hGH mutant in which Arg64 was replaced with Ala (R64A). This R64A mutant hormone has an approximately 20-fold decrease in receptor binding affinity compared to hGH [Kd values of 7.1 nM and 0.34 nM, respectively; Cunningham, Wells (supra)]. The titer of the R64A hGH-gene III fusion phagemid was comparable to that of the wild-type hGH phagemid. After one round of binding and elution (Table III), wild-type hGH phagemid is abundant 8-fold for phagemid R64A and -10 ⁴ for M13KO7 helper phage from a mixture of two phagemids and M13KO7. Was

Table III . Select hGH phagemid over relatively weak binding hGH mutant phagemid
hGHbp coated beads
Control beads hGHbp beads
Sample WT phagemid WT / R64A WT phagemid WT / R64A
/ Enrichment of all phagemids / Enrichment of all phagemids
First mixture 8/20 (1) 8/20 (1)
Supernatant ND-4 / 10 1.0
Eluate 1 (hGH) 7/20 0.8 17/20 8.5 * 3
Eluate 2 (pH 2.1) 11/20 1.8 21/27 5.2
* 1 Parent M13KO7 phage, wild-type hGH phagemid and R64A phagemid particles were mixed at a ratio of 10 ⁴ : 0.4: 0.6. Binding selection was performed using beads bound by BSA (control beads) or hGHbp (hGHbp beads) as described in Table II and the section on raw materials and methods. After each step, plasmid DNA was isolated from carbenicillin resistant colonies [Birnboim, HD, Doly, J., Nucleic Acids Res. 7: 1513-1523 (1979)] and analyzed by restriction analysis to determine that the DNA was wild-type. It was determined whether it contained the hGH or R64A hGH gene III fusion.
* 2 The enrichment of wild-type hGH phagemid over the R64A mutant is based on the ratio of phagemid present in the initial mixture of hGH phagemid present after each step to phagemid present (8/20), with a corresponding ratio of R64A phagemid. Calculated by dividing.
WT = wild type; ND = not measured.
* 3 enrichment of phagemid over total M13KO7 parental phage was 10 ⁴ after this step.

By displaying a mixture of wild-type gene III and gene III fusion proteins on phagemid particles, virions displaying large, properly folded proteins as fusions to gene III can be assembled and propagated. The copy number of the gene III fusion protein can be effectively controlled to avoid "chelation", which is still maintained at a sufficiently high level in the phagemid pool, and to screen large epitope libraries (> 10 ¹⁰ ). . We have shown that hGH (a 22 kD protein) can be displayed in its native, folded form. Binding selections eluted with free hGH and performed on receptor affinity beads efficiently enriched wild-type hGH phagemid over mutant hGH phagemid which was shown to have low receptor binding affinity . That is, phagemid particles having a reduced binding constant in the nanomolar range can be selected.
Protein-protein and antibody-antigen interactions are governed by discontinuous epitopes [Janin, J. et al., J. Mol . Biol. 204: 155-164 (1988); Argos, P., Prot . Eng . . 2: 101-113 (1988); Barlow, DJ , et al., Nature 322:. 747-748 (1987 ); and Davies, DR, et al., J.Biol.Chem 263: 10541-10544 (1988)]. That is, residues directly involved in binding are close together in the tertiary structure, but are separated by residues not involved in binding. The screening system provided by the present invention further advantageously analyzes protein-receptor interactions and allows the isolation of discontinuous epitopes in proteins with novel and high affinity binding It is.

Example VII Construction of a Selection Template for hGH Mutants from a Library Randomized at hGH Codons 172, 174, 176, 178 Mutants of the hGH-gene III fusion protein are described in Kunkel et al . [ Meth. Enzymol. 154: 367-382 (1987)]. As template DNA, the plasmid pS0132 (containing the native hGH gene fused to the carboxy-terminal half of M13 gene III under the control of the alkaline phosphatase promoter) is grown in CJ236 cells with the addition of M13-K07 phage as a helper. Was prepared by Single-stranded uracil-containing DNA was prepared for mutagenesis to assay (1) mutations in hGH that greatly reduced binding to hGH binding protein (hGHbp); and (2) assay parental background phage. Introduced a unique restriction site (KpnI) that could be used to select against it. T7 DNA polymerase and the following oligodeoxynucleotides:
Gly Thr
hGH codon: 178 179
5'-G ACA TTC CTG G G T A C C GTG CAG T-3 '
<KpnI>
Was used to perform oligonucleotide-directed mutagenesis. This oligo introduces a KpnI site (shown above) with a mutation (R178G, I179T) in hGH. Clones from mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. This resulting construct, used as a template for random mutagenesis, was named pHHO415.

Random Mutagenesis in hGH 4 of hGH Again using the method of Kunkel, codons 172, 174, 176, 178 were targeted for random mutagenesis in hGH. A single-stranded template was prepared from pHHO415 as described above and the following oligos:
hGH codon: 172 174
5'- GC TTC AGG AAG GAC ATG GAC NNS GTC NNS ACA-
Ile
176 178 179
- NNS CTG NNS A T C GTG CAG TGC CGC TCT GTG G-3 '
Mutagenesis was performed using a pool of As indicated above, this oligo pool reverted codon 179 to wild type (Ile), destroyed the unique KpnI site of pH0415, and randomized codons at positions 172, 174, 176 and 178 (NNS; where , N is A, G, C or T, and S is G or C). This codon choice cannot be used to create additional KpnI sites for the above sequences. The selection of this NNS synonymous sequence resulted in 32 possible codons at four sites (one “stop” codon and at least one codon for each amino acid), for a total of (32) ⁴ = 1,048 , 576 possible nucleotide sequences (12% of which contain at least one stop codon), or (20) ⁴ = 160,000 possible polypeptide sequences and 34,481 immature terminated sequences (ie, , A sequence containing at least one stop codon).

The growth mutagenesis product of the first library was extracted twice with phenol: chloroform (50:50) and ethanol precipitated using excess carrier tRNA to avoid the addition of salts that would complicate the subsequent electroporation step. I let it. Approximately 50 ng (15 fmoles) of DNA was injected into WJM101 cells (2.8 × ^{10 10} cells / ml) at a single pulse voltage setting of 2.49 kV (time constant = 4.7 msec) in a total volume of 45 μl in a 0.2 cm cuvette. ) Was introduced by electroporation.

The cells were allowed to recover for 1 hour at 37 ° C. with shaking and then mixed with 2YT medium (25 ml), 100 μg / ml carbenicillin, and M13-K07 (multiplicity of infection = 1000). By plating serial dilutions from this culture on a carbenicillin-containing medium, it was found that 8.2 × 10 ⁶ electrotransformants were obtained. After 10 minutes at 23 ° C., the culture was incubated overnight (15 hours) at 37 ° C. with shaking.

After overnight incubation, cells were pelleted and double-stranded DNA (dsDNA), designated pLIB1, was prepared by the alkaline lysis method. The supernatant was centrifuged once more to remove all remaining cells, and the phage designated phage pool φ1 was PEG precipitated and reconstituted in STE buffer [10 mM Tris (pH 7.6), 1 mM EDTA, 50 mM NaCl] (1 ml). Suspended. Phage titers, measured as the hGH-g3p gene III fusion (hGH-g ³⁾ For recombinant phagemid containing plasmid colony forming units (CFU), and the plaque forming units (PFU) for M13-K07 helper phage did.

Binding selection using immobilized hGHbp Binding: A portion of the phage pool φ1 (6 × 10 ⁹ CFU, 6 × 10 ⁷ PFU) was buffered with buffer A [phosphate buffered saline, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal]. Diluted 5-fold and mixed in a 1.5 ml silane-treated polypropylene test tube with a suspension (5 μl) of oxirane-polyacrylamide beads conjugated to hGHbp (350 fmol) containing the Ser237Cys mutation. As a control, an equal amount of phage was mixed with beads coated with BSA only in a separate tube. The phage were bound to the beads by incubating at room temperature (23 ° C.) for 3 hours with slow rotation (about 7 rpm). The following steps were performed at room temperature using a constant volume of 200 μl.

{2. Washing: The beads were spun for 15 seconds and the supernatant was removed (Supernatant 1). The beads were washed twice by resuspending in buffer A to remove non-specifically bound phage / phagemid and then pelleted. A final wash was performed by rotating the beads in buffer A for 2 hours.

{3. hGH elution: Phage / phagemid weakly bound to the beads were removed by stepwise elution with hGH. In the first step, the beads were spun with buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted, resuspended in buffer A containing 20 nM hGH, spun for 3 hours, and then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH, spun for 3 hours, and then pelleted.

4. Glycine elution: To remove the most tightly bound phagemid (ie, phagemid still bound after the hGH wash), suspend the beads in glycine buffer [1M glycine, pH 2.0 with HCl] and spin for 2 hours. And pelletized. This supernatant (fraction “G”; 200 μl) was neutralized by the addition of 1 M Tris base (30 μl).

The fraction G (1 × 10 ⁶ CFU, 5 × 10 ⁴ PFU) eluted from the hGHbp-beads was not substantially enriched in phagemid more than K07 helper phage. We attribute this to the fact that the packaged K07 phage displays an hGH-g3p fusion during propagation of the recombinant phagemid.

However, when compared to fraction G eluted from BSA-coated control beads, the hGHbp-beads gave a 14-fold higher CFU. This reflects the enrichment of phagemids displaying tightly bound hGH over non-specifically bound phagemids.

5. Proliferation: A part (4.3 × 10 ⁵ CFU) of fraction G eluted from the hGHbp-beads was used to infect log-growth WJM101 cells. Transduction was performed by mixing fraction G (100 μl) with WJM101 cells (1 ml), incubating at 37 ° C. for 20 minutes, and then adding K07 (multiplicity of infection = 1000). Cultures (25 ml of 2YT and carbenicillin) were grown as described above and a second pool of phage (library 1G, for primary glycine elution) was prepared as described above.

Phage from library 1G (FIG. 3) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained CFU 30 times higher in this selection than Fraction G eluted from BSA beads. Once again, a portion of fraction G was grown in WJM101 cells to obtain library 1G ² (glycine elution indicates that this library was selected twice). Double stranded DNA (pLIB 1G ² ) was also prepared from this culture.
KpnI test and restriction selection of dsDNA

To reduce the amount of background (KpnI ⁺⁾ template, was digested with pLIB part of 1G ² (about 0.5 [mu] g) KpnI, it was introduced electroporated into WJM101 cells. These cells were grown in the presence of K07 (multiplicity of infection = 100) as described for the original library and a new phage pool, pLIB3, was prepared (FIG. 3).

Further, a part (about 0.5 μg) of dsDNA derived from the first library (pLIB1) was digested with KpnI and directly electroporated into WJM101 cells. Transformants were recovered as described above, infected with M13-K07, and grown overnight to obtain a new phage library named phage library 2 (FIG. 3).

Sequential round selection (1) Excess (10 times CFU) purified K07 phage (not displaying hGH) was added into the bead binding cocktail; and (2) replacing the hGH step elution with a short wash of buffer A alone Except for this, phagemid binding, elution and propagation were performed in consecutive rounds on phagemid from both pLIB2 and pLIB3 as described above. In some cases, XL1-Blue cells were used for phagemid propagation.

An additional digestion of the dsDNA with KpnI was performed on pLIB 2G ³ and pLIB 3G ⁵ prior to the final round of bead binding selection (FIG. 3).

Selected phagemid DNA sequencing LIB 4G ⁴ from the four independently isolated clones and four independently isolated clones from LIB 5G ^6, was sequenced by dideoxy sequencing. All of these eight clones have identical DNA sequences:
hGH codon: 172 174 176 178
5'-AAG GTC TCC ACA TAC CTG AGG ATC-3 '
Had. That is, all of them encode the same mutant of hGH (E174S, F176Y). Residue 172 in these clones is Lys as in the wild type. Also, the codon selected for 172 is identical to wild type hGH. This is not surprising since AAG is the only lysine-codon possible from the set of synonymous "NNS" codons. In addition, residue 178-Arg is also identical to wild-type, except that the codon selected from the library was AAG and not the CGC found in wild-type hGH (the latter codon was also a set of "NNS" codons). Is possible).

Multiplicity of K07 infection The multiplicity of infection of K07 infection is an important parameter in the growth of recombinant phagemid. The multiplicity of infection of K07 must be high enough to ensure that virtually all cells transformed or transfected with the phagemid can package new phagemid particles. In addition, the concentration of wild-type gene III in each cell is maintained high, reducing the likelihood of multiple hGH-gene III fusion molecules being displayed on each phagemid particle, thereby reducing chelation at binding. Should be reduced. However, when the multiplicity of infection of K07 is too high, packaging of K07 will compete with packaging of the recombinant phagemid. We have found that acceptable phagemid yields with only 1-10% background K07 phage are obtained when the multiplicity of infection of K07 is 100.

