New molecular biology methods for protein engineering Mark J. Zoller Genentech, San Francisco, California, USA This review outlines recent advances in the application of molecular biological techniques to the study of protein structure and function. The chapter is divided into four main sections: methods for oligonucleotidedirected mutagenesis; mutational strategies for identifying functional residues and domains; systems for expression; and future developments. Few new methods were reported in 1990; however, a number of the papers that appeared represent refinements of previously reported strategies. This review is also published in Current Opinion in Structural Biology 1991, 1:605-610. Current Opinion in Biotechnology 1991, 2:526-531
Introduction
Point mutations
striction sites flanking the region of interest, and the limitation on the size (and perhaps expense) of the oligonudeotides that comprise the synthetic fragment. Enzymatic extension of mutagenic oligonudeotides hybridized to single-stranded DNA templates provides an alternative method that overcomes the restrictions of the cassette method [1]. The drawback of the primerextension method is that the efficiency of mutagenesis is generally less than 100 %. A number of methods, developed over 5 years ago, introduce genetic 'tricks' to obtain greater than 50% mutagenesis efficiency [2]. The method of Kunkel [3] is popular largely because of its simplicity and because it is now available in a kit form. The use of the highly accurate DNA polymerases from phage T4 or T7 results in virtually no extraneous mutations and has shortened the polymerization time required to copy the second strand to less than 2 h. These polymerases do not 'strand displace' the 5' end of the oligomer, and polymerization can be conducted at 37 °C. For slightly greater efficiency, the phosphothioate-incorporation method of Eckstein and colleagues [4] is gaining in popularity and is also available in kit form. Recently, this method has been adapted to operate on a double-stranded DNA template [5"]. This method has many more steps than the Kunkel procedure and is thus more tedious. A feature of the Eckstein methdd, however, is that the mutation encoded by the oligonucleotide is present in the resulting DNA molecule as a homoduplex. The features of this method have been exploited in doped-oligonucleotide mutagenesis experiments, which are discussed below.
Methods for the construction of point mutations in DNA have not changed greatly over the past few years. Cassette mutagenesis, in which a synthetic DNA fragment containing the desired sequence is ligated into the coding sequence is a simple method for which the efficiency of mutagenesis is virtually 100 %. The drawbacks of this method are, of course, the requirement for unique re-
The ease of the polymerase chain reaction (PER) procedure naturally spawned methods for site-directed mutagenesis [6,7.]. Generally, these methods use either one or two mutagenic primers and require two rounds of PeR amplification [2]. The advantage of a PCR method, as in cassette mutagenesis, is that the desired mutation is obtained with 100% efficiency. However, the need to ligate
Protein engineering is accomplished by the modification of an existing protein, d e n o v o peptide synthesis or the expression of genes encoding modified or new proteins. Each of these approaches has advantages and drawbacks. The use of molecular biology to create new proteins expands the range of tools available with which to study protein structure and function. The ability to construct genes encoding any desired amino acid sequence is now relatively simple. In experiments in which the desired mutant sequence is known (e.g. site-directed mutagenesis), the chosen method will almost certainly use an oligonucleotide. In other cases, in which the goal is to identify a protein with a desired activity, the amino acid sequence that yields this activity may not be known. In this situation, a random mutagenesis strategy provides a route to the desired protein. Thus, the decision to use a sitedirected or random approach depends largely on the questions that are asked. In either case, constructing the mutant DNA is only part of the problem. The vector for expression, the expression system, strategies for purification and assay must also be considered before embarking on protein mutagenesis and engineering.
Methods for oligonucleotide-directed mutagenesis
Abbreviations DHFR~ihydrofolate reductase; hGH--human growth hormone; PCR--polymerase chain reaction; ]-[--tetanus toxoid.
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New molecular biology methods for protein engineering 7oiler the PCR product into a vector and the documented inaccuracy of the Taq DNA polymerase hinder this approach. Unlike primer-extension mutagenesis using T4 DNA polymerase, the sequence of the entire amplified segment generated by PCR mutagenesis must be determined to be confident that there are no extraneous mutations. Until more accurate polymerases are developed, I think PCRbased mutagenesis is best applied in random-mutagenesis experiments and in the construction of complicated chimeric proteins.
Constructinggenes encoding chimeric proteins The analysis of chimeric proteins can be an efficient method of localizing functional domains. This approach has achieved great success in understanding the structure and function of seven-helix receptors [8]. In the past, genes encoding chimeric proteins were assembled following several approaches: d e n o r a gene synthesis; ligation Of oligonucleotide cassettes [9]; or 'loopout' deletion mutagenesis using an oligonucleotide [10]. Constructs in which an amino-terminal segment from one protein is fused to a carboxy-terminal segment from another protein are most easily accomplished by loop-out deletion mutagenesis. In preparing a complicated chimera, however, for example one that requires replacement of an internal segment, d e n o v o gene synthesis and oligonucleotide cassette ligation become tedious and costly. Several PCR-based methods provide a Simple means of constructing chimeras via cassette ligation. As in site-directed PCR mutagenesis, these methods use one or two mutagenic oligonucleotides and require two amplification cycles. However, two studies have been reported that combined PCR amplification with highefficiency primer-extension mutagenesis, and used only a single PCR amplification [11,12-].
Random mutagenesis Random mutagenesis has been used to search for proteins with desired properties or to study the effect of placing all possible residues at particular positions. For mutagenesis over a limited region, 'doped' oligonucleotides have been successfully used; these are synthesized using nucleotide precursors that are contaminated by a small amount of the other three nucleotides. This procedure yields a random distribution of point changes throughout the region defined by the oligonucleotide. Although this technique is not new [13,14], it has been improved by the inclusion of an efficient mutant-selection procedure [15,16]. The value of this technique is limited by the length of oligonucleotide that can be synthesized. Alternatively, a library of mutants throughout the protein can be prepared by using a series of doped oligomers that cover the entire coding sequence when placed endto-end [17"]. Using this method, however, mutations in more than one segment are difficult to obtain. Alternative strategies use PCR [18,19.] or thioate nucleotides [20.] to create randomly dispersed mutations throughout a region of DNA by misincorporation.
Strategies to identify functional residues and domains Systematic mutational analysisof protein interfaces The importance of protein-protein interactions in biological processes is well recognized. A common experimental approach to studying protein complexes is to mutate the cDNA for a protein, introduce the mutant DNA into cells, then assess the ability of the protein variant to complex with other proteins. The goal in these experiments is to identify domains that are important in protein-protein association. Following examples of 'promoter bashing' by deletion mutagenesis, proteincoding sequences have been subjected to similar abuses. For example, regions of the retinoblastoma anti-oncogene protein that complex with EIA oncoprotein and simian virus 40 large T antigen have been mapped by a PCR-based deletion-mutagenesis approach [21.]. Alternatively, oligonucleotide linkers that maintain the reading frame can be introduced into a coding sequence [22]. This strategy offers a crude means of identifying domains in a protein by locally disrupting structure but relies on the chance positioning of restriction sites. Although the successes of these crude approaches have surprised many structural protein chemists, there are now several strategies that provide a more rational way to attack this problem [23.]. Homolog-scanning mutagenesis has been used to identify segments of human growth hormone (hGH) that are important for binding to the hGH receptor by assaying chimeras containing segments of hGH and prolactin [24]. This approach can be taken when two structurally related proteins have different functions. Once an important region has been identified, it can be further dissected by alanine-scanning mutagenesis in which each amino acid is sequentially changed to alanine [25]. For many proteins, an identity-based strategy cannot be used and introduction of individual alanine substitutions at every position in the protein would be a tedious job. A simple yet effective approach termed charged-to-alanine scanning mutagenesis can be used to identify important regions within a protein [26.]; in this scheme, charged residues that cluster within a window of ~ 8 amino acids are changed to alanine. In a recent example of chargedto-alanine mutagenesis, a set of approximately 60 mutants constructed by this approach was used to identify functionally or structurally important regions of a protein kinase [27.]. When the 60 mutants were introduced into yeast, only one mutant was inactive and this contained a substitution of a conserved residue thought to be involved in catalysis. The rationale for this approach is that protein-protein interactions will occur via surfaceexposed residues. By inspection of protein structures, the charged residues are generally.located on the surface. For a protein of unknown structure, mutants made by this strategy will target the surface with high probability and, therefore, few variants will be misfolded. Substitution of alanine is expected to result in a loss of function by removal (not creation) of an interaction. By this ap-
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Proteinengineering proach, functional regions are quickly identified and can be subsequently studied in greater detail.
