New recombinant DNA methodology for protein engineering Mark J. Zoller A R I A D Pharmaceuticals, Cambridge, Massachusetts, USA Over the past year considerable progress has been made in the application of recombinant DNA technology to protein engineering. A number of new methods for gene synthesis and mutagenesis have been reported that simplify the construction of novel coding sequences. The polymerase chain reaction plays an increasingly important role in these methods. Amino acid diversity has been extended by the incorporation of unnatural amino acids via coupled in vitro transcription-translation methods. Novel random mutagenesis strategies have been developed that substitute amino acids with a desired chemical character at a given position, thereby generating a sophisticated library of protein variants. Current Opinion in Biotechnology 1992, 3:348-354
Introduction The use of recombinant DNA technology in the analysis of protein structure and function is n o w well established as I described in m y review in this issue last year [1]. Proteins present in nature at exceedingly small levels can n o w be expressed and purified with ease using molecular biology techniques. In addition, this technology has allowed the production of a vast array of variants, such as proteins that differ from the natural molecule by only a single amino acid, chimeric proteins, and proteins designed d e novo. All of this b e c a m e possible through advances in oligonucleotide synthesis technology, the isolation of enzymes that operate on DNA, research into the principles of g e n e expression, and recently, the d e v e l o p m e n t of the polymerase chain reaction (PCR). The techniques to produce recombinant proteins have b e c o m e more accessible with time. This is evident from the explosion in techniques books, courses that teach recombinant DNA methods, and kits for mutagenesis, PCR, and expression. On another front, sophisticated r a n d o m mutagenesis methodologies are being developed to generate libraries of antibodies, cytokines, and peptides. This review focuses on current strategies for gene construction, methods for generating random mutants, and applications of coupled in vitro transcription-translation.
Methods for gene synthesis and mutagenesis Putting genes together by template-assisted assembly Protein coding sequences can be obtained from cloned genomic DNA (introns m a y be a problem), from a
cloned cDNA, or by construction and cloning of a synthetic gene. A standard m e t h o d for gene synthesis (Fig. 1) is to anneal a set of overlapping oligonucleotides, then enzymatically link t h e m together using DNA ligase [2]. The synthetic oligonucleotides encode both the top and bottom strands of the gene. Thus, a gene encoding a 300-amino-acid protein would be 900 base pairs in length and would require a set of oligonucleotides comprised of 1800 nucleotides. In cases where a g e n e of related sequence exists, a method termed 'templateassisted assembly' can be used to reduce the n u m b e r of oligonucleotides b y half (Fig. 2). By this method, a series of oligonucleotides is synthesized from only o n e strand of the gene of interest. The oligonucleotides are annealed to a single-stranded DNA template containing the complementary strand of a related gene. Once annealed, the oligonucleotides lie head-to-tail along the template. The ends are ligated using DNA ligase, and the resulting long oligonucleotide is first isolated and then used in a standard oligonucleotide-directed m u tagenesis reaction. By this approach, Carter et al. [3"] used six oli,gonucleotides to construct a gene encoding the constant domain of a ~visotype light chain using a 1( light-chain template. These two genes share only 70% sequence identi W at the nucleotide level. The only limitations to this approach are the synthetic capabili W of current DNA synthesizers (they can only reliably synthesize oligomers up to 100 nucleotides) and the fact that the oligomers must b e designed so their ends are perfectly complementary to the template DNA. Daugherty et al. [4"] used PCR-based template assembly to construct a gene encoding a humanized monoclonal antibody. Alternatively, a set of mutagenic oligonucleotides can be annealed to a single-stranded template DNA and extended via standard primer-extension
Abbreviations cDNA--cytoplasmic DNA; HIV--human immunodeficiencyvirus; PCR--polymerasechain reaction.
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New recombinant DNA methodologyfor protein engineeringZoller 349 oligonucleotide mutagenesis [1]. However, a n u m b e r of clones usually need to b e sequenced to find one that has incorporated all of the oligonucleotides.