Table IV
　　　　　　　　　　　　　　　　　　　　　　 　　　　　　　　　　　　　
Phage moi (K07) enriched hGHbp / fraction KpnI
Pool CFU / PFU BSA beads
LIB1 1000 ND 14 0.44
LIB1G 1000 ND 30 0.57
LIB3 100 ND 1.7 0.26
LIB3G ³ 10 ND 8.5 0.18
LIB3G ⁴ 100 460 220 0.13
LIB5 100 ND 15 ND
LIB2 100 ND 1.7 <0.05
LIB2G 10 ND 4.1 <0.10
LIB2G ² 100 1000 27 0.18
LIB4 100 170 38 ND

The phage pool was labeled as previously shown (Figure 3). Multiplicity of infection (moi) means the multiplicity of K07 infection (PFU / cell) during phagemid propagation. Enrichment of CFU over PFU is shown when purified K07 is added to the coupling step. The ratio of CFU eluted from hGHbp beads over CFU eluted from BSA beads is shown. Fractionation of the KpnI-containing template (ie, pH0415) remaining in the pool is performed by digesting dsDNA with KpnI and EcoRI, running the product on a 1% agarose gel, and laser-scanning negative ethidium bromide stained DNA. Was measured by:

Affinity for receptor binding of the hormones hGH (E174S, F176Y) The fact that a single clone was isolated from two different alternative pathways (FIG. 3) indicates that the double mutant (E174S, F176Y) has an hGHbp Suggested that the binding is strong. To determine the affinity of this hGH mutant for hGHbp, we constructed this hGH mutant by site-directed mutagenesis using plasmid pB0720. This plasmid contains the wild-type hGH gene as a template, and the following oligonucleotides that change codons 174 and 176:
hGH codon: 172 174 176 178
Lys Ser Tyr Arg
5'- ATG GAC AAG GT G TC G ACA T A C CTG CGC ATC GTG -3 '
It contains. The resulting construct, pH0458B, was introduced into E. coli strain 16C9 for expression of the mutant hormone. Scatchard analysis of the competitive binding of hGH (E174S, F176Y) and ¹²⁵ I-hGH to hGHbp shows that this (E174S, F176Y) mutant has a binding affinity at least 5.0-fold stronger than that of wild-type hGH. It was found to have the property.

Example VIII Selection of hGH Variants from Helix 4 Random Cassette Library of Hormone-Phage Human growth hormone variants were prepared by the method of the invention using the phagemid described in FIG.

Construction of De-Fusionable Hormone-Phage Vectors We (1) improve the binding between hGH and the gene III part to make display of the hGH part more advantageous on phage; (2) substantially To limit the expression of the fusion protein to obtain a "monovalent representation"; (3) allow selection of restriction nucleases relative to the starting vector; (4) eliminate expression of the fusion protein from the starting vector; and (5) Cassette mutagenesis [Wells et al., Gene 34: 315-323 (1985)] and hGH with the aim of achieving easy expression of the corresponding free hormone from certain hGH-gene III fusion mutants. -A vector was designed for the expression of the gene III fusion protein.

It contains the pBR322 and f1 origins of replication, and is under the control of the E. coli phoA promoter [Bass et al., Proteins 8: 309-314 (1990)] under the control of the hGH-gene III fusion protein (hGH residues 1-191, one to Plasmid by oligonucleotide-directed mutagenesis of pS0132 (Kunkel et al., Methods Enzymol. 154: 367-382 (1987)) expressing a Gly residue followed by fusion to Pro-198 of gene III) . pS0643 was constructed (FIG. 9). Oligonucleotides:
5'-GGC-AGC-TGT-GGC-T TC-TAG-A GT-GGC-GGC-GGC-TCT-GGT-3 '
[This oligonucleotide introduces an XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH]. In the resulting construct, pS0643, a portion of gene III has been deleted, resulting in two silent mutagenesis (underlined), creating the following junction between hGH and gene III:
--- hGH ---------> Gene III ----->
187 188 189 190 191 am * 249 250 251 252 253 254
Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly
GGC AGC TGT GG A TTC TAG AGT GG C GGT GGC TCT GGT
This reduces the overall size of the fusion protein from 401 residues in pS0132 to 350 residues in pS0643. Experiments with monoclonal antibodies to hGH have shown that the hGH portion of the new fusion protein assembled into phage particles is more accessible than previous long fusions.

To grow phage displaying hormones, pS0643 and derivatives can be grown in E. coli amber-suppressor strains such as JM101 or XL1-Blue [Bullock et al., BioTechniques 5: 376-379 (1987)]. Shown above is a Glu substitution at the amber codon that occurs in the supE suppressor strain. Suppression by other amino acids is also possible in various well-known and commonly available E. coli strains.

P To express hGH (or mutant) that does not contain the gene III portion of the fusion, pS0643 and derivatives can simply be grown on non-suppressor strains such as 16C9. In this case, the amber codon (TAG) leads to the termination of translation and provides free hormone without the need for independent DNA assembly.

To create a site for cassette mutagenesis, pS0643 was modified with the oligonucleotide:
(1) 5'-CGG-ACT-GGG-C AG-ATA-T TC-AAG-CAG-ACC-3 '
[This destroys the unique BglII site of pS0643];
(2) 5'-CTC-AAG-AAC-TAC-G GG-TTA-CC C-TGA-CTG-CTT-CAG-GAA-GG-3 '
[This inserts a unique BstEII site, a one base frameshift, and a non-amber stop codon (TGA)]; and
(3) 5'-CGC-ATC-GTG-CAG-TGC- AGA-TCT -GTG-GAG-GGC-3 '
[This introduces a new BglII site]
To give the starting vector pH0509. Adding a frameshift with a TGA stop codon ensures that no Gene III fusion can be produced from the starting vector. The BstEII-BglII segment was excised from pH0509 and replaced with a mutated DNA cassette at the desired codon. Other restriction sites for cassette mutagenesis at other positions in hGH were also introduced into this hormone-phage vector.

Cassette Mutagenesis in hGH 4 of hGH Codons 172, 174, 176 and 178 of hGH were targeted for random mutagenesis. The reason for this is that all are present at or near the surface of hGH and contribute significantly to receptor binding [Cunningham and Wells, Science 244: 1081-1085 (1989)]; all of these are well-defined structures. And occupies two "turns" on the same side of helix 4; and because each of these has been replaced with at least one amino acid in a known evolutionary variant of hGH.

We chose the substitution of NNS (N = A / G / C / T; S = G / C) for each target residue. The selection of this NNS synonymous sequence yields 32 possible codons (codons for at least one each amino acid) at four sites, for a total of (32) ⁴ = 1,048,576 possible nucleotide sequences Or (20) ⁴ = 160,000 possible polypeptide sequences are obtained. The only stop codon, amber (TAG), is possible through this codon selection, and this codon can be suppressed as Glu1 in the supE strain of E. coli.

Two synonymous (degenerate) oligonucleotides with NNS at codons 172, 174, 176 and 178 were synthesized, phosphorylated and annealed to construct a mutation cassette:
5'-GT-TAC-TCT-ACT-GCT-TTC-AGG-AAG-GAC-ATG-GAC-NNS-GTC-NNS-ACA-NNS-CTG-NNS-ATC-GTG-CAG-TGC-A-3 '
And 5'-GA-TCT-GCA-CTG-CAC-GAT-SNN-CAG-SNN-TGT-SNN-GAC-SNN-GTC-CAT-GTC-CTT-CCT-GAA-GCA-GTA-GA-3 ' .
The vector was prepared by digesting pH0509 with BstEII followed by BglII. The product was run on a 1% agarose gel, the large fragment was excised, phenol extracted and ethanol precipitated. This fragment was treated with bovine intestinal phosphatase (Boehringer), then phenol: chloroform extracted, ethanol precipitated and resuspended for ligation with the mutation cassette.

After growth ligation of the first library in XL1-Blue cells , the reaction product was digested once more with BstEII, then phenol: chloroform extracted, ethanol precipitated, and resuspended in water [BstEII recognition site (GGTNACC). and the G and 174 to the 3-position of codon 172 is created in a cassette containing the ACC (Thr) codon. However, treatment with BstEII at this step should not select for all possible mutation cassettes. This is because virtually all cassettes are heteroduplex and cannot be cleaved by this enzyme]. About 150 ng (45 fmoles) of DNA was applied to XL1-Blue cells (1.8 × 10 ⁹ cells in 0.045 ml) in a 0.2 cm cuvette with a single pulse voltage setting of 2.49 kV (time constant = 4.7 m). Seconds), electroporation was introduced.

The cells were allowed to recover for 1 hour at 37 ° C. in SOC medium with shaking and then mixed with 2YT medium (25 ml), 100 mg / ml carbenicillin, and M13-K07 (moi = 100). After 10 minutes at 23 ° C., the culture was incubated overnight (15 hours) at 37 ° C. with shaking. By plating serial dilutions from this culture on a medium containing carbenicillin, it was found that 3.9 × 10 ⁷ electrotransformants were obtained.

After overnight incubation, cells were pelleted and double-stranded DNA (dsDNA), designated pH0529E (first library), was prepared by the alkaline lysis method. The supernatant was centrifuged once more to remove all remaining cells, and the phage named phage pool φH0529E (the first phage library) was PEG precipitated, and STE buffer [10 mM Tris (pH 7.6), 1 mM EDTA, 50 mM). NaCl] (1 ml). Phage titers were measured as colony forming units (CFU) for recombinant phagemid containing hGH-g3p. Approximately 4.5 × 10 ¹³ CFU was obtained from the starting library.

Synonym of departure library (degenerate)
Fifty-eight clones from the pool of electrotransformants were sequenced in the region of the BstEII-BglII cassette. Of these, 17% correspond to the starting vector, 17% contain at least one frameshift, and 7% have non-silent (non-stop) mutations outside the four target codons. Contained. We concluded that 41% of the clones had drawbacks from any of the above criteria, leaving a total functional pool of 2.0 × 10 ⁷ original transformants. Nevertheless, this number almost exceeds the number of possible DNA sequences by a factor of 20. Therefore, we are convinced that we have all the possible sequences shown in the starting library.

We examined the sequences of unselected phage to assess the degree of codon bias in mutagenesis (Table V). Although these results indicate that some codons (and amino acids) are more or less than random predictions, this library is quite diverse and evidence of a large amount of "brother" degeneracy Was found not to be present (Table VI).

Table V. Codon distribution (per 188 codons) of unselected hormone phage clones were sequenced from the starting library (pH0529E). All codons, including those from clones containing pseudomutations and / or frameshifts, were tabulated. *: Amber stop codon (TAG) is suppressed as Glu in XL1-Blue cells. Underlined codons were shown to be more / less than 50% or more.
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Residue Expected number Actual number Actual measurement / expected
Leu 17.6 18 1.0
Ser 17.6 26 1.5
Arg 17.6 10 0.57
Pro 11.8 16 1.4
Thr 11.8 14 1.2
Ala 11.8 13 1.1
Gly 11.8 16 1.4
Val 11.8 4 0.3
Ile 5.9 2 0.3
Met 5.9 1 0.2
Tyr 5.9 1 0.2
His 5.9 2 0.3
Trp 5.9 2 0.3
Phe 5.9 5 0.9
Cys 5.9 5 0.9
Gln 5.9 7 1.2
Asn 5.9 14 2.4
Lys 5.9 11 1.9
Asp 5.9 9 1.5
Glu 5.9 6 1.0
Amber * 5.9 6 1.0

Table VI . Not selected with open reading frame
(PH0529E) Clone designation, eg, TWGS, indicates hGH mutant 172T / 174W / 176G / 178S. The amber (TAG) codon translated as Glu in XL1-Blue cells is indicated by ε.
　　　　　　　　　　　　　　　　　　　　　　　　
KεNT KTEQ CVLQ
TWGS NNCR EASL
PεER FPCL SSKE
LPPS NSDF ALLL
SLDP HRPS PSHP
QQSN LSLε SYAP
GSKT NGSK ASNG
TPVT LTTE EANN
RSRA PSGG KNAK
LCGL LWFP SRGK
TGRL PAGS GLDG
AKAS GRAK NDPI
GNDD GTNG

Preparation of immobilized hGHbp and hPRLbp Reference [Bass et al., Proteins 8: 309-314 (1990), except that wild-type hGHbp [Fuh et al . , J. Biol. Chem. 265: 3111-3115 (1990)] was used. ], Immobilized hGHbp ("hGHbp-beads") was prepared. Competition binding experiments with [ ¹²⁵ I] hGH showed that 58 fmol of functional hGHbp bound per 1 μl of bead suspension.

Immobilized hPRLbp ("hPRLbp-beads") was prepared as described above using the 211-cell extracellular domain of the prolactin receptor [Cunningham et al., Science 250: 1709-1712 (1990)]. Competitive binding experiments with [ ¹²⁵ I] hGH in the presence of 50 μM zinc showed that 2.1 fmol of functional hPRLbp bound per 1 μl of bead suspension.

"Blank beads" were prepared by treating oxirane-acrylamide beads with 0.6M ethanolamine (pH 9.2) at 4 ° C for 15 hours.

Binding of hormone-phage to binding selection beads using immobilized hGHbp and hPRLbp was performed in any of the following buffers: Buffer A [PBS, 0.5% for selection using hGHbp and blank beads. BSA, 0.05% Tween 20, 0.01% Thimerosal]; Buffer B [50 mM Tris (pH 7.5), 10 mM MgCl ₂ , 0 for selection using hPRLbp in the presence of zinc (+ Zn ²⁺ ). Buffer C [PBS, 0.5% BSA, 0.5% BSA, 0.5% BSA, 0.5% Tween 20, 100 mM ZnCl ₂ ]; or, for selection using hPRLbp in the absence of zinc (+ EDTA). 05% Tween 20, 0.01% Thimerosal, 10 mM EDTA]. Binding selections were performed according to each of the following routes: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+ Zn ²⁺ ), (4) binding to hPRLbp-beads. Binding (+ EDTA), (5) two preadsorptions with hGHbp beads followed by binding of unadsorbed fraction to hPRLbp-beads (selection of "-hGHbp, + hPRLbp"), or (6) twice with hPRLbp-beads Preadsorption and subsequent binding of the unadsorbed fraction to hGHbp-beads (selection of "-hPRLbp, + hGHbp"). The latter two operations are expected to enrich mutants that bind to hPRLbp but do not bind to hGHbp, or mutants that bind to hGHbp but do not bind to hPRLbp. Phage binding and elution in each cycle was performed as follows:

1. Binding: A portion of the hormone phage (usually 10 ⁹ to 10 ¹⁰ CFU) is mixed with an equal volume of non-hormone phage (pCAT), diluted with an appropriate buffer (A, B or C), and In a polypropylene test tube, a total volume of 200 ml was mixed with a suspension of hGHbp, hPRLbp or blank beads (10 ml). The phage were bound to the beads by incubating for 1 hour at room temperature (23 ° C.) with slow rotation (about 7 rpm). The following steps were performed at room temperature using a constant volume of 200 μl.