Chemical modification of cysteine substitutions An interesting combination of site-directed mutagenesis and chemical modification has been reported in a study of ribulosebisphosphate carboxylase [28.]. Several variants were constructed that contained a single cysteine substitution at desired positions. The substituted proteins were subsequently treated with chemical reagents that selectively modified cysteines, resulting in the substitution of non-natural amino acids. In vivo functional screening or selection
Random mutagenesis has been employed to study enzyme mechanism, structure and evolution. In order to sample a large number of mutants, a genetic screen or selection can be used to identify functional, as opposed to non-functional, proteins. A genetic approach is the only practical way of identifying a particular protein variant encoded by one out of a million DNAs. In one approach, a library of variants of I repressor was constructed by ligating a degenerate oligonucleotide cassette into an expression vector [29"]. The library was introduced into Escberichia coli cells and the DNAs encoding functional and non-functional variants were identified and sequenced. The results showed the relative contributions of a particular amino acid to function and structure. In another example, random mutagenesis with a genetic selection procedure was used to improve the catalytic efficiency of a debilitated variant of triose-phosphate isomerase [17"]. A mutant library was prepared by primer-extension mutagenesis using doped oligonucleotides and introduced into cells lacking the enzyme. In order to enrich the library for mutant DNAS, the phosphothioate-incorporation method of Eckstein [4] was used. Other random-mutagenesis strategies have been discussed above. Despite the ease with which random mutations can be generated, the interpretation of the effect(s) of a particular substitution is often difficult. For example, most mutants will contain more than one amino acid substitution which must be separated in order to identify the key substitution responsible for the activity. In addition, the substitution of a particular residue, such as the removal of a cysteine or the introduction of a charged amino acid into the protein interior, can have a global effect on structure and function. Despite these drawbacks, random in vitro mutagenesis coupled to a genetic system continues to be a powerful means of generating proteins with altered (desirable) properties.
Systems for expression of protein variants Fusion proteins displayed on filamentous phage As described above, a library of mutants can be analysed by introduction into an E.. coli expression/sequencing
system. With either £-phage or plasmid-based expression vectors, technical problems limit the number of independent mutants that can be analysed to ,,~ 106. An exciting alternative system, using vectors derived from filamentous phage such as fd and M13, allows for the production and screening of > 1010 independent mutants. In the initial description of this method, degenerate oligonucleotides were ligated in frame with a minor phage coat protein encoded by gene III [30]. As the singlestranded vector DNA is packaged into virions, the gene III fusion protein becomes anchored to the phage partid e via the carboxy-terminal region of the gene 11Iprotein, thereby orientating the random sequences such that they are exposed to the solution. In this way, a library of fusion proteins is made such that each phage expresses a different exogenous protein. Selected phage can be bound to affinity columns containing a peptide receptor or an antipeptide antibody. The peptide sequence that binds the column can be determined by sequencing the isolated phage DNA. This system was initially applied to short ( < 50 amino acids) peptide segments [31"-33"]. This strategy has now been extended to the analysis of entire proteins. Proteins recently sequenced in this way include hGH [34"] and single-chain antibodies [35"]. This technology will allow us to select a protein ligand from a complex mixture of gene segments. Alternatively, fusion phage can be used to obtain ligands with higher, or more selective, alNaities for a receptor.
Expression of recombinant proteins in 'suppressor strains' Often, it is desirable to test the effect of substitution of all 20 amino acids at a particular position in a protein. The conventional way to do this is to prepare 20 different expression vectors encoding the wild-type and 19 mutant proteins. Several years ago, a scheme was proposed that would allow the 19 variants to be prepared from only one expression vector containing an amber stop codon at the position of interest. This would be done by preparing a suppressor tRNA for each amino acid. In order to produce a particular variant, one need only co-express in E. coli the suppressor tRNA for the amino acid to be inserted and the amber-containing expression vector. From a set of suppressor tRNAs constructed, 11 selectively incorporated only a single amino acid [36"]. Whether or not strains 'expressing these tRNAs will find general use is not yet known. Although the specificity of the 11 tRNAs appears to be quite high, an undetectably low level of mischarging could cloud the interpretation of the results. This strategy may provide a way to quickly screen through a library of variants to identify a subset that can be expressed by conventional means and studied in detail.
Cloning antibodies from libraries A promising new method is the application of PCR technology to antibody engineering. DNA encoding the variable regions of antibodies can be cloned by amplification of mRNA from spleen cells, lymphocytes or hybridoma
New molecular biology methodsfor protein engineering Zoller 529 cells. In one report, the amplified cDNAs for heavy and light chains were initially cloned separately then recombined randomly into a X-phage expression vector [37]. The phage DNA was packaged in vitro and, following infection of E. coli, the library of antibodies was expressed in plaques. Plaque lifts were screened to identify molecules that recognized a desired ligand. A recent report has used this approach to isolate an antibody that recognized the highly antigenic tetanus toxoid (TT) [38"]. It has been suggested that this approach may serve as an alternative to the standard methods for monoclonal antibody production. One of the problems with this approach is that the expression level in plaques is relatively low and, therefore, a sensitive assay is necessary. Perhaps a bigger problem is that the complexity of the library after mixing the heavy- and light-chain genes is low compared with the total number of possible combinations. In an alternative method, the variable light- and heavy-chain gene segments were ligated in vitro to form a singlechain molecule linked by a flexible spacer [39"]. Such an approach, coupled with fusion phage expression may increase the repertoire of these combinatorial libraries.
Perspectives and future developments The methodologies for altering protein-coding sequences have not changed much in the past year. Most reports in the literature involve the use of site-directed mutagenesis to test the function of a particular amino acid residue. It is my opinion that any of the primer-extension methods provide the simplest and quickest means of accomplishing this. Site-directed mutagenesis by PCR requires more manipulation of the DNA and is more likely to introduce extraneous mutations. However, PCR does provide a simple way of making chimeric molecules, though the inaccuracy of Taq polymerase is still a problem. With these ideas in mind, I have generated my 'wish list' for future developments: cheaper oligonucleotides ( < $0.50 per base) and preparation of reduced amounts (90 % of what is made is not used); longer oligonucleotides (at least 150 bases) with no mistakes; automated mutagenesis (from primer extension through DNA sequencing; see [40"]); more precise polymerases (especially for PCR); doped oligonucleotides that randomize codon trimers versus individual bases; and, perhaps the most bizarre wish, oligonucleotide-directed mutagenesis by in vitro gene conversion (addition of a mutagenic oligonucleotide to an extract that recombines the mutation into a template molecule in vitro w i t h 100 % efficiency).
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: . of interest ,, of outstanding interest 1.
ZOIIERMJ, Storm M: Oligonucleotide-Directed Mutagenesis Using M13-Derivcd Vectors: an Efficient and General Pro-
cedure for the Production of Point Mutations in Any Fragm e n t of DN& Nucleic Acids Res 1982, 10:64874500.
2. 3.
WE R, GROSSMAN L (eds): Methods in Enzymology, Vol 154 [book]. New York: Academic Press, 1987, pp 329-402. KUNKELT& Rapid and Efficient Site-Specific Mutagenesis W i t h o u t Phenotypic Selection. Proc Natl Acad Sci USA 1985,
82:488-492. 4.