Coding sequence of a related protein
14
Oligonucleotides~_./~/
Oligonucleotides
\ ~
~
Anneal
Anneal
(
"1
Single-stranded template DNA
1, DNA ligase
DNA ligase
(
/x
A
/x
)
DNA polymerase DNA ugase
Clone into expression vector /x
A
A
Fig. 1. Assembly of a synthetic gene by ligation of complementary overlapping oligonucleotides [2]. See text for details.
Putting genestogether by overlap extension Overlap extension PCR [5] can be used to synthesize a gene without template DNA. This approach was recently used to humanize a monoclonal antibody [4"] and to construct a synthetic gene encoding the human immunodeficiency virus (HIV)-2 Rev protein [6]. The gene is synthesized in two steps (Fig. 3). First, a mixture of the overlapping oligonucleotides is amplified for 7-25 cycles, then an aliquot of this reaction mixture is reamplified in the presence of two flanking primers. The ends of the internal segments overlap b y 18-20 base pairs. The authors note that a n u m b e r of cloned genes have to b e sequenced to identify one that is free of Taq polymerase misincorporation errors (discussed below). Dillon and Rosen [6] indicate that it is necessary to perform two rounds of PCR in order to obtain a PCR product of the desired size. Amplification with all of the primers in a single reaction results in a large n u m b e r of the DNA fragments that are shorter than the expected length.
PCR facilitates construction of chimeric genes
An important strategy to identify gene function is b y the analysis of chimeric proteins. Among the several ways to construct genes encoding chimeras are ligation of segments via c o m m o n restriction sites [7], loop-out deletion mutagenesis using oligonucleotides [8], and PCR amplification. While all three methods are fairly straightforward for construction of simple chimeras,
Introduce into
E. coli
Fig. 2. Creating chimeric genes via template-assembled oligonucleotides [3",4"]. See text for details. such as a gene encoding the N-terminal part of one protein and the C-terminal part of a second protein, the construction of a complex chimera, in which an internal segment of a protein is replaced with the analogous section from another protein, is more difficult. Here, PCR methods are particularly useful. In m y review last year [1] I described one such PCR method, termed 'sticky-feet directed mutagenesis' b y Clackson and Winter [9]. The segment to b e inserted is first p r e p a r e d by PCR amplification. Next, one strand of the PCR product is used in a standard primer extensionmutagenesis reaction using the other protein coding sequence as template. An alternative method, described more recently by Clackson et al. [10"] in an experiment to produce a library of single-chain antibodies, uses only PCR. This is a variation of overlap extension described above. First, the variable regions from the heavy and light chains were amplified b y PCR, then the two segments were joined b y a third PCR fragment that e n c o d e d a flexible linker. The overlap between the end of the linker fragment and the ends of the variable segments was 24 nucleotides.
350
Protein engineering
A residue) to the 3" end of the amplified fragment [14]. This extra nucleotide can b e removed by treatment of the amplified DNA with T4 DNA polymerase. Kuipers et al. [15] suggested the use of PCR primers that start one nucleotide 3' of a T in the target DNA. In this way, if the extra A residue is added it will not cause a mutation since the T-A base pair formed is found in the template. This method m a y not always be feasible and the additional nucleotide is not always added.
Oligonucleotides
18 bp overlap
IlllH
IIIlll
IIIIII a
III ~-__
m
llitll
_llllll P
IIIlll m
'
1 b
Fig. 3. Gene construction by overlap extension using multiple oligonucleotides [4",5,6]. (a). First round of PCR and (b) the second round of PCR. See text for details.