{2. Washing: The beads were spun for 15 seconds and the supernatant was removed. To reduce the number of non-specifically bound phage, the beads were washed five times by brief resuspension in a suitable buffer and then pelleted.

{3. hGH elution: Phage that bound weakly to the beads were removed by elution with hGH. The beads were spun with the appropriate buffer containing 400 nM hGH for 15-17 hours. This supernatant was used as “hGH eluate” and beads. The beads were washed briefly by resuspending in buffer and pelleting.

4. Glycine elution: To remove the most tightly bound phage (ie, phage still bound after the hGH wash), the beads were washed with glycine buffer [buffer A was supplemented with 0.2M glycine and pH was adjusted to 2.0 with HCl. And rotated for 1 hour to pelletize. This supernatant ("glycine eluate"; 200 μl) was neutralized by the addition of 1 M Tris base (30 ml) and stored at 4 ° C.

5. Proliferation: Under each set of conditions, a portion of the hGH eluate and glycine eluate from each set of beads was used to infect an independent culture of exponentially growing XL1-Blue cells. Transduction was performed by mixing the phage with XL1-Blue cells (1 ml), incubating at 37 ° C. for 20 minutes, and then adding K07 (moi = 100). Cultures (25 ml of 2YT and carbenicillin) were grown as described above and the next pool of phage was prepared as described above.

Phage binding, elution and propagation were performed in consecutive rounds according to the cycle described above. For example, phage amplified from hGH eluate from hGHbp beads was selected once more with hGHbp beads, eluted with hGH, and then used to infect a fresh culture of XL1-Blue cells. Five rounds of selection and three rounds of expansion were performed for each of the above selection methods.

DNA sequencing of selected phagemids A portion of the phage from the hGH and glycine elution steps of each cycle was used to inoculate XL1-Blue cells, which were plated on LB medium containing carbenicillin and tetracycline to derive from each phage pool. Independent clones were obtained. Single-stranded DNA was prepared from the isolated colonies, and the mutation cassette region was sequenced. The results of this DNA sequencing are summarized in FIG. 5, FIG. 6, FIG. 7, and FIG. 8 as deduced amino acid sequences.

DNA from sequenced clones was introduced into E. coli strain 16C9 to determine the expression of hGH mutants and the binding affinity of some of the selected hGH mutants to the assay hGHbp. As mentioned above, this strain is a non-suppressor strain and terminates protein translation after the last Phe-191 residue of hGH. Although single-stranded DNA was used for these transformations, even double-stranded DNA or whole phage can be easily electroporated into non-suppressor strains for free hormone expression.

Mutants of hGH were prepared from cells osmotically shocked by ammonium sulfate precipitation as described for hGH [Olson et al., Nature 293: 408-411 (1981)]. The protein concentration was measured by laser densitometry on a Coomassie-stained SDS-polyacrylamide gel electrophoresis gel using hGH as a standard [Cunningham and Wells, Science 244: 1081-1085 (1989)].

Binding affinity of each mutant, anti - using receptor monoclonal antibody (MAb 263), literature [Spencer et al., J.Biol.Chem 263:. 7862-7867 (1988 ); Fuh et al., J.Biol.Chem . 265: 3111-3115 (1990)] was determined by replacing the ¹²⁵ I hGH as described.

結果 The results of the number of hGH mutants selected by different routes (Figure 6) are shown in Table VII. Many of these mutants have a stronger binding affinity for hGHbp than wild-type hGH. The most improved mutant, KSYR, has a 5.6-fold higher binding affinity than wild-type hGH. The weakest mutant selected among these tested mutants had only about 10-fold lower binding affinity compared to hGH.

Binding assays can also be performed on mutants selected for hPRLbp-binding.

Table VII . Competitive binding to hGHbp Selected pools in which each mutant was found were identified as 1G (first glycine selection), 3G (third glycine selection), 3H (third hGH selection), 3 * (Third selection: did not bind to hPRLbp but did bind to hGHbp). The number of each mutant appearing in all sequenced clones is shown in parentheses.
　　　　　　　　　　　　　　　　　　　　　　 　　　　　　　　　　　　　
Mutant Kd (nM) Kd (mutant) Pool
/ Kd (hGH)
KSYR (6) 0.06 + 0.01 0.18 1G, 3G
RSFR 0.10 + 0.05 0.30 3G
RAYR 0.13 + 0.04 0.37 3 *
KTYK (2) 0.16 + 0.04 0.47 H, 3G
RSYR (3) 0.20 + 0.07 0.58 1G, 3H, 3G
KAYR (3) 0.22 + 0.03 0.66 3G
RFFR (2) 0.26 + 0.05 0.76 3H
KQYR 0.33 + 0.03 1.0 3G
KEFR = wt (9) 0.34 + 0.05 1.0 3H, 3G, 3 *
RTYH 0.68 + 0.17 2.0 3H
QRRYR 0.83 + 0.14 2.5 3 *
KKYK 1.1 +0.4 3.2 3 *
RSFS (2) 1.1 +0.2 3.3 3G, *
KSNR 3.1 +0.4 9.2 3 *

Additive and non-additive effects on binding At some residues, substitution of a particular amino acid has substantially the same effect, independent of surrounding residues. For example, substitution of F176Y in the background of 172R / 174S reduces binding affinity by 2.0-fold (RSFR vs. RSYR). Similarly, the binding affinity of the F176Y mutant (KAYR) in the 172K / 174A background is 2.9-fold weaker than the corresponding 176F mutant [KAFR; Cunningham and Wells (1989)].

On the other hand, the binding constants measured for some selected hGH mutants show the non-additive effects of some amino acid substitutions at residues 172, 174, 176 and 178. For example, in the 172K / 176Y background, substitution of E174S results in a mutant (KSYR) that binds hGHbp 3.7-fold more tightly than the corresponding mutant containing E174A (KAYR). . However, in the 172R / 176Y background, the effects of these E174 substitutions are reversed. In this case, the E174A mutant (RAYR) binds 1.5-fold more tightly than the E174S mutant (RSYR).

Such non-additive effects on the binding of substitutions at adjacent residues indicate the use of protein-phage binding selection as a means to select optimal mutants from a randomized library at several positions. Things. In the absence of detailed structural information, and without such a selection method, a large number of substitution combinations would be tried before finding the optimal mutant.

Example IX Selection of hGH Mutants from Helix 1 Random Cassette Library of Hormone-Phage

Using the method described in Example VIII, another region of hGH involved in binding to hGHbp and / or hPRLbp, i.e., residues 10, 14, 18, 21 of helix 1, was cloned into the phGHam-g3p vector (pS0643). (See Example VIII).

We chose to use the "amber" hGH-g3 construct (referred to as phGHam-g3p) because it appears to make the target protein hGH more accessible for binding. This is supported by data from a comparative ELISA assay for monoclonal antibody binding. Phages generated from both pS0132 [S. Bass, R. Greene, JA Wells, Proteins 8: 306 (1990)] and phGHam-g3 are known to have binding determinants near the carboxy terminus of hGH. Three antibodies (Medix2, 1B5.G2, and 5B7.C10) [BCCunningham, P. Jhurani, P.Ng, JAWells, Science 243: 1330 (1989); BCCunningham and JAWells, Science 244: 1081 (1989); L Jin and J. Wells (unpublished results)] and one antibody (Medix1) recognizing determinants in helices 1 and 3 [BCCunningham, P. Jhurani, P.Ng, JA Wells, Science 243: 1330] (1989); BCCunningham and JA Wells, Science 244: 1081 (1989)]. Phagemid particles from phGHam-g3 reacted more strongly with antibodies Medix2, 1B5.G2 and 5B7.C10 than phagemid particles from pS0132. In particular, the binding of pS0132 particles was reduced> 2000-fold for both Medix2 and 5B7.C10 and> 25-fold for 1B5.G2 compared to binding to Medix1. On the other hand, the binding of the phGHam-g3 phage is about 1.5-fold, 1.2-fold and 2.3-fold weaker against the Medix2, 1B5.G2 and 5B7.C10 antibodies, respectively, compared to the binding to Medix1. It was not too much.

Construction of a Helix 1 Library by Cassette Mutagenesis Alanine-scanning mutagenesis [BCCunningham, P. Jhurani, P. Ng, JA Wells, Science 243: 1330 (1989); BCCunningham and JA Wells, Science 244: 1081 (1989), Mutations were made to the residues in helix 1 previously identified by H.15, 16] to modulate the binding of the extracellular domains of the hGH and / or hPRL receptors (designated hGHbp and hPRLbp, respectively). . Cassette mutagenesis was performed essentially as described in the literature [JAWells, M. Vasser, DBPowers, Gene 34: 315 (1985)]. This library contains oligonucleotides:
5'-GCC TTT GAC A GG TAC C AG GAG TTT G-3 'and 5'-CCA ACT ATA CCA CT C TCG AG G TCT ATT CGA TAA C-3'
PhGHam-g3 [TAKunkel, JDRoberts, RAZakour, Methods Enzymol. 154] with unique KpnI (at hGH codon 27) and XhoI (at hGH codon 6) restriction sites (underlined) inserted by mutagenesis, respectively . : 367-382], using the mutagenesis of all four residues at once (see Example VIII). The latter oligo also introduced a +1 frameshift (the last G underlined), which terminated translation from the starting vector and minimized wild-type background in the phagemid library. This starting vector was named pH0508B. A helix 1 library in which hGH residues 10, 14, 18, 21 have been mutated contains complementary oligonucleotides:
5'-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG
CTG GCC TTT GAC ACG TAC-3 'and 5'-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT
GTC SNN GAG CC-3 '
Was prepared by ligating to the large XhoI-KpnI fragment of pH0508B. This KpnI site was broken at the junction of the ligation product, making it possible to use restriction enzyme digestion for analysis of the non-mutated background.

This library contains at least 10 ⁷ separate transformants, and when the library is completely random (10 ⁶ different codon combinations), on average about 10 copies of each possible mutant hGH The gene is obtained. Restriction analysis using KpnI showed that at least 80% of the helix 1 library construct contained the inserted cassette.

結合 Enrichment of hGH-phage binding from this library was performed using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described previously (Example VIII). Four residues in helix 1 (F10, M14, H18 and H21) were similarly mutated, resulting in a non-wild-type consensus after 4 and 6 cycles (Table VIII). Position 10 on the hydrophobic side of helix 1 tends to be hydrophobic, whereas positions 21 and 18 on the hydrophilic side are predominantly Asn dominant and no apparent consensus was found at position 14. (Table IX).

The binding constants of these hGH mutants to hGHbp are determined by expressing the free hormone mutant in non-suppressor E. coli strain 16C9, purifying the protein and competing displacement of labeled wt-hGH from hGHbp. (See Example VIII). As shown, some mutants bind more strongly to hGHbp than do wt-hGH.

Table VIII . Selection of hGH helix 1 mutant hGH variants expressed on phagemid particles (random mutations at residues F10, M14, H18, H21) were bound to hGHbp-beads and buffered with hGH (0.4 mM). And then by elution with glycine (0.2M, pH2) buffer (see Example VIII).
　　　　　　　　　　　　　　　　　　　　　　　　　　
Gly elution F10 M14 H18 H21
4 cycles
H G N N
A W D N (2)
Y TV N
ININ
L N SH
FSFG
6 cycles
H G N N (6)
F S FL
Consensus H G N N

Table IX . Consensus sequence from selected helix 1 library The frequency observed is the fraction of all sequenced clones with the indicated amino acids. Nominal frequencies were calculated based on the 32 codon degeneracy of the NNS. The maximum enrichment factor varies from 11 to 32 depending on the nominal frequency value for a residue. The value of [Kd (Ala mutant) / Kd (wt-hGH)] for a single alanine mutation is given in the literature [BCCunningham and JAWells, Science 244: 1081 (1989); BCCunningham, DJ Henner, JAWells, Science 247: 1461 (1990); BCCunningham and JA Wells, Proc. Natl. Acad. Sci. USA 88: 3407 (1991)].
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Wild-type Kd (Ala mutant) Selected frequency
Residue / Kd (wt-hGH) residue observation nominal enrichment F10 5.9 H 0.50 0.031 17
F 0.14 0.031 5
A 0.14 0.0622 2
M14 2.2 G 0.50 0.062 8
W 0.14 0.031 5
N 0.14 0.031 5
S 0.14 0.093 2
H18 1.6 N 0.50 0.031 17
D 0.14 0.031 5
F 0.14 0.031 5
H21 0.33 N 0.79 0.031 26
H 0.07 0.031 2

Table X. Binding of Purified hGH Helix 1 Mutant to hGHbp Competitive binding experiments were performed with [ ¹²⁵ I] hGH (wild type), hGHbp (extracellular receptor domain, including residues 1-238), and Mab263 [BCCunningham, P. Jhurani, P. Ng, JAWells, Science 243: 1330 (1989)]. The numerical value P indicates, as a fraction, the incidence of each mutant in all clones sequenced after one or more selection rounds.
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Sequence position P Kd (nM) ＼f (Kd mutant) Kd (wt-hGH)
10 14 18 21
H G N N 0.50 0.14 ± 0.04 0.42
A W D N 0.14 0.10 ± 0.03 0.30
wt = FMHH 0.34 ± 0.05 (1)
FSFL 0.07 0.68 ± 0.19 2.0
YTVN 0.07 0.75 ± 0.19 2.2
L N SH 0.07 0.82 ± 0.20 2.4
ININ 0.07 1.2 ± 0.31 3.4

Example X Selection of hGH Mutants from a Helix 4 Random Cassette Library Containing Mutations Found Previously by Hormone-Phage Enrichment
Designing muteins with improved binding by repeated selection using hormone-phage From our experiments with new non-binding homologues of hGH evolution variants identified by combining multiple independent amino acid substitutions It is suggested that cumulative improved mutants of hGH for binding to the receptor can be obtained [BCCunningham, DJ Henner, JA Wells, Science 247: 1461 (1990); BCCunningham and JA Wells, Proc. Natl. Acad. Sci. USA 88: 3407 (1991); HB Lowman, BCCunningham, JA Wells, J. Biol. Chem. 266, Printing (1991)].