TAYLORJW, OTr J, ECKSTEIN F: The Rapid Generation of Oligonucleotide-Directed Mutations at High Frequency Using Phosphorothioate-Modified DNA~ Nucleic Acids Res 1985, 13:8765-8785.
5. ,
OLSEN DB, ECKSTEIN F: High-Efficiency OligonucleotideDirected Plasmid Mutagenesis. Proc Natl Acad Sci USA 1990, 87:1451-1455. A highly efficient, though tedious, oligouudeotide-directed mutagenesis method that uses a double-stranded instead of single-stranded DNA template. The method is based on Eckstein's original thionucleotide selction procedure [4]. 6.
HIGUCHIR, KRUMMELB, SAIKI RK: A General Method of I n Vitro Preparation and Specific Mutagenesis of DNA Fragments: Study of Protein and DNA Interactions. Nucleic Acids Res 1988, 16:7351-7367.
7. .
PERR1NS, GImLAND G: Site-Specific Mutagenesis Using Asymmetric Polymerase Chain Reaction and a Single Mutant Primer. Nucleic Acids Res 1990, 18:7433-7438. A PCR method for oligo-directed mutagenesis based on a method of Itiguchi et al. [6] that uses only a single mutagenic primer and two amplification cycles. 8.
O'DOWDBF, HNATOWICH M, REGANJ'~, LEADER"0~i, CARON MG, LEFKOWITZRJ: Site-Directed Mutagenesis of the Cytoplasmic Domains of the Human Beta 2-Adrenergic Receptor. Localization of Regions Involved in G Protein-Receptor Coupling. J Biol Cbem 1988, 263:15985-15992.
9.
WELLSJA, VASSERM, POWERS DB: Cassette Mutagenesis: an Efficient Method for G e n e r a t i o n of Multiple Mutations at Defined Sites. Gene 1985, 34:315-323.
10.
CHANVL, SMITH M: In Vitro Generation of Specific Deletions in DNA Cloned in M13 Vectors Using Synthetic Oligodeoxyribonucleotides: Mutants in the 5'-Flanking Region of the Yeast Alcohol Dehydrogenase II. Nucleic Acids Res 1984, 12:2407-2419.
11.
CLACKSONT, WINTER G: 'Sticky Feet'-Directed Mutagenesis and its Application to Swapping Antibody Domains. Nucleic Acids Res 1989, 17:10163-10170.
12. .
XXrYCHOWSKIC, EMERSON SU, SILVERJ, FEINSTONE SM: Construction of Recombinant DNA Molecules by the Use of a Single Stranded DNA Generated by the Polymerase Chain Reaction: its Application to Chimeric Hepatitus A Virus/Poliovirus Subgenomic cDNA. Nucleic Acids Res 1990, 18:913-918. A procedure for construction of chimeric genes which is similar to that of Clackson and Winter [11] but uses the Eckstein oligo-mutagenesis method in the primer extension step. The efficiency of successful mutagenesis is only ~ 10%. 13.
MCNEILJB, SMITHM: Saccharomyces cerevisiae CYC1 mRNA 5'-End Positioning: Analysis by In Vitro Mutagenesis, Using Synthetic Duplexes with Random Mismatch Base Pairs. Mol Cell Biol 1985, 5:3545-3551.
14.
HUTCHISONCA, NORDEEN SK, VOGT K, EDGELL MH: A Complete Library of Point Substitution Mutations in the Glucocorticoid Response Element of Mouse Mammary T u m o r Virus. Proc Natl Acad Sci USA 1986, 83:710--714.
15.
HERMESJD, PAREKH SM, BLACKLOWSC, KOSTER H, KNOWLES JR: A Reliable Method for Random Mutagenesis: the Generation of Mutant Libraries Using Spiked Oligodeoxyribonucleotide Primers. Gene 1989, 84:143-151.
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LOEB DD, SWANSTROM1{, EVERITT L, MANCHESTERM, STAMPER SE, HUTCHISONCA: Complete Mutagenesis of t h e HIV-1 Protease. Nature 1989, 340:397-400.
17. •
HERMESJD, BLACKLOWSC, KNOWLESJR: Searching Sequence Space by Definably R a n d o m Mutagenesis: Improving the Catalytic Potency of an Enzyme. Proc Natl Acad Sci USA 1990, 87:696-700. Doped-oligonucleotide-directed mutagenesis is used to generate a library of triose-phosphate isomerase mutants. The goal of this experiment is to identify substitutions that improve catalytic efficiency. The starting template encodes an attenuated enzyme, and the mutant library is transformed into an E. coli strain that lacks the enzyme. Those plasraids encoding more active enzymes are isolated and sequenced. 18.
LEUNGD, CHEN, E, GOEDDEL, DV: A Method for Random Mutagenesis of a Defined Segment Using a Modified Polymerase Chain Reaction. Technique 1989, 1:11-15.
19.
LERNERCG, KOBAYASH/ T, INOUYE M: Isolation of Subtilisin • Pro-sequence Mutations that Affect Formation of Active Protease by Localized R a n d o m Polymerase Chain Reaction Mutagenesis. J Biol Chem 1990, 265:20085-20086. Random mutations in the pro-sequence of subtilisin are generated by standard PCR amplification. Following ligation of the mutagenized DNA fragment into the coding sequence, the library of mutants is introduced into E. coli and variants are detected phenotypical/y. The efficiency of successsful mutagenesis is ~ 4 %. HOLML, KOIVULAAK, LEHTOVAAP,A PM, HEMMINKIA, KNOWLES JKC: R a n d o m Mutagenesis Used to Probe the Structure and Function of Bacillus stearothermophilus a-Amylase. Pr~ rein Eng 1990, 3:181-191. A library of random mutants in cz-amylase is generated by misincorporation of ct-thionucleotides according to a previously published procedure [41]. A genetic screen is used to identify clones expressing active enzymes.
in Catalysis and Substrate Interactions. J Biol Cbem 1991, 266:8923-8931. Sixty mutants in the yeast cAMP-dependent protein kinase are prepared using the 'charged-to-alanine' scanning mutagenesis strategy. The mutants are a n t e d in vivo by introduction into a yeast strain that requires a functional protein kinase for growth, and in vitro for kinetic properties. This represents the first systematic mutational analysis of a protein kinase. 28. •
SMITHH13, LARLMERFW, HARTMANFC: An Engineered Change in Substrate Specificity of Ribulosebisphosphate Carboxylase/Oxygenase. J Biol Cbem 1990, 265:1243-1245. A cysteine substitution is first engineered into the enzyme, which is subsequently treated with reagents that selectively react with the side chain of cysteine. In this way, non-natural amino acids can be introduced into proteins. 29. •
REIDHAAR-OLSENJF, SAUER RT: Functionally Acceptable Sub-
30.
SMITHGP: Filamentous Fusion Phage: Novel Expression Vectors that Display Cloned Antigens o n t h e Virion Surface. Science 1985, 228:1315-1317.
stitutions in T w o ~x-Helical Regions of 1 Repressor. Proteins 1990, 7:306-316. A library of genes encoding protein variants is constructed by cassette mutagenesis. Specific residues are targeted using oligonucleotides conraining random codons at the positions of interest. A genetic selection procedure is used to identify clones expressing functional proteins. This analysis provides an experimental understanding of the roles of individual residues and expands the information obtained by inspection of the three-dimensional structure.
20. •
21. •
Hu 03 , DYSON N, HARLOWE: T h e Regions of t h e Retinoblastoma Protein N e e d e d for Binding to Adenovirus E1A or SV40 Large T Antigen are C o m m o n Sites for Mutations. EMBO J 1990, 9:1147-1155. Deletion mutants in retinoblastoma protein are prepared by PCR then expressed in cells to identify regions that associate with the viral oncogene proteins E1A and simian virus 40 large T antigen. Most of the retinobla.stoma variants in this study are stably expressed. For enzymes, however, this approach would probably not be very useful. 22.