D N A polymerases differ in their fidelity
Once Taq DNA polymerase b e c a m e widely used in cloning experiments, researchers found that DNA synthesis was not error free. This is not a problem where the aggregate PCR product will b e analyzed by sequencing or by restriction digestion. On the other hand, experiments in which a particular DNA molecule is cloned from a PCR reaction m a y give rise to an undesirable mutation, for example o n e that changes a codon. The p r o b l e m of errors in PCR has b e e n extensively investigated by Eckert and Kunkel [11"] and by K e o h a v o n g and Thilly [12]. In surveying the literature, Eckert and Kunkel found that DNA amplified by Taq polymerase contained on average one mistake per 10000 nucleotides per cycle. Experiments performed by Eckert and Kunkel [13"] s h o w e d that a combination of low concentrations of deoxyribonucleotides and magnesium, a buffer with a p H of 6, high levels of target DNA, and a low number of cycles, yielded the highest fidelity of DNA synthesis w h e n using Taq. It was noted, however, that these conditions may not b e optimal for the amount of DNA produced by PCR. The Taq polymerase has another problem: it adds a non-template encoding nucleotide (usually an
New thermostable D N A polymerases improve fidelity
Biochemical studies of Taq polymerase s h o w e d that the major source of errors during DNA synthesis is the lack of proof-reading activity. Unlike the DNA polymerases from Escherichia coli or p h a g e T4, the Taq DNA polymerase lacks the 3'-5' exonuclease activity found in these more accurate polymerases. Although E. coli DNA polymerase can be used in PCR, the use of thermostable DNA polymerases is simpler and resuits in higher yields of product. To improve the fidelity of DNA synthesis in PCR, researchers b e g a n to search for thermostable DNA polymerases that exhibit proof-reading activity. The fidelity of synthesis by the DNA polymerase from Thermococcus litoralis (sold commercially as Vent polymerase) w a s s h o w n to b e approximately 3-5 times higher than that with Taq polymerase [16,17]. Recently the thermostable DNA polymerase from Pyrococcus furiosus has b e c o m e available and has b e e n reported to exhibit a 12-fold higher fidelity c o m p a r e d with Taq [18]. Care should b e taken to follow the suggested buffer and nucleotide conditions for these other polymerases as they m a y differ from those optimized for Taq polymerase. As noted by Eckert and Kunkel [11.'], using a more accurate polymerase will reduce but not eliminate the chance of synthesis errors. Other factors, such as unbalanced concentration of nucleotides and DNA damage at high temperature, can contribute to undesired mutations in the amplified DNA. It is clear from polymerase fidelity studies that the PCR conditions and choice of polymerase can greatly affect the resultant fidelity of synthesis and the yield of DNA. The presence of an u n w a n t e d codon change can have drastic effects on the property of the encoded protein. Thus, as reco m m e n d e d b y Eckert and Kunkel Ill-q, conditions that provide a low error rate yet result in a workable yield of DNA for cloning should be chosen. The question naturally arises: should the cloned PCR amplified fragment be sequenced? Experience indicates that u n d e r optimal conditions - - low magnesium and nucleotide concentration, pH 6-7, a large amount (over 50 ng) of template DNA, and only 10-15 amplification cycles - - it w o u l d be unlikely for a Taq polymerase-generated PCR fragment of about 500 base pairs in length to contain a mutation. But to be absolutely sure, sequencing is the safest option. Some researchers take the approach of assaying two clones of each construct generated in separate PCRs. This same advice is applicable in experiments that use mutant genes generated by primer extension mutagenesis. However, as the er-
New recombinant DNA methodology for protein engineering Zoller 351 ror rates of T7 or T4 DNA polymerases are 10-50 times lower than thermostable polymerases, sequencing the entire coding sequence is not as necessary.