In an attempt to further improve a helix 4 double mutant (E174S / F176Y; see Example VIII) selected from a helix 4a library found to bind more tightly to the hGH receptor, the helix 4b library It was created. Mutations were made to residues around positions 174 and 176 on the hydrophilic surface of helix 4, using the E174S / F176Y hGH mutant as the background starting hormone.

Construction of Helix 4b Library by Cassette Mutagenesis Cassette mutagenesis was performed essentially as described in the literature [JAWells, M. Vasser, DBPowers, Gene 34: 315 (1985)]. Using a mutant of phGHam-g3 (Example VIII) in which only one BstEII and BglII restriction site has been inserted, using cassette mutagenesis to mutate all four residues at once (see Example VIII) A helical 4b library was created in which residues 167, 171, 175 and 179 were mutated in the E174S / F176Y background. Complementary oligonucleotide:
5'-pG TTA CTC TAC TGC TTC NNS AAG GAC ATG NNS AAG GTC AGC NNS TAC
CTG CGC NNS GTG CAG TGC A-3 'and 5'-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC CTT SNN CAT GTC
CTT SNN GAA GCA GTA GA-3 '
Was inserted into the BstEII-BglII site of the vector. This BstEII site was deleted in the ligated cassette. Fifteen unselected clones from this helix 4b library were sequenced. Of these, none lacked the cassette insert, 20% were frameshifted and 7% had non-silent mutations.

Results of hGHbp enrichment Binding enrichment of hGH-phages from this library was performed using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described previously (Example VIII). Was. After 6 cycles of binding, a reasonable and clear consensus emerged (Table XI). Interestingly, all positions tended to contain polar residues, especially Ser, Thr and Asn (Table XII).

Assay of hGH mutants The binding constants of some of these hGH mutants to hGHbp were determined by expressing the free hormone mutant in non-suppressor E. coli strain 16C9, purifying the protein, and labeling the labeled wt from hGHbp. Determined by assaying by competitive displacement of -hGH (see Example VIII). As shown, the binding affinities of some helix 4b mutants for hGHbp were stronger than those of wt-hGH (Table XIII).

hGH Variant Receptor Selectivity Finally, we began the analysis of the binding affinities of some more robust hGHbp binding mutants for their ability to bind to hPRLbp. The E174S / F176Y mutant binds 200 times weaker to hPRLbp than hGH. Each of the E174T / F176Y / R178K and R167N / D171S / E174S / F176Y / I179T mutants binds> 500-fold weaker to hPRLbp than hGH. That is, a novel receptor-selective mutant of hGH can be obtained by the phage display method.

Information content of specific residues identified by hormone-phagemid selection Of the 12 residues mutated in the three hGH-phagemid libraries (Examples VIII, IX, X), four were wild-type residues It exhibited strong (but not exclusive) conservation (K172, T175, F176 and R178). Not surprisingly, there were residues that when converted to Ala caused the greatest disruption (4-60 fold) in binding affinity for hGHbp. There is a group of four other residues (F10, M14, D171 and I179) where the Ala substitution causes a weaker effect on binding (2-7 fold) and these positions have only a small wild type consensus. Not shown. Finally, another four residues (H18, H21, R167 and E174) that promoted binding to hPRLbp but not hGHbp were identified by the selection with hGHbp-beads against the wild-type hGH sequence. No such consensus was shown. In fact, two residues (E174 and H21) increase binding affinity for hGHbp by a factor of 2 to 4 when substituted for Ala [BCCunningham, P. Jhurani, P. Ng, JA Wells, Science 243: 1330 (1989). BCCunningham and JAWells, Science 244: 1081 (1989); BCCunningham, DJ Henner, JAWells, Science 247: 1461 (1990); BCCunningham and JAWells, Proc. Natl. Acad. Sci. USA 88: 3407 (1991)]. That is, the alanine-scanning mutagenesis data is reasonably related to the variability of replacing each position. In fact, the reduced binding affinity caused by alanine substitution [BCCunningham, P. Jhurani, P. Ng, JA Wells, Science 243: 1330 (1989); BCCunningham and JA Wells, Science 244: 1081 (1989); BCCunningham, DJ Henner, JA Wells. USA 1988, Science 247: 1461 (1990); BCCunningham and JA Wells, Proc. Natl. Acad. Sci. USA 88: 3407] show the percentage of wild-type residues found in the phagemid pool after 3-6 rounds of selection. Is reasonably predicted. Alanine-scanning information is useful for targeting side chains that modulate binding, and phage selection can be used to optimize side chains and the variability of each site (and / or combination of sites) for substitution. Suitable for defining The combination of scanning mutagenesis (BCCunningham, P. Jhurani, P. Ng, JA Wells, Science 243: 1330 (1989); BCCunningham and JA Wells, Science 244: 1081 (1989)) and phage display provides And a powerful tool for studying molecular evolution in vitro.

Mutations in the Repeated Enrichment of the Hormone-Phagemid Library Known from hormone-phagemid enrichment to improve binding if the combination mutation in hGH has an additive effect on binding affinity for the receptor The resulting mutations can be combined simply by cleavage and ligation or mutagenesis of restriction fragments to yield cumulatively optimized mutants of hGH.

On the other hand, mutations in one region of hGH that optimize receptor binding may be structurally or functionally incompatible with mutations in overlapping or different regions of the molecule. In such cases, hormone-phagemid enrichment can be achieved by any of several variations of the iterative enrichment methods listed below:
(1) Random DNA libraries can be created in each of two (or possibly more) regions of the molecule by cassettes or other mutagenesis methods: then the restriction from these two DNA libraries Mixed libraries can be created by ligation of fragments; (2) hGH variants that have been optimized for binding by mutations in one region of the molecule can be linked to the second of the molecule, as in the example of the helix 4b library. (3) Two or more random libraries can be partially selected for improved binding by hormone-phagemid enrichment: this optimized binding After "coarse selection" of the sites, still another partial library was Remixing can result in a single library that is partially different in two or more regions of the molecule, which is then further selected for optimized binding using hormone-phagemid enrichment can do.

Table XI . Mutated phagemids of hGH selected from helix 4b library after 4 and 6 cycles of enrichment hGH helix 4b mutants each containing the E174S / F176Y double mutation (random at residues 167,171,175,179) Mutations) were selected by binding to hGHbp-beads and elution with hGH (0.4 mM) buffer followed by glycine (0.2 M, pH2) buffer. One mutant (+) contained the pseudomutation R178H.

　　　　　　　　　　　　　　　　　　　　　　　　　　　
R167 D171 T175 I179
4 cycles
N STT
KSTT
S N T T
DSTT
DSTT +
DS AT
DSAN
TDTT
NDTN
A NTN
A STT
6 cycles
NSTT (2)
N NTT
NSTQ
D S ST
ESTI
K STL
consensus
N STT
DN

Table XII . Consensus sequences from selected libraries The observed frequency is the fraction of all sequenced clones with the indicated amino acids. Nominal frequencies were calculated based on the 32 codon degeneracy of the NNS. The maximum enrichment factor varies from 11 to 16 to 32 depending on the nominal frequency value for a residue. The value of [Kd (Ala mutant) / Kd (wt-hGH)] for a single alanine mutation was taken from the following literature; for position 175 only the value of the T175S mutant [BCCunningham, P. Jhurani, P. Ng, JAWells, Science 243: 1330 (1989); BCCunningham and JAWells, Science 244: 1081 (1989); BCCunningham, DJ Henner, JAWells, Science 247: 1461 (1990); BCCunningham and JAWells, Proc. Natl . Acad . Sci . USA 88: 3407 (1991)].

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Wild-type Kd (Ala mutant) Selected frequency
Residue / Kd (wt-hGH) residue observation nominal enrichment R167 0.75 N 0.35 0.031 11
D 0.24 0.031 8
K 0.12 0.031 4
A 0.12 0.0622
D171 7.1 S 0.76 0.093 8
N 0.18 0.031 6
D 0.12 0.031 4
T175 3.5 T 0.88 0.062 14
A 0.12 0.031 4
I179 2.7 T 0.71 0.062 11
N 0.18 0.031 6

Table XIII . Binding of Purified hGH Mutants to hGHbp Competitive binding experiments were performed using [ ¹²⁵ I] hGH (wild type), hGHbp (extracellular receptor domain, including residues 1-238), and Mab263 (11). Done. The numerical value P indicates, as a fraction, the incidence of each mutant in all clones sequenced after one or more selection rounds. Note that the helix 4b mutation (*) is in the background of hGH (E174S / F176Y). In this list of helix 4b mutations, the E174S / F176Y mutant (*) with wt residues at 167, 171, 175, 179 is underlined.

　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Sequence location P Kd (nM) Kd (Ala mutant)
/ Kd (wt-hGH)
* * * *
167 171 175 179
NSTTT 0.18 0.04 ± 0.02 0.12
ESTI 0.06 0.04 ± 0.02 0.12
KSTL 0.06 0.05 ± 0.03 0.16
N NTT T 0.06 0.06 ± 0.03 0.17
R D T I 0 0.06 ± 0.01 (0.18)
NSTQ 0.06 0.26 ± 0.11 0.77

Example XI Assembly of Fab Molecules on Phagemid Surface Plasmid Construction Plasmid pDH188 contains DNA encoding the Fab portion of a humanized IgG antibody called 4D5 that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR101 and has been deposited with the ATCC [Rockville, MD].

Briefly, this plasmid was prepared as follows. The starting plasmid is pS0132, containing the alkaline phosphatase promoter described above. The DNA encoding human growth hormone was excised and a series of operations were performed to make the ends of the plasmid compatible for ligation, and then the DNA encoding 4D5 was inserted. This 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain and contains DNA encoding the stII signal sequence at its 5 'end. The second gene contains four parts, the first of which is the DNA encoding the stII signal sequence at the 5 'end. This is followed by the DNA encoding the variable domain of the heavy chain, followed by the DNA encoding the first domain of the heavy chain constant region, followed by the DNA encoding the M13 gene III. The salient features of this construct are shown in FIG. The sequence of the DNA encoding 4D5 is shown in FIG.

Transformation of E. coli and generation of phage Plasmids were introduced into SR101 cells using both polyethylene glycol (PEG) and electroporation. PEG-competent cells were prepared and transformed according to the method of Chung and Miller [ Nucleic Acids Res. 16: 3580 (1988)]. Cells competent for electroporation were prepared according to the method of Zabarovsky and Winberg [ Nucleic Acids Res. 18: 5912 (1990)] and then transformed by electroporation. After inoculating the cells in an SOC medium [described by Sambrook et al. (Supra)] (1 ml), the cells were grown at 37 ° C. for 1 hour with shaking. At this point, cell concentration was measured using OD ₆₀₀ light dispersion. The titrated KO7 phage stock was added to give a multiplicity of infection (MOI) of 100, and the phage was allowed to attach to the cells for 20 minutes at room temperature. This mixture was then diluted in 25 ml of 2YT broth (described by Sambrook et al., Supra) and incubated overnight at 37 ° C. with shaking. The next day, cells were pelleted by centrifugation at 5000 xg for 10 minutes, the supernatant was collected, and phage particles were precipitated with 0.5M NaCl and 4% PEG (final concentration) for 10 minutes at room temperature. The phage particles were pelleted by centrifugation at 10,000 × g for 10 minutes, resuspended in TEN [1 mM Tris (pH 7.6), 1 mM EDTA, and 150 mM NaCl] (1 ml) and stored at 4 ° C.

Preparation of antigen-coated plates 0.5 mg / ml solution of BSA (control antigen) or 0.1 mg / ml solution of extracellular domain (EDC) of HER-2 antigen in 0.1 M sodium bicarbonate (pH 8.5) The wells of a Falcon 12 well tissue culture plate were coated with 0.5 ml aliquots from After applying the solution to the wells, the plate was incubated overnight at 4 ° C. on a rocking platform. The plate was then blocked by removing the first solution, applying blocking buffer [30 mg / ml BSA in 0.1 M sodium bicarbonate] (0.5 ml) and incubating at room temperature for 1 hour. Finally, the blocking buffer was removed, buffer A [PBS, 0.5% BSA, and 0.05% Tween 20] (1 ml) was added, and the plates were incubated at 4 ° C. until used for phage selection. Stored for up to 10 days.

Phage Selection Approximately 10 ⁹ phage particles were mixed with a 100-fold excess of KO7 helper phage and buffer A (1 ml). The mixture was divided into two 0.5 ml portions, one of which was applied to ECD coated wells and the other to BSA coated wells. The plates were incubated for 1-3 hours at room temperature with shaking, then washed three times for 30 minutes each with buffer A (1 ml each). Elution of the phage from the plate was performed at room temperature in one of two ways: (1) a first overnight incubation of 0.025 mg / ml purified Mu4D5 antibody (murine) followed by acid elution buffer. Incubation for 30 minutes with [0.2 M glycine (pH 2.1), 0.5% BSA, and 0.05% Tween 20] (0.4 ml), or (2) incubation with acid elution buffer alone. The eluate was then neutralized with 1 M Tris base and TEN (0.5 ml each) was added. These samples were then grown, titrated and stored at 4 ° C.

Proliferation of phage A portion of the eluted phage was added to 2YT broth (0.4 ml) and mixed with about 10 ⁸ mid-log phase male E. coli strain SR101. Phage were allowed to attach to cells for 20 minutes at room temperature and then added to 5 ml of 2YT broth containing 50 μg / ml carbenicillin and 5 μg / ml tetracycline. The cells were grown for 4-8 hours at 37 ° C. until they reached mid-log phase. The OD ₆₀₀ was measured and the cells were superinfected with KO7 helper phage for phage production. After obtaining the phage particles, they were titrated to determine the number of colony forming units (cfu). This was accomplished by taking a portion of a serial dilution of a phage stock, infecting mid-log phase SR101, and plating on LB plates containing 50 vg / ml carbenicillin.