STONEJC, ATKINSONT, SMITH M, PAWSON T: Identification of Functional Regions in t h e Transforming Protein of Fujinami Sarcoma Virus by in-Phase Insertion Mutagenesis. Cell 1984, 37:549-558.
23. WELLSJA: Systematic Mutational Analyses of Protein-Protein • Interfaces. Methods Enzymol 1991, in press. A review of systematic approaches to constructing mutations in proteins in order to identify structural and functional determinants. 24.
CUNNINGHAMBC, JHURANI P, NG P, WELLS JA: Receptor and Antibody Epitopes in H u m a n G r o w t h H o r m o n e Identiffed by Homolog-Scanning Mutagenesis. Science 1989, 243:1330-1336.
25.
CUNNINGHAMBC, WELTSJA: High-Resolution Epitope Mapping of h G H - R e c e p t o r Interactions by Alanine-Scanning Mutagenesis. Science 1989, 244:1081-1085.
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BASs S, MULKERIN M, WELLS JA: A Systematic Mutational Analysis of Hormone-Binding D e t e r m i n a n t s in t h e H u m a n Growth H o r m o n e Receptor. Proc Natl Acad Sci USA 1991, 88:4498-4502. 'Charged-to-alanine' scanning mutagenesis is developed in order to identify regions of hGH receptor that interact with the ligand. 27. •
GIBBS CS, ZOLLER MJ: Rational Scanning Mutagenesis of a Protein Kinase Identifies Functional Regions Involved
31. •
CWlRLASE, PETERS EA, BARRETTRW, DOWER WJ: Peptides o n Phage: a Vast Library of Peptides for Identifying Ligands. Proc Natl Acad Sci USA 1990, 87:637845382. A library of random hexapeptides is generated by ligating degenerate oligoncleotide into the gene HI protein of a filamentous phage vector. From 108 recombinants, 51 clones are selected that bind to a monoclonal antibody recognizing a peptide from 13-endorphin. 32. •
DEVLINJJ, PANGANIBANLC, DEVLIN PE: R a n d o m Peptide Libraries: a Source of Specific Protein Binding Molecules. Science 1990, 249:404406. A library of 15-residue peptides is expressed on fusion phage. An ollgonucleotide containing 15 degenerate codons is ligated into the amino terminus of gene III in a filamentous phage vector. Phage are selected that bind to streptavidin: all contain a core sequence His-Pro-Gin. 33. SCOTTJK, SMITH GP: Searching for Peptide Ligands w i t h an • Epitope Library. Science 1990, 249:386-390. An application of the phage-display technology reported originally by Smith [30]. A library of random hexapeptides is generated by llgation of a degenerate oligonucleotide into a phage vector. Phage that bind to two monoclonal antibodies are selected. 34. •
BASS S, GREENE R, WELLSJ& H o r m o n e Phage: an Enrichment Method for Variant Proteins w i t h Altered Binding Properties. Proteins 1990, 8:309-314. A variation of pha$e display in which the entire gene encoding hGH is fused to gene HI. H u m a n growth h o r m o n e phage is selectively b o u n d to an hGH receptor affinity colunm. The vector used in this study is a phagemid. A helper phage, M13KO7, is used to package the phagemid DNA: This results in a mixture of wild-type gene 111 protein (necessary for phage infection) and the gene 11I fusion protein being present on the phage surface. This mixture maintains the infectivity of the fusion phage and limits the n u m b e r of fusion proteins b o u n d to a phage. This last feature is vital to the successful selection of proteins/peptides with high affinities. 35. •
MCCAFFERTYJM, GRIFFITHSAD, WINTER G, CHISWELLDJ: Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains. Nature 1990, 348:552-554. A fusion phage is prepared that expresses on the phage surface the variable regions of an antilysozyme antibody. A single-chain antibody-gene Ill fusion gene is constructed that consists of the heavy-chain gene linked to the light chain gene by a flexible peptide segment (GtY4Ser) 3. Phage expressing the single-chain antibody is selectively b o u n d to a lysozyme afl'mity column.
New molecular biology methods for protein engineering Zoller NORMANLYJ, KLEINA LG, MASSONJ-M, ABELSON J, MIH.FR JH: Construction of Escherichia coli Amber Suppressor tRNA Genes llI: Determination of tRNA Specificity. J Mol Biol 1990, 213:719-726. A set of suppressor tRNAs is tested for its ability to selectively insert the specificed amino acid into a protein. A gene for dihydrofolate reductase (DHFR) containing an amber codon at position 10 is co-expressed with each of the suppressor tRNAs. The expressed DHFR and the selectivity of each tRNA is determined by sequencing to determine the amino acid(s) present at position 10. Transfer RNAs selectively insert a single amino acid into DHFR. Eleven other tRNAs fail to be selective and insert either lysine or glutamine. 36. •
37.
HUSE WD, SASTRY L, IVERSON SA, KANG AS, ALTING-MEES M, BURTON DR, BENKOVIC SJ, LERNER RA: Generation of a Large Combinatorial Library of t h e Immunogiobulin Repertoire in Phage Lambda [See C o m m e n t s ] Science 1989, 246:1275-1289.
38. •
MULIaMAXRL, GROSS F ~ AMBERG JR ET AL.: Identification of H u m a n Antibody Fragment Clones Specific for T e t a n u s Toxoid in a Bacteriophage ~ I m m u n o e x p r e s s i o n Library. Proc Natl Acad Sci USA 1990, 87:8095-8099. An application of a procedure described previously [37] for cloning genes for antibodies to a specified ligand. Lymphocyte mRNA is isolated from humans that have been immunized with TF. Following PCR amplification, genes encoding the heavy and light chains are cloned separately, then recombined into a single 1 phage vector. The phage library is plated onto a lawn of E. colg, and phage expressing antibodies that bind T r are identified by screening plaque lifts using radiolabelled TF. 39.
CHAUDHARY VK, BATRA JK, GALLO MG, WILLINGHAM MC, FITZGERALD DJ, PASTAN I: A Rapid Method of Cloning Functional Variable-Region Antibody G e n e s in Escherichia coli as Single-Chain Immunotoxins. Proc Natl Acad Sci USA 1990, 87:1066-1070.
This paper describes a strategy for cloning the genes encoding antibody variable domains into an E. coli expression vector. PCR amplification is used to isolate variable light (VL) and heavy (VH) chain coding sequences from hybridoma mRNA- The PCR primers are designed so that the VII and VL segments can be ligated in frame to encode a single-chain antibody. The antibody encoding DNA is ligated into an expression vector so that it is produced as a fusion protein linked to a bacterial exotoxin, thus forming a 'single-chain immunotoxin'. This paper contains useful information for designing PCR primers and linkers that can be used to clone antibody coding sequences. 40. •
HULTMANT, MURBY M, STAHL S, HORNES E, UIMEN M: Solid Phase In Vitro Mutagenesis Using Plasmid DNA Template. Nucleic Acids Res 1990, 18:5107-5112. This paper reports a method for oligonucleotide-directed mutagenesis in which the DNA template is b o u n d to a solid support, in this case magnetic beads. In the first step, PCR is used to prepare a long singlestranded DNA molecule that contains the mutant oligonucleotide and to prepare a single-stranded vector molecule with ends that are complementary to the mutant ollgomer. In the second step, the two DNAs are annealed and simply transformed into E. coli. This method uses technology from an earlier report describing the preparation of templates for DNA sequencing. Unlike most PCR-based mutagenesis methods, this procedure only requires a single amplification cycle. However, the PCR step in which single-stranded vector DNA is prepared may introduce undesirable mutations. However, this approach suggests that highefficiency automated mutagenesis will eventually be possible. 41.