Methods to generate amino acid diversity Mutant libraries are prepared using degenerate oligonucleotides There are several mutational strategies used in the analysis of protein structure and function [19]. Site-directed mutagenesis, in which a specific substitution is made in a coding sequence, is used to ascertain the role of a particular residue. In contrast, r a n d o m mutagenesis is used to identify functionally important regions or residues. Historically, this has been done in vitro by chemical modification, enzymatic misincorporation, insertion of linkers or enzymatic deletion. Often used in conjunction with a genetic selection or simple screen, random mutagenesis can help to narrow d o w n the number of residues for further analysis or to identify a variant with a desired property. As I described previously [1], in the absence of genetic selection, systematic site-directed approaches, such as homolog-scanning or charged-to-alanine-scanning mutagenesis, have proved to be efficient methods to identify a region to study further. Once a region of interest has been identified, functionally important residues can be found by conducting an alanine scan. The key residues can then be analyzed individually in more detail by testing the effects of all 19 amino acids substituted at a site. Wells and coworkers [20] used cassette mutagenesis with degenerate oligonucleotides in their pioneering experiments on the enzyme subtilisin. In this case, an oligonucleotide cassette was synthesized with NNN at the c o d o n of interest, where N is derived from an equimolar, mixture of A,C,G,T. The degenerate cassette was ligated into the subtilisin coding sequence and clones encoding the 19 substitution mutants were isolated and used to direct synthesis of the various mutant enzymes (NNG/C can be used instead of NNN to reduce the frequency of stop codons to 1 in 32). When more than one c o d o n is mutated using this approach the number of encoded sequences increases exponentially. Thus, two randomized codons encode 400 (202) different combinations, three encode 8000 (203), and so on. While isolation of clones that encode the twenty different codons might take a bit of effort, isolation of clones that would encode every combination of two randomized codons would take a long time. Thus, for practical reasons, large mutant libraries are screened using a genetic strategy. Sauer and coworkers [21] used this approach to probe functionally and structurally important amino acids in a bacteriophage repressor. Recently, Pielak and coworkers [22"1 applied this approach to yeast cytochrome c. As I described last year [1], the display of random peptides on phage uses libraries of mutants generated by cloning random cassettes into M13 phage. Wells and coworkers [23"q have randomized a segment of the growth hormone cod-
ing sequence involved in receptor binding and have screened the library for tighter binding human growth h o r m o n e variants using the phage display selection technique. This approach yielded a variant that exhibited about 10-times tighter binding than the wild type. The ability to transform E. coli limits the size of these libraries to about 108-109 transformants per microgram of DNA. Therefore with NNG/C at each codon, it is practical to mutate only six codons and still be able to sample the entire library (206 = 6.4x 107 coding sequences within 326 = i x 109 DNA molecules). Degenerate oligonucleotides prepared using NNG/C encode all 20 amino acids. Arkin and Youvan [24°'] have described a set of nucleotide mixtures that encode a subset of amino acids. For example, NTG/C encodes the hydrophobic amino acids phenylalanine, isoleucine, leucine, methionine and valine. Other mixtures can be designed that encode primarily hydrophilic, small amphipathic or aromatic amino acids. These mixtures could be used to prepare degenerate oligonucleotides to mutate a defined segment of a protein coding region. Knowledge of the chemical nature of the amino acids that form the region of interest would be used to tailor a particular nucleotide mixture for each amino acid to be mutated (or to prevent the substitution of an undesired amino acid). The advantage of this approach over the NNG/C method is that it reduces the degeneracy in a w a y that w o u l d be functionally acceptable. For example, substitution of a lysine for a buried phenylalanine w o u l d most likely have a catastrophic effect on protein structure and/or stability. By substituting only hydrophobic amino acids, there is a greater chance of producing an active protein that is not denatured. Reducing the degeneracy of the library allows for more than six codons to be mutated given current transformation frequencies. Roberts et al. [25] used oligonucleotides synthesized with limited nucleotide mixtures to prepare a library of bovine pancreatic trypsin inhibitor mutants in a phage display vector. In a second example, Garrard and coworkers [3"] mutated the complementarity-determining regions of an antibody using degenerate oligonucleotides biased at certain positions to encode only a subset of amino acids.
Suppressor strains simplify testing multiple substitutions Last year Normanly et al. [26] described a set of E. coli strains containing amber suppressor tRNAs that insert different amino acids. These strains could be used to test the effects of a variety of amino acids at one position of a protein. An amber c o d o n is introduced into the desired position of the protein coding sequence cloned in an expression vector. This vector is introduced into each of the different strains and the protein is expressed and isolated from each one. In this way, each strain produces a protein with a different amino acid substitution. In an exhaustive example of this approach [27"q, variants of bacteriophage T4 lysozyme (164 amino acids) were expressed in amber suppressor strains of Salmonella t y p h i m u r i u m . An amber c o d o n
352 Proteinengineering was placed at every position of the coding sequence except the initiator methionine. The 163 amber-encoding mutants were introduced into 13 suppressor strains allowing analysis of o v e r 2000 single amino acid substitution variants. From the analysis of plaque formation, which is d e p e n d e n t o n the activity of T4 lysozyme, the tolerance to substitution could be correlated with structure and activity. Although the intepretation of the data was complex, one simple conclusion is that lysine is the most frequently unacceptable substitution and alanine the least deleterious. Such an approach m a y be used as an initial screen to identify interesting variants to study further.