Measurement of RIA Affinity The affinities of the Fab phage and the h4D5 Fab fragment for the ECD antigen were measured using a competitive receptor-binding RIA [Burt, DR, Receptor Binding in Drug Research , O'Brien, RA (eds.), Pp. 3-29, Dekker, New York (1986)]. This ECD antigen was labeled with ¹²⁵ I using the continuous chloramine-T method [De Larco, JE et al . , J. Cell. Physiol. 109: 143-152 (1981)]. This resulted in a radioactive tracer having a specific activity of 14 μCi / μg into which 0.47 mol of iodine was introduced per mol of receptor. A series of 0.2 ml solutions containing 0.5 ng (by ELISA) of Fab or Fab phage, 50 nCi of ¹²⁵ I-ECD tracer, and a range of unlabeled ECD (6.4 ng-3277 ng) were prepared. For overnight. Labeled ECD-Fab or ECD-Fab phage complexes were separated from unbound labeled antigen by forming an assembly complex induced by the addition of anti-human IgG (Fitzgerald 40-GH23) and 6% PEG8000. The complex was pelleted by centrifugation (15,000 × g for 20 minutes), and the amount of labeled ECD (cpm) was measured with a gamma counter. The dissociation constant (Kd) is determined by an improved version of the program LIgAND using Scatchard analysis [Scatchard, G., Ann. NY Acad. Sci. 51: 660-672 (1949)] [Munson, P. and Rothbard, D., Anal .Biochem. 107: 220-239 (1980)]. This Kd value is shown in FIG.

Competitive Cell Binding Assay The murine 4D5 antibody was labeled with ¹²⁵ I to a specific activity of 40-50 μCi / μg using the rhodogen method. A solution containing a fixed amount of labeled antibody and increasing amounts of unlabeled mutant Fab was prepared and added to almost the entire SK-BR-3 cell culture in a 96-well microtiter dish (final concentration of labeled antibody). Was 0.1 nM). After overnight incubation at 4 ° C., the supernatant was removed, the cells were washed, and the radioactivity bound to the cells was measured with a gamma counter. Kd values were determined by analyzing the data using a modified version of the program LIGAND [Munson, P. and Rothbard, D., supra].

Deposit of plasmid pDH188 (ATCC No. 68663) has been made under the provisions and regulations of the Budapest Treaty on the International Recognition of Microbial Deposits in Patent Procedures (Budapest Treaty). This ensures the maintenance of viable microorganisms for 30 years from the date of deposit. This microorganism is available from the ATCC under the terms of the Budapest Treaty and with the consent of Genentech, Inc. and the ATCC, and has issued the relevant US patents or any US or foreign The earlier of the publication of the patent application, it is guaranteed that the progeny of this culture will be made available permanently and unrestrictedly available to anyone, and that US patents granted under 35 USC §122 It is assured that the descendants will be available to those determined by the Commissioner of Patents and Trademarks and the regulations pursuant thereto (including 37 CFR §1.14 which specifically refers to 886 OG 638).

The assignee of the present application will, upon notification, promptly replace a live sample of the culture when the deposited microorganism is dead, lost, or destroyed when cultured under appropriate conditions. I agree. The availability of a deposited culture should not be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with national patent laws.

It is believed that the above specification is sufficient to enable one skilled in the art to practice the invention. The deposited embodiment is intended as an independent illustration of a particular embodiment of the invention, and the present invention is not limited by the deposited microorganisms, as any functionally equivalent culture is within the scope of the invention. Is not limited. The deposit of materials herein has acknowledged that the statements contained herein are inappropriate to enable the practice of any aspect of the present invention, including the best mode. It is not intended to limit the scope of the claims to that particular example. Indeed, various modifications of the invention in addition to those described and shown herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. It is.

Although the invention has necessarily been described in connection with a preferred embodiment, those skilled in the art after reading the above description will appreciate that various modifications may be made to the subject matter described herein without departing from the spirit and scope of the invention. Changes, equivalent substitutions, and mutations could be made. That is, the present invention can be implemented by methods other than those specifically described in the present specification. Accordingly, the protection afforded by this patent is limited only by the appended claims and equivalents thereof.

Example XII Selection of hGH Mutants from Helix 1 and Helix 4 Hormone-Phage Mutant Combinations
Generation of Additive Mutants of hGH According to the principle of additivity [JA Wells, Biochemistry 29: 8509 (1990)], even when mutations in different parts of the protein do not interact, It is expected that the combination will result in an additive change in the free energy of binding to another molecule (the change is additive in terms of ΔΔG _binding or K d = exp [−ΔG / RT ] Is multiplicative). That is, a mutation that gives a two-fold increase in binding affinity, when combined with a second mutation that causes a three-fold increase, creates a double mutation with a six-fold increased affinity of the starting mutant. It is expected to give.

To test whether the multiple mutations obtained by hGH-phage selection have a cumulatively good effect on hGHbp (hGH-binding protein; extracellular domain of hGH receptor) binding, the helix 1 library was tested. Mutations found in the three most strongly binding mutants of hGH derived (Example IX: F10A / M14W / H18D / H21N, F10H / M14G / H18N / H21N, and F10F / M14S / H18F / H21L) , The three most tightly binding variants found in the helix 4b library (Example X: R167N / D171S / T175 / I179T, R167E / D171S / T175 / I179, and R167N / D171N / T175 / I179T). Combined with mutations.

HGH-phagemid double-stranded DNA (dsDNA) was isolated from each of the 1 helix variants and digested with restriction enzymes EcoRI and BstXI. The larger fragment was then isolated from each helix 4b variant and ligated with the smaller fragment from each helix 1 mutant to yield the novel two-helix variants shown in Table XIII. In addition, all of these mutants contained the E174S / F176Y mutation obtained in the previous hGH-phage binding selection (see Example X for details).

Construction of a Selective Combination Library of hGH The additive principle seems to apply to a large number of mutation combinations, but some combinations (eg, E174S with F176Y) are clearly non-additive ( See Examples VIII and X). For example, to reliably identify the most tightly binding variant with four mutations in helix 1 and four mutations in helix 4, ideally all eight residues should be abruptly changed at once. Mutations would then select the pool of mutants that bind most tightly overall. However, such a pool consists of 1.1 × ^{10 12} DNA sequences (using NNS codon degeneracy) encoding 2.6 × ^{10 10} different polypeptides. Obtaining a random phagemid library large enough to ensure the display of all mutants (probably 10 ¹³ transformants) is not practical with current transformation methods.

We first address this challenge by using sequential rounds of mutagenesis to take the most tightly binding mutations from one library and then mutate the other residues to further improve binding. Worked (Example X). In a second method, we combine the best mutations from two independently selected libraries using the principle of additivity to create multiple mutants with improved binding (Above). Here, we create a combinatorial library of random or partially random mutants, thereby combining possible mutations at positions 10, 14, 18, 21, 167, 171, 175, and 179 in hGH. Was further explored. Pooled phagemids from the helix 1 library (independently 0, 2 or 4 cycle selection; Example IX) and pools from the helix 4b library (independently 0, 2 or 4 cycle selection; Example X) Were used to generate three different combinatorial libraries of hGH-phagemid, and this combinatorial mutant pool was screened for hGHbp binding. Since a certain amount of sequence diversity is present in each of these pools, the resulting combinatorial library can examine more sequence combinations than can be made by hand (eg, Table XIII).

The hGH-phagemid double-stranded DNA (dsDNA) was isolated from each of the 1 helix library pools (0, 2 or 4 round selection) and digested with the restriction enzymes AccI and BstXI. The large fragment from each helix 1 mutant pool was then isolated and ligated with the small fragment from each helix 4b mutant pool, and the three combinatorial libraries pH0707A (unselected unselected as described in Examples IX and X). Helix 1 and helix 4b pool), pH0707B (twice selected helix 1 pool and twice selected helix 4b pool), and pH0707C (four selected helix 1 pool and four selected helix 4b pool) were obtained. In addition, the same ligation was performed with less DNA and was named pH0707D, pH0707E and pH0707F (corresponding to 0, 2 and 4 rounds of starting library, respectively). Also, all of these mutant pools contained the mutation E174S / F176Y obtained from the previous hGH-phage binding selection (see Example X for details).

The selection ligation products pH0707A-F of the combinatorial library of hGH-phage variants were processed and electrotransformed into XL1-Blue cells as described (Example VIII). Based on colony forming units (CFU), the number of transformants obtained from each pool was as follows: pH0707A to 2.4 × 10 ⁶ , pH0707B to 1.8 × 10 ⁶ , pH0707C to 1.6 × 10 ^6. , 4x10 8x10 ^5, 3x10 from pH0707E ^5, and from pH0707F from pH0707D ^5. hGH-phagemid particles were prepared and selected for hGHbp-binding for 2-7 cycles as described in Example VIII.

Rapid selection of hGH-phagemid libraries In addition to screening phagemid libraries for tightly binding protein variants as measured by equilibrium binding affinity, the on-rate of binding to receptors or other molecules (k It is also important to screen for mutants that have altered either _on ) or off-rate (k _off ). From thermodynamics, these rates are related to the equilibrium dissociation constant _{Kd = (k off / k on} ). We assume that some variants of a particular protein have similar K d for binding, but have very different k _on and k _off . Conversely, changes in Kd of a variant to another variant, effects on k _on, will be those effects, or by both, for k _off. The pharmacological properties of the protein may depend _{on the} binding affinity or k _on or k _off (depending on the detailed mechanism of action). Here we sought to identify hGH variants with higher on-rate and to examine the effect of changing k _on . We postulate that selection can be selectively weighted towards k _off by increasing the binding time and by increasing the washing time and / or enrichment with the cognate ligand (hGH). .

Time course analysis of wild-type hGH-phagemid binding to immobilized hGHbp shows less than 10% of all hGH-phagemid particles that can be eluted in the final pH2 wash (see Example VIII for total binding and elution protocol). Binds after 1 minute incubation, but more than 90% appear to bind after 15 minutes incubation.

For "rapid binding selection", phagemid particles from the pH0707B pool (independently selected twice for helices 1 and 4) were incubated with immobilized hGHbp for only 1 min, and then with binding buffer (1 ml). Six washes were performed (the hGH washing step was eliminated and the remaining hGH-phagemid particles were eluted with a pH 2 (0.2 M glycine in binding buffer) wash). Enrichment of hGH-phagemid particles relative to non-displayed particles is due to the use of short-term binding and mutations in which hGH-phagemid binding selection selects tightly from the randomized pool, even when no cognate ligand (hGH) challenge is used. Showed to sort the body.

Assays for hGH mutants The binding constants of some of these hGH mutants to hGHbp were determined by expressing free hormone mutants in non-suppressor E. coli strain 16C9 or 34B8, purifying the protein, and performing a radioimmunoprecipitation assay. Determined by competitive displacement of labeled wt-hGH from hGHbp in the assay (see Example VIII). In the following Table XIII-A, all variants in addition to phenylalanine ₁₇₆ substituted by glutamate ₁₇₄ and tyrosine ₁₇₆ substituted (E174S and F176Y) by serine _174, the hGH amino acids 10,14,18,21, It has the additional substitutions shown in the table at positions 167, 171, 175 and 179.

Table XIII-A . HGH by addition of helix 1 and helix 4b mutations
Mutant
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Helix 1 Helix 4
Wild-type residue: F10 M14 H18 H21 R167 D171 T175 I179
Mutant H0650AD H G N N N S T T
H0650AE H G N N E S T I
H0650AF H G N N N N T T
H0650BD A W D N N S T T
H0650BE A W D N E S T I
H0650BF A W D N N N T T
H0650CD F S F L N S T T
H0650CD F S F L E S T I
H0650CD F S F L N N T T

H In Table XIV below, hGH variants were selected from the combinatorial library by phagemid binding selection. All hGH variants in Table XIV contain two background mutations (E174S / F176Y). The hGH-phagemid pool from the libraries pH0707A (Part A), pH0707B and pH0707E (Part B) or pH0707C (Part C) was selected in 2-7 cycles for binding to hGHbp. The numerical value P indicates the fractional incidence of each variant type in a set of clones sequenced from each pool.

Table XIV . HGH variants from hormone-phagemid binding selection of combinatorial libraries
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Helix 1 Helix 4
Wild-type residue: F10 M14 H18 H21 R167 D171 T175 I179
P variant Part A: 4 cycles:
0.60 H0714A.1 H G N N N S T N
0.40 H0714A.4 A N D A N N T N *
Part B:
Two cycles:
0.13 H0712B.1 F S F G H S T T
0.13 H0712B.2 H Q T S A D N S
0.13 H0712B.4 H G N N N A T T
0.13 H0712B.5 F S F L S D T T
0.13 H0712B.6 A S T N R D T I
0.13 H0712B.7 Q Y N N H S T T
0.13 H0712B.8 W G S S R D T I
0.13 H0712E.1 F L S S K N T V
0.13 H0712E.2 W N N S H S T T
0.13 H0712E.3 A N A S N S T T
0.13 H0712E.4 P S D N R D T I
0.13 H0712E.5 H G N N N N T S
0.13 H0712E.6 F S T G R D T I
0.13 H0712E.7 M T S N Q S T T
0.13 H0712E.8 F S F L T S T S
4 cycles:
0.17 H0714B.1 A W D N R D T I
0.17 H0714B.2 A W D N H S T N
0.17 H0714B.3 M Q M N N S T T
0.17 H0714B.4 H Y D H R D T T
0.17 H0714B.5 L N S H R D T I
0.17 H0714B.6 L N S H T S T T
7 cycles:
0.57 H0717B.1 A W D N N A T T
0.14 H0717B.2 F S T G R D T I
0.14 H0717B.6 A W D N R D T I
0.14 H0717B.7 I Q E H N S T T
0.50 H0717E.1 F S L A N S T V
Part C:
4 cycles:
0.67 H0714C.2 F S F L K D T T
* = Also contained mutations L15R, K168R.