LEHTOVAARAPM, KOIVULAAK, BAMFORDJ, KNOWLESJK: A N e w M e t h o d for Random Mutagenesis of Complete Genes: Enzymatic Generation of Mutant Libraries I n Vitro. Protein Eng 1988, 2:63-68.
MJ Zoller, Department of Protein Engineering, Genentech, 460 Pt. San Bruno Blvd., South San Francisco, California 94080, USA.
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Introduction
Point mutations
striction sites flanking the region of interest, and the limitation on the size (and perhaps expense) of the oligonudeotides that comprise the synthetic fragment. Enzymatic extension of mutagenic oligonudeotides hybridized to single-stranded DNA templates provides an alternative method that overcomes the restrictions of the cassette method [1]. The drawback of the primerextension method is that the efficiency of mutagenesis is generally less than 100 %. A number of methods, developed over 5 years ago, introduce genetic 'tricks' to obtain greater than 50% mutagenesis efficiency [2]. The method of Kunkel [3] is popular largely because of its simplicity and because it is now available in a kit form. The use of the highly accurate DNA polymerases from phage T4 or T7 results in virtually no extraneous mutations and has shortened the polymerization time required to copy the second strand to less than 2 h. These polymerases do not 'strand displace' the 5' end of the oligomer, and polymerization can be conducted at 37 °C. For slightly greater efficiency, the phosphothioate-incorporation method of Eckstein and colleagues [4] is gaining in popularity and is also available in kit form. Recently, this method has been adapted to operate on a double-stranded DNA template [5"]. This method has many more steps than the Kunkel procedure and is thus more tedious. A feature of the Eckstein methdd, however, is that the mutation encoded by the oligonucleotide is present in the resulting DNA molecule as a homoduplex. The features of this method have been exploited in doped-oligonucleotide mutagenesis experiments, which are discussed below.
Methods for the construction of point mutations in DNA have not changed greatly over the past few years. Cassette mutagenesis, in which a synthetic DNA fragment containing the desired sequence is ligated into the coding sequence is a simple method for which the efficiency of mutagenesis is virtually 100 %. The drawbacks of this method are, of course, the requirement for unique re-
The ease of the polymerase chain reaction (PER) procedure naturally spawned methods for site-directed mutagenesis [6,7.]. Generally, these methods use either one or two mutagenic primers and require two rounds of PeR amplification [2]. The advantage of a PCR method, as in cassette mutagenesis, is that the desired mutation is obtained with 100% efficiency. However, the need to ligate
Protein engineering is accomplished by the modification of an existing protein, d e n o v o peptide synthesis or the expression of genes encoding modified or new proteins. Each of these approaches has advantages and drawbacks. The use of molecular biology to create new proteins expands the range of tools available with which to study protein structure and function. The ability to construct genes encoding any desired amino acid sequence is now relatively simple. In experiments in which the desired mutant sequence is known (e.g. site-directed mutagenesis), the chosen method will almost certainly use an oligonucleotide. In other cases, in which the goal is to identify a protein with a desired activity, the amino acid sequence that yields this activity may not be known. In this situation, a random mutagenesis strategy provides a route to the desired protein. Thus, the decision to use a sitedirected or random approach depends largely on the questions that are asked. In either case, constructing the mutant DNA is only part of the problem. The vector for expression, the expression system, strategies for purification and assay must also be considered before embarking on protein mutagenesis and engineering.
Methods for oligonucleotide-directed mutagenesis
Abbreviations DHFR~ihydrofolate reductase; hGH--human growth hormone; PCR--polymerase chain reaction; ]-[--tetanus toxoid.
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New molecular biology methods for protein engineering 7oiler the PCR product into a vector and the documented inaccuracy of the Taq DNA polymerase hinder this approach. Unlike primer-extension mutagenesis using T4 DNA polymerase, the sequence of the entire amplified segment generated by PCR mutagenesis must be determined to be confident that there are no extraneous mutations. Until more accurate polymerases are developed, I think PCRbased mutagenesis is best applied in random-mutagenesis experiments and in the construction of complicated chimeric proteins.
Constructinggenes encoding chimeric proteins The analysis of chimeric proteins can be an efficient method of localizing functional domains. This approach has achieved great success in understanding the structure and function of seven-helix receptors [8]. In the past, genes encoding chimeric proteins were assembled following several approaches: d e n o r a gene synthesis; ligation Of oligonucleotide cassettes [9]; or 'loopout' deletion mutagenesis using an oligonucleotide [10]. Constructs in which an amino-terminal segment from one protein is fused to a carboxy-terminal segment from another protein are most easily accomplished by loop-out deletion mutagenesis. In preparing a complicated chimera, however, for example one that requires replacement of an internal segment, d e n o v o gene synthesis and oligonucleotide cassette ligation become tedious and costly. Several PCR-based methods provide a Simple means of constructing chimeras via cassette ligation. As in site-directed PCR mutagenesis, these methods use one or two mutagenic oligonucleotides and require two amplification cycles. However, two studies have been reported that combined PCR amplification with highefficiency primer-extension mutagenesis, and used only a single PCR amplification [11,12-].
Random mutagenesis Random mutagenesis has been used to search for proteins with desired properties or to study the effect of placing all possible residues at particular positions. For mutagenesis over a limited region, 'doped' oligonucleotides have been successfully used; these are synthesized using nucleotide precursors that are contaminated by a small amount of the other three nucleotides. This procedure yields a random distribution of point changes throughout the region defined by the oligonucleotide. Although this technique is not new [13,14], it has been improved by the inclusion of an efficient mutant-selection procedure [15,16]. The value of this technique is limited by the length of oligonucleotide that can be synthesized. Alternatively, a library of mutants throughout the protein can be prepared by using a series of doped oligomers that cover the entire coding sequence when placed endto-end [17"]. Using this method, however, mutations in more than one segment are difficult to obtain. Alternative strategies use PCR [18,19.] or thioate nucleotides [20.] to create randomly dispersed mutations throughout a region of DNA by misincorporation.
Strategies to identify functional residues and domains Systematic mutational analysisof protein interfaces The importance of protein-protein interactions in biological processes is well recognized. A common experimental approach to studying protein complexes is to mutate the cDNA for a protein, introduce the mutant DNA into cells, then assess the ability of the protein variant to complex with other proteins. The goal in these experiments is to identify domains that are important in protein-protein association. Following examples of 'promoter bashing' by deletion mutagenesis, proteincoding sequences have been subjected to similar abuses. For example, regions of the retinoblastoma anti-oncogene protein that complex with EIA oncoprotein and simian virus 40 large T antigen have been mapped by a PCR-based deletion-mutagenesis approach [21.]. Alternatively, oligonucleotide linkers that maintain the reading frame can be introduced into a coding sequence [22]. This strategy offers a crude means of identifying domains in a protein by locally disrupting structure but relies on the chance positioning of restriction sites. Although the successes of these crude approaches have surprised many structural protein chemists, there are now several strategies that provide a more rational way to attack this problem [23.]. Homolog-scanning mutagenesis has been used to identify segments of human growth hormone (hGH) that are important for binding to the hGH receptor by assaying chimeras containing segments of hGH and prolactin [24]. This approach can be taken when two structurally related proteins have different functions. Once an important region has been identified, it can be further dissected by alanine-scanning mutagenesis in which each amino acid is sequentially changed to alanine [25]. For many proteins, an identity-based strategy cannot be used and introduction of individual alanine substitutions at every position in the protein would be a tedious job. A simple yet effective approach termed charged-to-alanine scanning mutagenesis can be used to identify important regions within a protein [26.]; in this scheme, charged residues that cluster within a window of ~ 8 amino acids are changed to alanine. In a recent example of chargedto-alanine mutagenesis, a set of approximately 60 mutants constructed by this approach was used to identify functionally or structurally important regions of a protein kinase [27.]. When the 60 mutants were introduced into yeast, only one mutant was inactive and this contained a substitution of a conserved residue thought to be involved in catalysis. The rationale for this approach is that protein-protein interactions will occur via surfaceexposed residues. By inspection of protein structures, the charged residues are generally.located on the surface. For a protein of unknown structure, mutants made by this strategy will target the surface with high probability and, therefore, few variants will be misfolded. Substitution of alanine is expected to result in a loss of function by removal (not creation) of an interaction. By this ap-
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Proteinengineering proach, functional regions are quickly identified and can be subsequently studied in greater detail.