Expression of protein variants in vitro Expression of recombinant proteins is not limited to the standard systems of bacteria, yeast or mammalian ceils [28]. Insect ceils are an excellent system for the expression of a n u m b e r of proteins such as eukaryotic tyrosine kinases a n d viral oncogenes [29]. Channel proteins have b e e n studied b y the microinjection of channel mRNA into frog oocytes [30"]. Once inside the cell, the RNA is translated and a functional channel forms at the oocyte plasma membrane. Channel cDNA cloned into a plasmid containing a p r o m o t e r for an RNA polymerase from a bacteriophage such as SP6, T3 or T7 provides a convenient source of the RNA [31]. Protein variants can be p r o d u c e d by making the desired mutations in the DNA template. For s o m e applications, there is n o need to use any cellular system and instead protein synthesis can b e accomplished i n vitro. RNA can be synthesized i n v i t r o then added to an i n v i t r o translation system [32] or DNA templates can be a d d e d to a coupled transcription-translation extract [33]. The protein p r o d u c e d can be assayed directly or purified before enzymatic or structural analysis. Lesley e t al. [34] have recently described a clever modification of this system in w h i c h protein is synthesized i n v i t r o from PCR generated DNA templates. The promoter for bacteriophage T7 is positioned in front of the coding sequence of interest b y PCR with a primer that contains the T7 promoter, then the PCR fragment is added to an E. c o l i S-30 extract [33]. Noren e t al. [35] have exploited i n v i t r o transcriptiontranslation to incorporate unnatural amino acids into proteins. An a m b e r mutation is introduced into the coding sequence at the desired position. This plasmid is added to the transcription-translation extract, s u p p l e m e n t e d with a suppressor tRNA to which the desired unnatural amino acid has b e e n chemically coupled. This system has b e e n used to prepare site-specific variants of ~qactamase and recently, a light-activated lysozyme [36"']. The ability to incorporate amino acids other than the standard 20 greatly expands the repertoire of engineered proteins. However, this system has b e e n criticized for the rather low yield of protein. Yields tend to vary depending on the particular pro-
tein of interest, but lysozyme was obtained at levels b e t w e e n 40-50 I.tg m l - 1
Conclusion There is n o w a plethora of methods at hand to synthesize or mutate a coding sequence, many of which came about as a result of the application of the PCR. The low fidelity of DNA replication in a PCR is being i m p r o v e d by the use of n e w thermostable DNA polymerases. As protein engineers find applications for the n e w random mutagenesis methods, it is expected that sophisticated DNA synthesis machines will b e developed. The study of i n v i t r o synthesized proteins could b e greatly e x p a n d e d b y i m p r o v e m e n t in protein yield. Important technologies that I did not cover in this rev i e w are the use of fusion proteins [37], epitope tags [38] and metal-binding peptides [39"] to facilitate protein purification. T w o emerging areas to watch with interest are engineering n e w proteases with unique specificities [40] and the d e v e l o p m e n t of peptide ligases for the generation of n e w protein variants [41"].
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest
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4.
DAUGHERTYB, DEMARTINO J, LAW M-F, KAWKA D, SINGER I, MARK G: Polytnerase Chain Reaction Facilitates the C l o n i n g , CDR-grafting, and Rapid Expression o f a M u r i n e M o n o c l o n a l A n t i b o d y D i r e c t e d a g a i n s t the
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5.
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21
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27. •.