H In Table XV below, hGH variants were selected from the combinatorial library by phagemid binding selection. All hGH variants in Table XV contain two background mutations (E174S / F176Y). The number P is the fractional incidence of a particular variant in all clones sequenced after four cycles of rapid binding selection.

Table XV . hGH variants from rapid hGHbp binding selection of a combined hGH-phagemid library
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Helix 1 Helix 4
Wild-type residue: F10 M14 H18 H21 R167 D171 T175 I179
P mutant 0.14 H07BF4.2 W G S S R D T I
0.57 H07BF4.3 M A D N N S T T
0.14 H07BF4.6 A W D N S S V T ≠
0.14 H07BF4.7 H Q T S R D T I
≠ = also contained the mutation Y176F (wild-type hGH also contained F176).

In Table XVI below, binding constants were determined by competitive displacement of ¹²⁵ I-labeled hormone H0650BD or labeled hGH using hGHbp (1-238) and either Mab5 or Mab263. Mutant H0650BD appears to bind 30-fold more tightly than wild-type hGH.

Table XVI . Equilibrium binding constants of selected hGH variants
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
hGH mutant Kd (mutant) / Kd (H0650BD) Kd (mutant) / Kd (hGH) Kd (pM)
hGH 32-1-340 ± 50
H0650BD -1- 0.031 10 ± 3
H0650BF 1.5 0.045 15 ± 5
H0714B.6 3.4 0.099 34 ± 19
H0712B.7 7.4 0.22 74 ± 30
H0712E.2 16 0.48 60 ± 70

Example XIII Selective enrichment of hGH-phage and non-substrate phage containing protease substrate sequences As described in Example I, plasmid pS0132 was constructed using gene Proprotein residue Pro198 with insertion of an extra glycine residue. Contains the hGH gene fused to Using this plasmid, hGH-phage particles in which the hGH-gene III fusion product is monovalently displayed on the phage surface can be obtained (Example IV). This fusion protein contains the entire hGH protein fused to the carboxy-terminal domain of gene III by a variable linker sequence.

To test the possibility of using phage display to select a preferred substrate sequence for a protein hydrolase, a genetically engineered mutant of subtilisin BPN 'was used [Carter, P. et al., Proteins: Structure , function and genetics 6: 240-248 (1989)]. This mutant (hereinafter referred to as A64SAL subtilisin) contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Because this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly limited and certain histidine-containing substrates are preferentially hydrolyzed [Carter et al., Science 237: 394-399. (1987)].

Construction of hGH-Substrate-Phage Vector The sequence of the linker region in pS0132 was determined using oligonucleotides:
5'-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3 '
To create a substrate sequence for A64SAL subtilisin. This gives the protein sequence:
Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp
Was introduced into the linker region between hGH and the carboxy-terminal domain of gene III (the first Phe residue in the above sequence is Phe191 of hGH). Array:
Ala-Ala-His-Tyr-Thr-Arg-Gln
Is known to be a good substrate for A64SAL subtilisin [Carter et al. (1989); supra]. The resulting plasmid was named pS0640.

Phagemid particles derived from the hGH-substrate-phage selective enrichment pS0132 and pS0640 were generated as described in Example I. In a first experiment, each phage pool (10 μl each) was prepared using oxirane beads (as described in Example II) in a buffer (100 μl) containing 20 mM Tris-HCl (pH 8.6) and 2.5 M NaCl. Preparation; 30 μl). The coupling and washing steps were performed as described in Example VII. The beads were then resuspended in the same buffer (400 μl) with or without 50 nM A64SAL subtilisin. After incubation for 10 minutes, the supernatant was collected and the phage titer (cfu) was determined. Table XVII shows that about 10-fold more substrate-containing phagemid particles (pS0640) were eluted in the presence of the enzyme than in the absence of the enzyme, and that of the non-substrate phagemid (pS0132) in the presence or absence of the enzyme. It shows that it elutes more than a case. Increasing enzyme, phagemid or bead concentrations did not change this ratio.

In an attempt to reduce the non-specific elution of the immobilized immobilized phagemid of the selection enrichment method , tightly binding hGH variants were introduced into pS0132 and pS0640 instead of the wild-type hGH gene. The hGH variant used is the variant described in Example XI (pH 0650 bp) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr. This results in two novel phagemids: pDM0390 (containing tightly binding hGH and no substrate sequence) and pDM0411 (tightly binding hGH and substrate sequence Ala-Ala-His-Tyr-Thr-Arg-Gln). ) Were constructed. Also, the binding wash and elution protocol was changed as follows:
(i) Binding: COSTAR 12-well tissue culture plates were coated with 2 μg / ml hGHbp in 0.5 ml / well sodium carbonate buffer (pH 10.0) for 16 hours. The plate was then incubated with 1 ml / well of blocking buffer [phosphate buffered saline (PBS) containing 0.1% w / v bovine serum albumin) for 2 hours and 10 mM Tris-HCl (pH 7.5). ) Washed with assay buffer containing 1 mM EDTA and 100 mM NaCl. Phagemid was prepared again as described in Example I, the phage pool was diluted 1: 4 with the above assay buffer, and 0.5 ml phage per well was incubated for 2 hours.
(Ii) Washing: The plate was thoroughly washed with PBS + 0.05% Tween 20, and incubated with the washing buffer (1 ml) for 30 minutes. This washing step was repeated three times.
(Iii) Elution: The plates are incubated for 10 minutes in an elution buffer consisting of 20 mM Tris-HCl (pH 8.6) +100 mM NaCl, then the buffer (0.5 ml) with or without 500 nM A64SAL subtilisin ) To elute the phage.

Table XVII shows that the proportion of specifically eluted substrate-phagemid particles was dramatically increased compared to the method described above for pS0640 and pS0132. This result may be due to the tight binding of the hGH mutant having a significantly lower off-rate for binding to the hGH binding protein compared to wild-type hGH.

Table XVII . Specific Elution of Substrate-Phagemid by A64SAL Subtilisin Colony-forming units by plating 10-fold dilutions of phage (10 μl) onto 10 μl spots of XL1-Blue cells on LB agar plates containing 50 μg / ml carbenicillin (Cfu) was evaluated.
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
(I) Wild-type hGH gene: phagemid bound to hGHbp-oxirane beads + 50 nM without A64SAL enzyme
pS0640 (substrate) 9 × 10 ⁶ cfu / 10 μl 1.5 × 10 ⁶ cfu / 10 μl
pS0132 (non-substrate) 6 × 10 ⁵ cfu / 10 μl 3 × 10 ⁵ cfu / 10 μl
(Ii) pH0650 bp mutant hGH gene: phagemid bound to hGHbp coated plate + no 50 nM A64SAL enzyme
pDM0411 (substrate) 1.7 × 10 ⁵ cfu / 10 μl 2 × 10 ³ cfu / 10 μl
pDM0390 (non-substrate) 2 × 10 ³ cfu / 10 μl 1 × 10 ³ cfu / 10 μl

Example XIV Identification of Preferred Substrates for A64SAL Subtilisin Using Selective Enrichment of a Substrate Sequence Library Attempt to identify good substrate sequences from a library of random substrate sequences using the selective enrichment method described in Example XIII .
Construction of a Vector for Insertion of a Randomized Substrate Cassette A vector suitable for introducing a randomized substrate cassette and subsequent expression of a substrate sequence library was designed. The starting point was the vector pS0643 described in Example VIII. Oligonucleotides:
5'-AGC-TGT-GGC-TTC- GGG-CCC -GCC-GCC-GC G-TCG-AC T-GGC-GGT-GGC-TCT-3 '
Was used to introduce site-directed mutagenesis, which introduced ApaI (GGGCCC) and SalI (GTCGAC) restriction sites between hGH and gene III. This new construct was named pDM0253 (the actual sequence of pDM0253 was
5'-AGC-TGT-GGC- TTC-GGG-CCC-GCC- C CC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3 '
And the underlined base substitutions are due to spurious errors in the mutant oligonucleotide). Also, by exchanging the fragment derived from pDM0411 (Example XIII), the tightly binding hGH mutant described in the Examples was introduced. The resulting library vector was named pDM0454.

Preparation of Library Cassette Vector and Insertion of Mutation Cassette To introduce the library cassette, pDM0454 was digested with ApaI and then with SalI, then precipitated with 13% PEG8000 + 10 mM MgCl ₂ and washed twice with 70% ethanol. And resuspended. This precipitates the vector efficiently, but leaves small ApaI-SalI fragments in solution [Paithankar, KR and Prasad, KSN, Nucleic Acids Research 19: 1346]. The product was run on a 1% agarose gel, the ApaI-SalI digested vector was excised, purified using the Bandprep kit (Pharmacia), and resuspended for ligation with the mutation cassette.

The inserting cassette contained a sequence similar to the DNA sequence in the linker region of pS0640 and pDM0411, but had the codons of histidine and tyrosine residues in the substrate sequence replaced by randomized codons. We chose to replace NNS (N = G / A / T / C; S = G / C) at each of the randomized positions as described in Example VIII. The oligonucleotide used in the mutation cassette was
5'-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3 '(encryption chain) and 5'-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA -GGG-CC-3 '(non-encrypted chain)
Met. Also, this cassette destroys the SalI site, so that digestion with SalI can be used to reduce vector background. These oligonucleotides did not phosphorylate prior to insertion into the Apa-Sal cassette site. This is because we feared that subsequent oligomerization of the cassette subpopulations could lead to spurious results with multiple cassette inserts. After annealing and ligation, the reaction product was phenol: chloroform extracted, ethanol precipitated and resuspended in water. Initially, no digestion with SalI was performed to reduce the background vector. As described in Example VIII, about 200 ng was electroporated into XL1-Blue cells to prepare a phagemid library.

Selection of susceptible substrates from the substrate library The selection method used is the same as that described for pDM0411 and pDM0390 in Example XIII. After each round of selection, the eluted phages were transformed by transducing fresh XL1-Blue cell cultures and growing a new phagemid library as described for hGH-phages in Example VIII. Propagated. The progress of the selection procedure was monitored by measuring the titer of eluted phage and sequencing individual clones after each round of selection.

Table A shows continuous phage titers for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection. The ratio of specifically eluted phage: non-specifically eluted phage (ie, phage eluted with enzyme: phage eluted without enzyme) increases dramatically from round 1 to round 3. It is clear that this suggests that the good substrate population is increasing with each selection round.

配 列 Sequencing of 10 isolates from the starting library indicated that all of them consisted of the wild-type pDM0464 sequence. This is due to the SalI site being very close to the end of the DNA fragment after digestion with ApaI, thus leading to a less efficient digestion. Nevertheless, there are only 400 possible sequences in the library, so this population should still be noted.

Tables B1 and B2 show the sequences of the isolates obtained after round 2 and round 3 selection. After two rounds of selection, there is clearly a high occurrence of histidine residues. This was exactly what was expected (as described in Example XIII, A64SAL subtilisin requires a histidine residue in the substrate because it uses a substrate-assisted catalytic mechanism. To do that). After three rounds of selection, each of the 10 clones sequenced has histidine in the randomized cassette. However, it should be noted that two of these sequences are of pDM0411 and were not present in the starting library and are therefore impurities.

Table A. Elution from initial phage pool and 3 rounds of selection enrichment Phage titration A 10-fold dilution of phage (10 μl) was plated on a 10 μl spot of XL1-Blue cells on an LB agar plate containing 50 μg / ml carbenicillin. The colony forming units (cfu) were evaluated by plating.
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Round 1
Starting library: 3 × 10 ¹² cfu / ml
Library: + 500nM A64SAL: 4 × 10 ³ cfu / 10μl
Without enzyme: 3 × 10 ³ cfu / 10μl
pDM0411: +500 nM A64SAL: 2 × 10 ⁶ cfu / 10 μl
(Control) No enzyme: 8 × 10 ³ cfu / 10 μl
Round 2
Round 1 library: 7 × 10 ¹² cfu / ml
Library: + 500nM A64SAL: 3 × 10 ⁴ cfu / 10μl
Without enzyme: 6 × 10 ³ cfu / 10μl
pDM0411: +500 nM A64SAL: 3 × 10 ⁶ cfu / 10 μl
(Control) No enzyme: 1.6 × 10 ⁴ cfu / 10μl
Round 3
Round 2 library: 7 × 10 ¹¹ cfu / ml
Library: +500 nM A64SAL: 1 × 10 ⁵ cfu / 10 μl
No enzyme: <10 ³ cfu / 10μl
pDM0411: +500 nM A64SAL: 5 × 10 ⁶ cfu / 10 μl
(Control) No enzyme: 3 × 10 ⁴ cfu / 10 μl

Table B1 . Sequence of eluted phage after two rounds of selective enrichment All protein sequences should be in the form of AA ** TRQ, where * represents a randomized codon. In the table below, the randomized codons and amino acids are underlined.
　　　　　　　　　　　　　　　　　　　　　　　　 　
After Round 2 :
Number of array occurrences * *
AA H Y TRQ
... GCT GCT CAC TAC ACC CGG CAA ... 2

AA H M TRQ
... GCT GCT CAC ATG ACC CGG CAA ... 1

AA L H TRQ
... GCT GCT CTC CAC ACC CGG CAA ... 1

AA L H TRQ
... GCT GCT CTG CAC ACC CGG CAA ... 1

AA H T RQ
... GCT GCT CAC ACC CGG CAA ... 1 #

AA ? H TRQ
... GCT GCT ??? CAC ACC CGG CAA 1 ##

... wild type pDM0454 3
　　　　　　　　　　　　　　　　　　　　　　　　　
# False deletion of one codon in cassette ## Ambiguous sequence