Chemical modification of cysteine substitutions An interesting combination of site-directed mutagenesis and chemical modification has been reported in a study of ribulosebisphosphate carboxylase [28.]. Several variants were constructed that contained a single cysteine substitution at desired positions. The substituted proteins were subsequently treated with chemical reagents that selectively modified cysteines, resulting in the substitution of non-natural amino acids. In vivo functional screening or selection
Random mutagenesis has been employed to study enzyme mechanism, structure and evolution. In order to sample a large number of mutants, a genetic screen or selection can be used to identify functional, as opposed to non-functional, proteins. A genetic approach is the only practical way of identifying a particular protein variant encoded by one out of a million DNAs. In one approach, a library of variants of I repressor was constructed by ligating a degenerate oligonucleotide cassette into an expression vector [29"]. The library was introduced into Escberichia coli cells and the DNAs encoding functional and non-functional variants were identified and sequenced. The results showed the relative contributions of a particular amino acid to function and structure. In another example, random mutagenesis with a genetic selection procedure was used to improve the catalytic efficiency of a debilitated variant of triose-phosphate isomerase [17"]. A mutant library was prepared by primer-extension mutagenesis using doped oligonucleotides and introduced into cells lacking the enzyme. In order to enrich the library for mutant DNAS, the phosphothioate-incorporation method of Eckstein [4] was used. Other random-mutagenesis strategies have been discussed above. Despite the ease with which random mutations can be generated, the interpretation of the effect(s) of a particular substitution is often difficult. For example, most mutants will contain more than one amino acid substitution which must be separated in order to identify the key substitution responsible for the activity. In addition, the substitution of a particular residue, such as the removal of a cysteine or the introduction of a charged amino acid into the protein interior, can have a global effect on structure and function. Despite these drawbacks, random in vitro mutagenesis coupled to a genetic system continues to be a powerful means of generating proteins with altered (desirable) properties.
Systems for expression of protein variants Fusion proteins displayed on filamentous phage As described above, a library of mutants can be analysed by introduction into an E.. coli expression/sequencing
system. With either £-phage or plasmid-based expression vectors, technical problems limit the number of independent mutants that can be analysed to ,,~ 106. An exciting alternative system, using vectors derived from filamentous phage such as fd and M13, allows for the production and screening of > 1010 independent mutants. In the initial description of this method, degenerate oligonucleotides were ligated in frame with a minor phage coat protein encoded by gene III [30]. As the singlestranded vector DNA is packaged into virions, the gene III fusion protein becomes anchored to the phage partid e via the carboxy-terminal region of the gene 11Iprotein, thereby orientating the random sequences such that they are exposed to the solution. In this way, a library of fusion proteins is made such that each phage expresses a different exogenous protein. Selected phage can be bound to affinity columns containing a peptide receptor or an antipeptide antibody. The peptide sequence that binds the column can be determined by sequencing the isolated phage DNA. This system was initially applied to short ( < 50 amino acids) peptide segments [31"-33"]. This strategy has now been extended to the analysis of entire proteins. Proteins recently sequenced in this way include hGH [34"] and single-chain antibodies [35"]. This technology will allow us to select a protein ligand from a complex mixture of gene segments. Alternatively, fusion phage can be used to obtain ligands with higher, or more selective, alNaities for a receptor.
Expression of recombinant proteins in 'suppressor strains' Often, it is desirable to test the effect of substitution of all 20 amino acids at a particular position in a protein. The conventional way to do this is to prepare 20 different expression vectors encoding the wild-type and 19 mutant proteins. Several years ago, a scheme was proposed that would allow the 19 variants to be prepared from only one expression vector containing an amber stop codon at the position of interest. This would be done by preparing a suppressor tRNA for each amino acid. In order to produce a particular variant, one need only co-express in E. coli the suppressor tRNA for the amino acid to be inserted and the amber-containing expression vector. From a set of suppressor tRNAs constructed, 11 selectively incorporated only a single amino acid [36"]. Whether or not strains 'expressing these tRNAs will find general use is not yet known. Although the specificity of the 11 tRNAs appears to be quite high, an undetectably low level of mischarging could cloud the interpretation of the results. This strategy may provide a way to quickly screen through a library of variants to identify a subset that can be expressed by conventional means and studied in detail.
Cloning antibodies from libraries A promising new method is the application of PCR technology to antibody engineering. DNA encoding the variable regions of antibodies can be cloned by amplification of mRNA from spleen cells, lymphocytes or hybridoma
New molecular biology methodsfor protein engineering Zoller 529 cells. In one report, the amplified cDNAs for heavy and light chains were initially cloned separately then recombined randomly into a X-phage expression vector [37]. The phage DNA was packaged in vitro and, following infection of E. coli, the library of antibodies was expressed in plaques. Plaque lifts were screened to identify molecules that recognized a desired ligand. A recent report has used this approach to isolate an antibody that recognized the highly antigenic tetanus toxoid (TT) [38"]. It has been suggested that this approach may serve as an alternative to the standard methods for monoclonal antibody production. One of the problems with this approach is that the expression level in plaques is relatively low and, therefore, a sensitive assay is necessary. Perhaps a bigger problem is that the complexity of the library after mixing the heavy- and light-chain genes is low compared with the total number of possible combinations. In an alternative method, the variable light- and heavy-chain gene segments were ligated in vitro to form a singlechain molecule linked by a flexible spacer [39"]. Such an approach, coupled with fusion phage expression may increase the repertoire of these combinatorial libraries.
Perspectives and future developments The methodologies for altering protein-coding sequences have not changed much in the past year. Most reports in the literature involve the use of site-directed mutagenesis to test the function of a particular amino acid residue. It is my opinion that any of the primer-extension methods provide the simplest and quickest means of accomplishing this. Site-directed mutagenesis by PCR requires more manipulation of the DNA and is more likely to introduce extraneous mutations. However, PCR does provide a simple way of making chimeric molecules, though the inaccuracy of Taq polymerase is still a problem. With these ideas in mind, I have generated my 'wish list' for future developments: cheaper oligonucleotides ( < $0.50 per base) and preparation of reduced amounts (90 % of what is made is not used); longer oligonucleotides (at least 150 bases) with no mistakes; automated mutagenesis (from primer extension through DNA sequencing; see [40"]); more precise polymerases (especially for PCR); doped oligonucleotides that randomize codon trimers versus individual bases; and, perhaps the most bizarre wish, oligonucleotide-directed mutagenesis by in vitro gene conversion (addition of a mutagenic oligonucleotide to an extract that recombines the mutation into a template molecule in vitro w i t h 100 % efficiency).
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: . of interest ,, of outstanding interest 1.
ZOIIERMJ, Storm M: Oligonucleotide-Directed Mutagenesis Using M13-Derivcd Vectors: an Efficient and General Pro-
cedure for the Production of Point Mutations in Any Fragm e n t of DN& Nucleic Acids Res 1982, 10:64874500.
2. 3.
WE R, GROSSMAN L (eds): Methods in Enzymology, Vol 154 [book]. New York: Academic Press, 1987, pp 329-402. KUNKELT& Rapid and Efficient Site-Specific Mutagenesis W i t h o u t Phenotypic Selection. Proc Natl Acad Sci USA 1985,
82:488-492. 4.
TAYLORJW, OTr J, ECKSTEIN F: The Rapid Generation of Oligonucleotide-Directed Mutations at High Frequency Using Phosphorothioate-Modified DNA~ Nucleic Acids Res 1985, 13:8765-8785.