Table B2 . Sequence of eluted phage after three rounds of selection enrichment

All protein sequences should be in the form of AA ** TRQ, where * represents a randomized codon. In the table below, the randomized codons and amino acids are underlined.
　　　　　　　　　　　　　　　　　　　　　　　　 　
After Round 3 :
Number of array occurrences
* *
AA H Y TRQ
... GCT GCT CAC TAT ACG CGT CAG ... 2 #

AA L H TRQ
... GCT GCT CTC CAC ACC CGG CAA ... 2

AA Q H TRQ
... GCT GCT CAG CAC ACC CGG CAA ... 1

AA T H TRQ
... GCT GCT ACG CAC ACC CGG CAA ... 1

AA H S RQ
... GCT GCT CAC TCC CGG CAA ... 1

AA H H TRQ
... GCT GCT CAT CAT ACC CGG CAA 1 ##

AA H F RQ
... GCT GCT CAC TTC CGG CAA ... 1

AA H T RQ
... GCT GCT CAC ACC CGG CAA ... 1
　　　　　　　　　　　　　　　　　　　　　　　　　
# Contaminated sequence from pDM0411 ## Contain "illegal" codon CAT: T cannot appear at position 3 of codon

Sequence table (1) General information (i) Patent applicant: Genentech, Inc.
Gailard, Liza Jay
Henner, Dennis Jay
Bath, Stephen
Green, Ronald
Rowman, Henry Bee
Wells, James A
Matthews, David Jay (ii) Title of the Invention: Method for enriching mutant proteins with altered binding properties (iii) Number of sequences: 27
(Iv) Contact:
(A) Address: Genentech, Inc. (B) Street: Point San Bruno Boulevard 460 (C) City: South San Francisco (D) State: California (E) Country: United States (F) ZIP: 94080
(V) Computer reading ceremony:
(A) Medium type: 5.25 inch, 360 Kb floppy disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS
(D) Software: patin (Genentech)
(Vi) Data of the present application:
(A) Application number: To be determined (B) Filing date: To date (C) Classification: To be determined (vii) Priority application data:
(A) Application number: 07/743614
(B) Filing date: August 9, 1991 (vii) Priority claim application data:
(A) Application No. 07/715300
(B) Filing date: June 14, 1991 (vii) Priority application data:
(A) Application No. 07/683400
(B) Filing date: April 10, 1991 (vii) Priority application data:
(A) Application number: 07/621667
(B) Filing date: December 3, 1990 (viii) Patent attorney / agent Information:
(A) Name: Benson, Robert H. (B) Registration number: 30,446
(C) Reference / reference number: 645P4
(Ix) Telephone contact information:
(A) Phone number: 415 / 266-1489
(B) Fax number: 415 / 952-9881
(C) Telex number: 910 / 371-7168
(2) Information of SEQ ID NO: 1 (i) Sequence characteristics:
(A) Length: 36 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 1:

GGCAGCTGTG GCTTCTAGAG TGGCGGCGGC TCTGGT 36

(2) Information of SEQ ID NO: 2 (i) Sequence characteristics:
(A) Length: 36 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 2:

AGCTGTGGCT TCGGGCCCTT AGCATTTAAT GCGGTA 36

(2) Information of SEQ ID NO: 3 (i) Sequence characteristics:
(A) Length: 33 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 3:

TTCACAAACG AAGGGCCCCT AATTAAAGCC AGA 33

(2) Information of SEQ ID NO: 4 (i) Sequence characteristics:
(A) Length: 30 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 4:

CAATAATAAC GGGCTAGCCA AAAGAACTGG 30

(2) Information of SEQ ID NO: 5 (i) Sequence characteristics:
(A) Length: 24 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 5:

CACGACAGAA TTCCCGACTG GAAA 24

(2) Information of SEQ ID NO: 6 (i) Sequence characteristics:
(A) Length: 23 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 6:

CTGTTTCTAG AGTGAAATTG TTA 23

(2) Information of SEQ ID NO: 7 (i) Sequence features:
(A) Length: 21 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 7:

ACATTCCTGG GTACCGTGCA G 21

(2) Information of SEQ ID NO: 8 (i) Sequence characteristics:
(A) Length: 63 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 8

GCTTCAGGAA GGACATGGAC NNSGTCNNSA CANNSCTGNN SATCGTGCAG 50
TGCCGCTCTG TGG 63

(2) Information of SEQ ID NO: 9 (i) Sequence characteristics:
(A) Length: 24 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 9

AAGGTCTCCA CATACCTGAG GATC 24

(2) Information of SEQ ID NO: 10 (i) Sequence characteristics:
(A) Length: 33 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 10:

ATGGACAAGG TGTCGACATA CCTGCGCATC GTG 33

(2) Information of SEQ ID NO: 11 (i) Sequence characteristics:
(A) Length: 36 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 11:

GGCAGCTGTG GCTTCTAGAG TGGCGGCGGC TCTGGT 36

(2) Information of SEQ ID NO: 12 (i) Sequence characteristics:
(A) Length: 36 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 12:

GGCAGCTGTG GATTCTAGAG TGGCGGTGGC TCTGGT 36

(2) Information of SEQ ID NO: 13 (i) Sequence characteristics:
(A) Length: 12 amino acids (B) Type: amino acids (D) Topology: linear (xi) Sequence: SEQ ID NO: 13:

Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly
1 5 10 12

(2) Information of SEQ ID NO: 14 (i) Sequence characteristics:
(A) Length: 27 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 14:

CGGACTGGGC AGATATTCAA GCAGACC 27

(2) Information of SEQ ID NO: 15 (i) Sequence characteristics:
(A) Length: 38 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 15:

CTCAAGAACT ACGGGTTACC CTGACTGCTT CAGGAAGG 38

(2) Information of SEQ ID NO: 16 (i) Sequence characteristics:
(A) Length: 30 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 16:

CGCATCGTGC AGTGCAGATC TGTGGAGGGC 30

(2) Information of SEQ ID NO: 17 (i) Sequence characteristics:
(A) Length: 66 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 17:

GTTACTCTAC TGCTTTCAGG AAGGACATGG ACNNSGTCNN SACANNSCTG 50
NNSATCGTGC AGTGCA 66

(2) Information of SEQ ID NO: 18 (i) Sequence characteristics:
(A) Length: 64 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 18:

GATCTGCACT GCACGATSNN CAGSNNTGTS NNGACSNNGT CCATGTCCTT 50
CCTGAAGCAG TAGA 64

(2) Information of SEQ ID NO: 19 (i) Sequence characteristics:
(A) Length: 25 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 19:

GCCTTTGACA GGTACCAGGA GTTTG 25

(2) Information of SEQ ID NO: 20 (i) Sequence characteristics:
(A) Length: 33 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 20:

CCAACTATAC CACTCTCGAG GTCTATTCGA TAA 33

(2) Information of SEQ ID NO: 21 (i) Sequence characteristics:
(A) Length: 66 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 21

TCGAGGCTCN NSGACAACGC GNNSCTGCGT GCTNNSCGTC TTNNSCAGCT 50
GGCCTTTGAC ACGTAC 66

(2) Information of SEQ ID NO: 22 (i) Sequence characteristics:
(A) Length: 58 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 22:

GTGTCAAAGG CCAGCTGSNN AAGACGSNNA GCACGCAGSN NCGCGTTGTC 50
SNNGAGCC 58

(2) Information of SEQ ID NO: 23 (i) Sequence characteristics:
(A) Length: 65 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 23

GTTACTCTAC TGCTTCNNSA AGGACATGNN SAAGGTCAGC NNSTACCTGC 50
GCNNSGTGCA GTGCA 65

(2) Information of SEQ ID NO: 24 (i) Sequence characteristics:
(A) Length: 64 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 24:

GATCTGCACT GCACSNNGCG CAGGTASNNG CTGACCTTSN NCATGTCCTT 50
SNNGAAGCAG TAGA 64

(2) Information of SEQ ID NO: 25 (i) Sequence characteristics:
(A) Length: 2178 bases (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear (xi) Sequence: SEQ ID NO: 25

ATGAAAAAGA ATATCGCATT TCTTCTTGCA TCTATGTTCG TTTTTTCTAT 50
TGCTACAAAC GCGTACGCTG ATATCCAGAT GACCCAGTCC CCGAGCTCCC 100
TGTCCGCCTC TGTGGGCGAT AGGGTCACCA TCACCTGCCG TGCCAGTCAG 150
GATGTGAATA CTGCTGTAGC CTGGTATCAA CAGAAACCAG GAAAAGCTCC 200
GAAACTACTG ATTTACTCGG CATCCTTCCT CTACTCTGGA GTCCCTTCTC 250
GCTTCTCTGG ATCCAGATCT GGGACGGATT TCACTCTGAC CATCAGCAGT 300
CTGCAGCCGG AAGACTTCGC AACTTATTAC TGTCAGCAAC ATTATACTAC 350
TCCTCCCACG TTCGGACAGG GTACCAAGGT GGAGATCAAA CGAACTGTGG 400
CTGCACCATC TGTCTTCATC TTCCCGCCAT CTGATGAGCA GTTGAAATCT 450
GGAACTGCCT CTGTTGTGTG CCTGCTGAAT AACTTCTATC CCAGAGAGGC 500
CAAAGTACAG TGGAAGGTGG ATAACGCCCT CCAATCGGGT AACTCCCAGG 550
AGAGTGTCAC AGAGCAGGAC AGCAAGGACA GCACCTACAG CCTCAGCAGC 600
ACCCTGACGC TGAGCAAAGC AGACTACGAG AAACACAAAG TCTACGCCTG 650
CGAAGTCACC CATCAGGGCC TGAGCTCGCC CGTCACAAAG AGCTTCAACA 700
GGGGAGAGTG TTAAGCTGAT CCTCTACGCC GGACGCATCG TGGCCCTAGT 750
ACGCAAGTTC ACGTAAAAAG GGTATCTAGA GGTTGAGGTG ATTTTATGAA 800
AAAGAATATC GCATTTCTTC TTGCATCTAT GTTCGTTTTT TCTATTGCTA 850
CAAACGCGTA CGCTGAGGTT CAGCTGGTGG AGTCTGGCGG TGGCCTGGTG 900
CAGCCAGGGG GCTCACTCCG TTTGTCCTGT GCAGCTTCTG GCTTCAACAT 950
TAAAGACACC TATATACACT GGGTGCGTCA GGCCCCGGGT AAGGGCCTGG 1000
AATGGGTTGC AAGGATTTAT CCTACGAATG GTTATACTAG ATATGCCGAT 1050
AGCGTCAAGG GCCGTTTCAC TATAAGCGCA GACACATCCA AAAACACAGC 1100
CTACCTGCAG ATGAACAGCC TGCGTGCTGA GGACACTGCC GTCTATTATT 1150
GTTCTAGATG GGGAGGGGAC GGCTTCTATG CTATGGACTA CTGGGGTCAA 1200
GGAACCCTGG TCACCGTCTC CTCGGCCTCC ACCAAGGGCC CATCGGTCTT 1250
CCCCCTGGCA CCCTCCTCCA AGAGCACCTC TGGGGGCACA GCGGCCCTGG 1300
GCTGCCTGGT CAAGGACTAC TTCCCCGAAC CGGTGACGGT GTCGTGGAAC 1350
TCAGGCGCCC TGACCAGCGG CGTGCACACC TTCCCGGCTG TCCTACAGTC 1400
CTCAGGACTC TACTCCCTCA GCAGCGTGGT GACTGTGCCC TCTAGCAGCT 1450
TGGGCACCCA GACCTACATC TGCAACGTGA ATCACAAGCC CAGCAACACC 1500
AAGGTGGACA AGAAAGTTGA GCCCAAATCT TGTGACAAAA CTCACACAGG 1550
GCCCTTCGTT TGTGAATATC AAGGCCAATC GTCTGACCTG CCTCAACCTC 1600
CTGTCAATGC TGGCGGCGGC TCTGGTGGTG GTTCTGGTGG CGGCTCTGAG 1650
GGTGGTGGCT CTGAGGGTGG CGGTTCTGAG GGTGGCGGCT CTGAGGGAGG 1700
CGGTTCCGGT GGTGGCTCTG GTTCCGGTGA TTTTGATTAT GAAAAGATGG 1750
CAAACGCTAA TAAGGGGGCT ATGACCGAAA ATGCCGATGA AAACGCGCTA 1800
CAGTCTGACG CTAAAGGCAA ACTTGATTCT GTCGCTACTG ATTACGGTGC 1850
TGCTATCGAT GGTTTCATTG GTGACGTTTC CGGCCTTGCT AATGGTAATG 1900
GTGCTACTGG TGATTTTGCT GGCTCTAATT CCCAAATGGC TCAAGTCGGT 1950
GACGGTGATA ATTCACCTTT AATGAATAAT TTCCGTCAAT ATTTACCTTC 2000
CCTCCCTCAA TCGGTTGAAT GTCGCCCTTT TGTCTTTAGC GCTGGTAAAC 2050
CATATGAATT TTCTATTGAT TGTGACAAAA TAAACTTATT CCGTGGTGTC 2100
TTTGCGTTTC TTTTATATGT TGCCACCTTT ATGTATGTAT TTTCTACGTT 2150
TGCTAACATA CTGCGTAATA AGGAGTCT 2178

(2) Information of SEQ ID NO: 26 (i) Sequence characteristics:
(A) Length: 237 amino acids (B) Type: amino acids (D) Topology: linear (xi) Sequence: SEQ ID NO: 26:

Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe
1 5 10 15
Ser Ile Ala Thr Asn Ala Tyr Ala Asp Ile Gln Met Thr Gln Ser
20 25 30
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
35 40 45
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln
50 55 60
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
65 70 75
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser
80 85 90
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
95 100 105
Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr
110 115 120
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
125 130 135
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
140 145 150
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
155 160 165
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
170 175 180
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
185 190 195
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
200 205 210
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
215 220 225
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
230 235 237