5. ,
OLSEN DB, ECKSTEIN F: High-Efficiency OligonucleotideDirected Plasmid Mutagenesis. Proc Natl Acad Sci USA 1990, 87:1451-1455. A highly efficient, though tedious, oligouudeotide-directed mutagenesis method that uses a double-stranded instead of single-stranded DNA template. The method is based on Eckstein's original thionucleotide selction procedure [4]. 6.
HIGUCHIR, KRUMMELB, SAIKI RK: A General Method of I n Vitro Preparation and Specific Mutagenesis of DNA Fragments: Study of Protein and DNA Interactions. Nucleic Acids Res 1988, 16:7351-7367.
7. .
PERR1NS, GImLAND G: Site-Specific Mutagenesis Using Asymmetric Polymerase Chain Reaction and a Single Mutant Primer. Nucleic Acids Res 1990, 18:7433-7438. A PCR method for oligo-directed mutagenesis based on a method of Itiguchi et al. [6] that uses only a single mutagenic primer and two amplification cycles. 8.
O'DOWDBF, HNATOWICH M, REGANJ'~, LEADER"0~i, CARON MG, LEFKOWITZRJ: Site-Directed Mutagenesis of the Cytoplasmic Domains of the Human Beta 2-Adrenergic Receptor. Localization of Regions Involved in G Protein-Receptor Coupling. J Biol Cbem 1988, 263:15985-15992.
9.
WELLSJA, VASSERM, POWERS DB: Cassette Mutagenesis: an Efficient Method for G e n e r a t i o n of Multiple Mutations at Defined Sites. Gene 1985, 34:315-323.
10.
CHANVL, SMITH M: In Vitro Generation of Specific Deletions in DNA Cloned in M13 Vectors Using Synthetic Oligodeoxyribonucleotides: Mutants in the 5'-Flanking Region of the Yeast Alcohol Dehydrogenase II. Nucleic Acids Res 1984, 12:2407-2419.
11.
CLACKSONT, WINTER G: 'Sticky Feet'-Directed Mutagenesis and its Application to Swapping Antibody Domains. Nucleic Acids Res 1989, 17:10163-10170.
12. .
XXrYCHOWSKIC, EMERSON SU, SILVERJ, FEINSTONE SM: Construction of Recombinant DNA Molecules by the Use of a Single Stranded DNA Generated by the Polymerase Chain Reaction: its Application to Chimeric Hepatitus A Virus/Poliovirus Subgenomic cDNA. Nucleic Acids Res 1990, 18:913-918. A procedure for construction of chimeric genes which is similar to that of Clackson and Winter [11] but uses the Eckstein oligo-mutagenesis method in the primer extension step. The efficiency of successful mutagenesis is only ~ 10%. 13.
MCNEILJB, SMITHM: Saccharomyces cerevisiae CYC1 mRNA 5'-End Positioning: Analysis by In Vitro Mutagenesis, Using Synthetic Duplexes with Random Mismatch Base Pairs. Mol Cell Biol 1985, 5:3545-3551.
14.
HUTCHISONCA, NORDEEN SK, VOGT K, EDGELL MH: A Complete Library of Point Substitution Mutations in the Glucocorticoid Response Element of Mouse Mammary T u m o r Virus. Proc Natl Acad Sci USA 1986, 83:710--714.
15.
HERMESJD, PAREKH SM, BLACKLOWSC, KOSTER H, KNOWLES JR: A Reliable Method for Random Mutagenesis: the Generation of Mutant Libraries Using Spiked Oligodeoxyribonucleotide Primers. Gene 1989, 84:143-151.
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LOEB DD, SWANSTROM1{, EVERITT L, MANCHESTERM, STAMPER SE, HUTCHISONCA: Complete Mutagenesis of t h e HIV-1 Protease. Nature 1989, 340:397-400.
17. •
HERMESJD, BLACKLOWSC, KNOWLESJR: Searching Sequence Space by Definably R a n d o m Mutagenesis: Improving the Catalytic Potency of an Enzyme. Proc Natl Acad Sci USA 1990, 87:696-700. Doped-oligonucleotide-directed mutagenesis is used to generate a library of triose-phosphate isomerase mutants. The goal of this experiment is to identify substitutions that improve catalytic efficiency. The starting template encodes an attenuated enzyme, and the mutant library is transformed into an E. coli strain that lacks the enzyme. Those plasraids encoding more active enzymes are isolated and sequenced. 18.
LEUNGD, CHEN, E, GOEDDEL, DV: A Method for Random Mutagenesis of a Defined Segment Using a Modified Polymerase Chain Reaction. Technique 1989, 1:11-15.
19.
LERNERCG, KOBAYASH/ T, INOUYE M: Isolation of Subtilisin • Pro-sequence Mutations that Affect Formation of Active Protease by Localized R a n d o m Polymerase Chain Reaction Mutagenesis. J Biol Chem 1990, 265:20085-20086. Random mutations in the pro-sequence of subtilisin are generated by standard PCR amplification. Following ligation of the mutagenized DNA fragment into the coding sequence, the library of mutants is introduced into E. coli and variants are detected phenotypical/y. The efficiency of successsful mutagenesis is ~ 4 %. HOLML, KOIVULAAK, LEHTOVAAP,A PM, HEMMINKIA, KNOWLES JKC: R a n d o m Mutagenesis Used to Probe the Structure and Function of Bacillus stearothermophilus a-Amylase. Pr~ rein Eng 1990, 3:181-191. A library of random mutants in cz-amylase is generated by misincorporation of ct-thionucleotides according to a previously published procedure [41]. A genetic screen is used to identify clones expressing active enzymes.
in Catalysis and Substrate Interactions. J Biol Cbem 1991, 266:8923-8931. Sixty mutants in the yeast cAMP-dependent protein kinase are prepared using the 'charged-to-alanine' scanning mutagenesis strategy. The mutants are a n t e d in vivo by introduction into a yeast strain that requires a functional protein kinase for growth, and in vitro for kinetic properties. This represents the first systematic mutational analysis of a protein kinase. 28. •
SMITHH13, LARLMERFW, HARTMANFC: An Engineered Change in Substrate Specificity of Ribulosebisphosphate Carboxylase/Oxygenase. J Biol Cbem 1990, 265:1243-1245. A cysteine substitution is first engineered into the enzyme, which is subsequently treated with reagents that selectively react with the side chain of cysteine. In this way, non-natural amino acids can be introduced into proteins. 29. •
REIDHAAR-OLSENJF, SAUER RT: Functionally Acceptable Sub-
30.
SMITHGP: Filamentous Fusion Phage: Novel Expression Vectors that Display Cloned Antigens o n t h e Virion Surface. Science 1985, 228:1315-1317.
stitutions in T w o ~x-Helical Regions of 1 Repressor. Proteins 1990, 7:306-316. A library of genes encoding protein variants is constructed by cassette mutagenesis. Specific residues are targeted using oligonucleotides conraining random codons at the positions of interest. A genetic selection procedure is used to identify clones expressing functional proteins. This analysis provides an experimental understanding of the roles of individual residues and expands the information obtained by inspection of the three-dimensional structure.
20. •
21. •
Hu 03 , DYSON N, HARLOWE: T h e Regions of t h e Retinoblastoma Protein N e e d e d for Binding to Adenovirus E1A or SV40 Large T Antigen are C o m m o n Sites for Mutations. EMBO J 1990, 9:1147-1155. Deletion mutants in retinoblastoma protein are prepared by PCR then expressed in cells to identify regions that associate with the viral oncogene proteins E1A and simian virus 40 large T antigen. Most of the retinobla.stoma variants in this study are stably expressed. For enzymes, however, this approach would probably not be very useful. 22.
STONEJC, ATKINSONT, SMITH M, PAWSON T: Identification of Functional Regions in t h e Transforming Protein of Fujinami Sarcoma Virus by in-Phase Insertion Mutagenesis. Cell 1984, 37:549-558.
23. WELLSJA: Systematic Mutational Analyses of Protein-Protein • Interfaces. Methods Enzymol 1991, in press. A review of systematic approaches to constructing mutations in proteins in order to identify structural and functional determinants. 24.
CUNNINGHAMBC, JHURANI P, NG P, WELLS JA: Receptor and Antibody Epitopes in H u m a n G r o w t h H o r m o n e Identiffed by Homolog-Scanning Mutagenesis. Science 1989, 243:1330-1336.
25.
CUNNINGHAMBC, WELTSJA: High-Resolution Epitope Mapping of h G H - R e c e p t o r Interactions by Alanine-Scanning Mutagenesis. Science 1989, 244:1081-1085.
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BASs S, MULKERIN M, WELLS JA: A Systematic Mutational Analysis of Hormone-Binding D e t e r m i n a n t s in t h e H u m a n Growth H o r m o n e Receptor. Proc Natl Acad Sci USA 1991, 88:4498-4502. 'Charged-to-alanine' scanning mutagenesis is developed in order to identify regions of hGH receptor that interact with the ligand. 27. •
GIBBS CS, ZOLLER MJ: Rational Scanning Mutagenesis of a Protein Kinase Identifies Functional Regions Involved
31. •
CWlRLASE, PETERS EA, BARRETTRW, DOWER WJ: Peptides o n Phage: a Vast Library of Peptides for Identifying Ligands. Proc Natl Acad Sci USA 1990, 87:637845382. A library of random hexapeptides is generated by ligating degenerate oligoncleotide into the gene HI protein of a filamentous phage vector. From 108 recombinants, 51 clones are selected that bind to a monoclonal antibody recognizing a peptide from 13-endorphin. 32. •
DEVLINJJ, PANGANIBANLC, DEVLIN PE: R a n d o m Peptide Libraries: a Source of Specific Protein Binding Molecules. Science 1990, 249:404406. A library of 15-residue peptides is expressed on fusion phage. An ollgonucleotide containing 15 degenerate codons is ligated into the amino terminus of gene III in a filamentous phage vector. Phage are selected that bind to streptavidin: all contain a core sequence His-Pro-Gin. 33. SCOTTJK, SMITH GP: Searching for Peptide Ligands w i t h an • Epitope Library. Science 1990, 249:386-390. An application of the phage-display technology reported originally by Smith [30]. A library of random hexapeptides is generated by llgation of a degenerate oligonucleotide into a phage vector. Phage that bind to two monoclonal antibodies are selected. 34. •
BASS S, GREENE R, WELLSJ& H o r m o n e Phage: an Enrichment Method for Variant Proteins w i t h Altered Binding Properties. Proteins 1990, 8:309-314. A variation of pha$e display in which the entire gene encoding hGH is fused to gene HI. H u m a n growth h o r m o n e phage is selectively b o u n d to an hGH receptor affinity colunm. The vector used in this study is a phagemid. A helper phage, M13KO7, is used to package the phagemid DNA: This results in a mixture of wild-type gene 111 protein (necessary for phage infection) and the gene 11I fusion protein being present on the phage surface. This mixture maintains the infectivity of the fusion phage and limits the n u m b e r of fusion proteins b o u n d to a phage. This last feature is vital to the successful selection of proteins/peptides with high affinities. 35. •
MCCAFFERTYJM, GRIFFITHSAD, WINTER G, CHISWELLDJ: Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains. Nature 1990, 348:552-554. A fusion phage is prepared that expresses on the phage surface the variable regions of an antilysozyme antibody. A single-chain antibody-gene Ill fusion gene is constructed that consists of the heavy-chain gene linked to the light chain gene by a flexible peptide segment (GtY4Ser) 3. Phage expressing the single-chain antibody is selectively b o u n d to a lysozyme afl'mity column.
New molecular biology methods for protein engineering Zoller NORMANLYJ, KLEINA LG, MASSONJ-M, ABELSON J, MIH.FR JH: Construction of Escherichia coli Amber Suppressor tRNA Genes llI: Determination of tRNA Specificity. J Mol Biol 1990, 213:719-726. A set of suppressor tRNAs is tested for its ability to selectively insert the specificed amino acid into a protein. A gene for dihydrofolate reductase (DHFR) containing an amber codon at position 10 is co-expressed with each of the suppressor tRNAs. The expressed DHFR and the selectivity of each tRNA is determined by sequencing to determine the amino acid(s) present at position 10. Transfer RNAs selectively insert a single amino acid into DHFR. Eleven other tRNAs fail to be selective and insert either lysine or glutamine. 36. •
37.
HUSE WD, SASTRY L, IVERSON SA, KANG AS, ALTING-MEES M, BURTON DR, BENKOVIC SJ, LERNER RA: Generation of a Large Combinatorial Library of t h e Immunogiobulin Repertoire in Phage Lambda [See C o m m e n t s ] Science 1989, 246:1275-1289.
38. •
MULIaMAXRL, GROSS F ~ AMBERG JR ET AL.: Identification of H u m a n Antibody Fragment Clones Specific for T e t a n u s Toxoid in a Bacteriophage ~ I m m u n o e x p r e s s i o n Library. Proc Natl Acad Sci USA 1990, 87:8095-8099. An application of a procedure described previously [37] for cloning genes for antibodies to a specified ligand. Lymphocyte mRNA is isolated from humans that have been immunized with TF. Following PCR amplification, genes encoding the heavy and light chains are cloned separately, then recombined into a single 1 phage vector. The phage library is plated onto a lawn of E. colg, and phage expressing antibodies that bind T r are identified by screening plaque lifts using radiolabelled TF. 39.
CHAUDHARY VK, BATRA JK, GALLO MG, WILLINGHAM MC, FITZGERALD DJ, PASTAN I: A Rapid Method of Cloning Functional Variable-Region Antibody G e n e s in Escherichia coli as Single-Chain Immunotoxins. Proc Natl Acad Sci USA 1990, 87:1066-1070.
This paper describes a strategy for cloning the genes encoding antibody variable domains into an E. coli expression vector. PCR amplification is used to isolate variable light (VL) and heavy (VH) chain coding sequences from hybridoma mRNA- The PCR primers are designed so that the VII and VL segments can be ligated in frame to encode a single-chain antibody. The antibody encoding DNA is ligated into an expression vector so that it is produced as a fusion protein linked to a bacterial exotoxin, thus forming a 'single-chain immunotoxin'. This paper contains useful information for designing PCR primers and linkers that can be used to clone antibody coding sequences. 40. •
HULTMANT, MURBY M, STAHL S, HORNES E, UIMEN M: Solid Phase In Vitro Mutagenesis Using Plasmid DNA Template. Nucleic Acids Res 1990, 18:5107-5112. This paper reports a method for oligonucleotide-directed mutagenesis in which the DNA template is b o u n d to a solid support, in this case magnetic beads. In the first step, PCR is used to prepare a long singlestranded DNA molecule that contains the mutant oligonucleotide and to prepare a single-stranded vector molecule with ends that are complementary to the mutant ollgomer. In the second step, the two DNAs are annealed and simply transformed into E. coli. This method uses technology from an earlier report describing the preparation of templates for DNA sequencing. Unlike most PCR-based mutagenesis methods, this procedure only requires a single amplification cycle. However, the PCR step in which single-stranded vector DNA is prepared may introduce undesirable mutations. However, this approach suggests that highefficiency automated mutagenesis will eventually be possible. 41.
LEHTOVAARAPM, KOIVULAAK, BAMFORDJ, KNOWLESJK: A N e w M e t h o d for Random Mutagenesis of Complete Genes: Enzymatic Generation of Mutant Libraries I n Vitro. Protein Eng 1988, 2:63-68.
MJ Zoller, Department of Protein Engineering, Genentech, 460 Pt. San Bruno Blvd., South San Francisco, California 94080, USA.
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