(2) Information of SEQ ID NO: 27 (i) Sequence characteristics:
(A) Length: 461 amino acids (B) Type: amino acids (D) Topology: linear (xi) Sequence: SEQ ID NO: 27

Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe
1 5 10 15
Ser Ile Ala Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser
20 25 30
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
35 40 45
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
50 55 60
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr
65 70 75
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg
80 85 90
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
95 100 105
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
110 115 120
Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
125 130 135
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
140 145 150
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
155 160 165
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
170 175 180
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
185 190 195
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
200 205 210
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
215 220 225
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
230 235 240
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Gly Pro Phe Val
245 250 255
Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro Val
260 265 270
Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu
275 280 285
Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
290 295 300
Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr
305 310 315
Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala
320 325 330
Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser
335 340 345
Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp
350 355 360
Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala
365 370 375
Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser
380 385 390
Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln
395 400 405
Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr
410 415 420
Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val
425 430 435
Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser
440 445 450
Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser
455 460 461

FIG. 1 shows a method for displaying (revelating) large proteins on the surface of filamentous phage and a method for enriching for modified receptor binding. A plasmid phGH-M13gIII was constructed in which the entire coding sequence of hGH was fused to the carboxy-terminal domain of M13 gene III. Transcription of this fusion protein is under the control of the lac promoter / operator sequence and secretion is dictated by the stII signal sequence. Phagemid particles are obtained by infection with "helper" phage M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor. The wild-type gene III (derived from the M13KO7 phage) is shown by multiple arrows at the top of the phage with 4-5 copies, and the fusion protein (derived from the phagemid phGH-M13gIII) is indicated by the hGH fold (replace the head of the arrow). Illustrated. FIG. 2: Immunoblot of all phage particles shows that hGH co-migrate with phage. Phagemid particles purified with a cesium chloride gradient were applied to duplicate wells and electrophoresed on a 1% agarose gel in 375 mM Tris, 40 mM glycine (pH 9.6) buffer. The gel is immersed in transfer buffer containing 2% SDS and 2% β-mercaptoethanol [25 mM Tris (pH 8.3), 200 mM glycine, 20% methanol] for 2 hours, and then in transfer buffer for 6 hours. Rinse. The proteins in the gel were then electroblotted onto Immobilon membrane (Millipore). Membranes containing one set of samples were stained with Coomassie blue to indicate the location of phage proteins (A). The membranes were immunostained for hGH by reacting the duplicate membrane with a polyclonal rabbit anti-hGH antibody and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (B). Lane 1 contained the M13KO7 parental phage, which could only be observed on Coomassie blue stained membranes due to the lack of hGH. Lanes 2 and 3 contained another preparation of hormonal phagemid particles and could be observed on both Coomassie and hGH immunostaining. The differences in migration distance between the parent M13KO7 phage and the hormone phagemid particles reflect differences in the size of the internally packaged genomes (8.7 kb and 5.1 kb, respectively). FIG. 3 is a diagram summarizing each step in a method for selecting an hGH-phage library in which codons 172, 174, 176 and 178 are randomized. A template molecule, pH0415, containing the hGH (R178G, I179T) gene and a unique KpnI restriction site, was mutagenized as described herein, and electrotransfected into E. coli strain WJM101 to produce an initial phagemid library (library). 1) was obtained. A portion (about 2%) of Library 1 was used directly in the first round of selection as described herein to obtain Library 1G. On the other hand, double-stranded DNA (dsDNA) was prepared from Library 1, digested with the restriction enzyme KpnI to eliminate the background of the template, and electrotransferred into WJM101 to obtain Library 2. Subsequent rounds of selection (or KpnI digestion; shaded boxes) and subsequent phagemid expansion were performed as indicated by the arrows, according to the methods described herein. Were sequenced by dideoxy sequencing the four independent clones from four independent clones and libraries 5G ⁶ from the library 4G ^4. All of these clones had identical DNA sequences corresponding to the hGH mutant (Glu 174 Ser, Phe 176 Tyr). FIG. 4: SGH structural model derived from the 2.8 ° fold diagram of porcine growth hormone determined by crystallography. Residues in hGH that strongly modulate binding to hGH-binding proteins are indicated in shaded circles. Alanine substitutions that cause a 10-fold or greater decrease in binding affinity (solid circles), a 4- to 10-fold decrease (•), an increase (O), or a 2- to 4-fold decrease (•) are shown. Spiral ring projections in the α-helical region revealed these two polarities. The darkened, shaded, or unshaded residues indicate charge, polarity, or non-polarity, respectively. In helix 4, the most important residues for mutation are on the hydrophilic side. Figure 5: hGH 172, 174, 176 and 178 found after sequencing a number of clones from rounds 1 and 3 of the selection step for the indicated pathway (hGH elution, glycine elution, or glycine elution after pre-adsorption). Indicates the amino acid substitution at the position (eg, the designation KSYR represents the hGH mutant 172K / 174S / 176Y / 178R). Non-functional sequences (ie, vector background, or other immature and / or frameshifted mutants) are indicated as "NF". Functional sequences containing non-silent pseudomutations (ie, other than the set of target residues listed above) are indicated by a "+". Protein sequences that appear more than once in all sequencing clones but have different DNA sequences are indicated by "#". Protein sequences which appear more than once in the sequencing clones and have the same DNA sequence are indicated by a "*". It should be noted that two different contaminating sequences were found after three rounds of selection (these clones did not match the cassette mutant, but did match the previously constructed hormone phage). The pS0643 contaminant is consistent with wild-type hGH-phage (hGH "KEFR"). The pH0457 contaminant abundant in the third round of glycine-selected phage pool is consistent with the previously identified hGH mutant "KSYR". The amplification of these contaminants highlights the ability of the hormone-phage selection method to select for rarely occurring mutants. Also, the convergence of these sequences was significant in all three pathways (ie, R or K occurred most frequently at positions 172 and 178, Y or F occurred most frequently at position 176, and S , T, A and other residues occur at position 174). Figure 6: Sequence from phage selected on hPRLbp-beads in the presence of zinc. The display is the same as that described in FIG. Here the sequence convergence is unpredictable, but under the most stringent (glycine) selection conditions, there appears to be a bias towards hydrophobic sequences (L, W and P residues are found more in this pool). . FIG. 7: Sequence from phage selected on hPRLbp-beads in the absence of zinc. The display is the same as that described in FIG. In contrast to the sequence of FIG. 6, the sequence here appears to be relatively hydrophilic. After four rounds of selection by hGH elution, the two clones (ANHQ and TLDT / 171V) are abundant in the pool. FIG. 8: Sequence from phage selected on blank beads. The display is the same as that described in FIG. After three rounds of selection by glycine elution, no siblings were observed and background levels of non-functional sequences were retained. Figure 9: Construction of phagemid fl origin (ori) from pHHO415. This vector for cassette mutagenesis and expression of the hGH-gene III fusion protein was constructed as follows. It contains the pBR322 and fl origins of replication, and under the control of the E. coli phoA promoter, the hGH-gene III fusion protein (hGH followed by one Gly residue, which is fused to the Pro-198 of gene III). Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132, which expresses Mutagenesis was performed using the following oligonucleotides: 5'-GGC-AGC-TGT-GGC- TTC-TAG -AGT-GGC-GGC-GGC-TCT-GGT-3 '. This oligonucleotide introduced an XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH. FIG. 10: A depicts a plasmid pDH188 insert containing DNA encoding the light and heavy chains (variable and constant domain 1) of a Fab humanized antibody directed to the HER-2 receptor. is there. _VL and _VH are the variable regions of the light and heavy chains, respectively. C _k is the constant region of the human kappa light chain. CH1 _G1 is the first constant region of the human γ1 chain. Both coding regions start with a bacterial stII signal sequence. B illustrates the entire plasmid pDH188 containing the insert shown in 5A. After introducing this plasmid into E. coli SR101 cells and adding helper phage, the plasmid is packaged into phage particles. Some of these particles display Fab-pIII fusions, where pIII is the protein encoded by M13 gene III DNA. The segment in this plasmid diagram corresponds to the insert shown in 5A. FIG. 11A: Shows the nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11B: Shows the nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11C shows the nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11D: Shows the nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11E: Nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11F shows the nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11G: Nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 11H: Nucleotide sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface (SEQ ID NO: 25). The amino acid sequence of the light chain (SEQ ID NO: 26) is shown as well as the amino acid sequence of the heavy chain pIII fusion (SEQ ID NO: 27). FIG. 12: Enrichment of wild-type 4D5 Fab phagemid from mutant Fab phagemid. A mixture of wild-type phagemid and mutant 4D5 Fab phagemid in a ratio of 1: 1,000 was selected on plates coated with the extracellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid was infected into E. coli and plasmid DNA was prepared. The plasmid DNA was then digested with EcoRV and PstI, separated on a 5% polyacrylamide gel and stained with ethidium bromide. Bands were visualized under UV light. Bands resulting from wild-type and mutant plasmids are indicated by arrows. The first round of selection eluted only under acid conditions. Subsequent rounds were eluted either by acid elution (left side of the figure) or by a humanized 4D5 antibody washing step using the method described in Example VIII followed by acid elution (right side of the figure). Three mutant 4D5 Fab molecules were prepared. That is, H91A (amino acid histidine at position 91 of the _VL chain was mutated to alanine; shown in the “A” lane in the figure), Y49A (amino acid tyrosine at position 49 of the _VL chain was mutated to alanine. ; Shown in the "B" lane in the figure), and Y92A (the amino acid tyrosine at position 92 of the _VL chain was mutated to alanine; shown in the "C" lane in the figure). Amino acid position counting was performed according to Kabat et al. [“Protein Sequence of Immunological Inserts”, 4th edition, USDept of Health and Human Services, Public Health Service, Nat'l.Institute of Health, Bethesda, MD (1987)]. FIG. 13: Scatchard analysis of RIA affinity measurements described during the experimental protocol is shown. The amount of labeled ECD antigen bound is shown on the x-axis and the amount of bound divided by the amount released is shown on the y-axis. The slope of the line indicates Ka, and the calculated Kd is 1 / Ka.

Claims (10)

A phagemid expression vector containing a transcriptional regulatory element operably linked to a gene fusion encoding a fusion protein, said gene fusion comprising a first gene encoding a polypeptide and a phage coat protein Containing a second gene encoding at least a portion of
(A) the vector does not contain a gene encoding a mature coat protein,
(B) does not contain a complete phage genome, or (c) transforming a host cell with the vector does not produce phage particles in the absence of helper phage, and (d) encodes an mRNA repressible stop codon. A phagemid expression vector in which the replacing DNA triplet codon is inserted between the fusion ends of the first and second genes or is substituted for the amino acid-encoded triplet codon adjacent to the gene fusion junction . The phagemid vector according to claim 1, wherein the stop codon is UAG, UAA or UGA. The phagemid expression vector according to claim 1 or 2, wherein the phage coat protein is encoded by gene III of filamentous phage. The phagemid expression vector according to claim 3, wherein the mammalian protein is a human protein. The protein is growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A chain, insulin B chain, proinsulin, relaxin A chain, relaxin B chain , Prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptor, calcitonin, glucagon, factor VIII, antibodies, lung surfactant, urokinase, streptokinase, Human tissue type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hematopoietic growth factor, tumor necrosis factors α and β, enkephalinase, human serum albumin, Muller inhibitor, mouse gonadotropin-related peptide, β-lactamase, tissue Child protein, inhibin, activin, vascular endothelial growth factor, integrin receptor, thrombopoietin, protein A and D, rheumatoid factor, nerve growth factor (NGF-β), platelet growth factor, transforming growth factor (TGF); TGF-α And TGF-β, insulin-like growth factors I and II, insulin-like growth factor binding protein, CD-4, DNase, latency-related peptide, erythropoietin, osteoinductive factors, interferon α, β and γ, colony stimulating factor (CSF ), M-CSF, GM-CSF and G-CSF, interleukin (IL), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay-accelerating factor, viral antigen, HIV envelope protein GP120 and GP140, atrial natriuretic peptides A, B and C, more than one of the above proteins The phagemid expression vector according to any one of claims 1 to 4, which is selected from the group consisting of a protein having a unit, an immunoglobulin, and a fragment. A host cell containing the phagemid expression vector according to any one of claims 1 to 5, and a helper phage having a gene encoding a coat protein. A phagemid particle comprising at least a part of the phagemid expression vector according to any one of claims 1 to 5, wherein the amount or number of phagemid particles displaying two or more copies of a fusion protein on the particle surface is less than 20%. A group of particles. A method for selecting a novel binding polypeptide, comprising:
(A) constructing a family of phagemid expression vectors according to any of claims 1 to 5 (where the phagemid expression vector contains a mutant first gene encoding a mutant polypeptide);
(B) transforming a suitable host cell with the phagemid expression vector;
(C) infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein sufficient to produce recombinant phagemid particles;
(D) culturing the transformed and infected host cells under conditions suitable for the formation of recombinant phagemid particles containing at least a portion of the expression vector and capable of transforming the host (wherein The amount or number of phagemid particles displaying two or more copies of the fusion protein on the particle surface is less than 20%);
(E) contacting the recombinant phagemid particles with a target molecule to bind at least a portion of the phagemid particles to the target molecule; and (f) separating phagemid particles that bind to the target molecule from unbound particles;
A method comprising a step. A group of phagemid particles produced by the method according to claim 8. 9. The method of claim 8, wherein the first gene encodes a polypeptide linked to a linker amino acid sequence, and the phagemid expression vector contains a mutant first gene encoding a mutant linker amino acid sequence. Step (f),
(F) contacting the bound phagemid particles with a protease capable of hydrolyzing at least a portion of the bound amino acid sequence of the bound phagemid particles; and (g) isolating the hydrolyzed phagemid particles; and, optionally,
(H) further contacting a suitable host cell with the hydrolyzed phagemid particles and repeating steps (d)-(g);
9. The method of claim 8, wherein

Images