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MELTONDA, KREIG PA, REBAGLIATIMR, MANIATIS T, Z1NN K, GREEN MR: Efficient I n Vitro Syrtthesis o f Biologically Active RNA a n d RNA H y b r i d i z a t i o n P r o b e s f r o m P l a s m i d s C o n t a i n i n g a B a c t e r i o p h a g e SP6 Promoter. Nucleic Acids Res 1984, 12:7035-7056.
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MACKOW ER, YAMANAKA MY, DANG MN, GREENBERG HB: DNA Anapllftcation-restrieted T r a n s c r i p t i o n - t r a n s lation: Rapid Analysis o f R h e s u s R o t a v i r u s Neutralizat i o n Sites, Proc Natl A c a d Sci U S A 1990, 87:518-522.
33.
YANGH-L, IVASHKIVL, CHEN H-Z, ZUBAYG, CASHELM: Ce llfree C o u p l e d T r a n s c r i p t i o n - t r a n s l a t i o n S y s t e m for Inv e s t i g a t i o n o f Linear DNA S e g m e n t s . Proc Natl Acad Sci U S A 1980, 77:7029-7033.
34,
LESLEY S, BROW M, BURGESS R: Use o f I n Vitro Protern S y n t h e s i s f r o m P o l y m e r a s e C h a i n Reaction-generated T e m p l a t e s to Study I n t e r a c t i o n o f E s c h e r i c h i a coli T r a n s c r i p t i o n Factors w i t h Core RNA P o l y m e r a s e a n d f o r Epitope M a p p i n g o f M o n o c l o n a l Antibodies. J Biol Chem 1991, 266:2632-2638.
22.
354
Protein engineering 35.
NOREN CJ, ANTHONY-CAHILLSJ, GRIFFITH MC, SCHULTZ PG: A General Method for Site-specific Incorporation o f U n n a t u r a l A m i n o A c i d s i n t o P r o t e i n s . Science 1989, 244:182-188.
MENDELD, ELLMANJ, SCHULTZP: C o n s t r u c t i o n o f a Lightactivated Protein b y Unnatural A m i n o Acid Mutagenesis. J A m Chem Soc 1991, 113:2758-2760. A variant of T4 lysozyme containing an aspartyl ~-nitrobenzyl ester at the active site was prepared by cell-free coupled transcriptiontranslation. In the absence of light the modified protein was inactive. Upon irradiation, the nitrobenzyl ester w a s converted to the free acid and nitrobenzaldehyde, t h e r e b y resulting in a fully active enzyme. 36. ..
37.
SMITH DB, JOHNSON KS: S i n g l e - s t e p P u r i f i c a t i o n o f Polypeptides Expressed i n E s c h e r i c b i a c o l i a s Fusions w i t h G l u t a t h i o n e - S - t r a n s f e r a s e . Gene 1988, 6 7 : 3 1 4 0 .
38.
FIELD J, NIKAWA J, BROEK D, MACDONALD R, RODGERS L, WILSON IA, LERNER RA, WIGLER M: P u r i f i c a t i o n o f a RAS-responsive Adenylyl Cyclase C o m p l e x f r o m Saccharomyces cerevisiae b y Use o f a n Epitope Addition Method. Mol Cell Biol 1988, 8:2159-2165.
39.
AR_NOLDFH, HAYMOREBL: E n g i n e e r e d M e t a l - b i n d i n g P r o t e i n s : Purification to P r o t e i n F o l d i n g . Science 1991, 252:1796-1797. A short review on the introduction of metal-binding sites into proteins by addition of histidine residues in a certain spatial arrangement. 40.
CARTER I°~ ABRAI-LMSEN L, WELLS JA: P r o b i n g the Mecha n i s m a n d I m p r o v i n g the Rate o f Substrate-assisted Catalysis i n S u b t i l i s i n BPN'. Biochemistry 1991, 30:614245148.
41.
ABRAHMSEN L, TOM J, BURNIER J, BUTCHER KA, KOSSIAKOFF A, WELLS JA: E n g i n e e r i n g S u b t i l i s i n and Its Substrates for Efficient L i g a t i o n o f Peptide Bonds in Aqueous Solution. Biochemistry 1991, 30:4151~i159. T h o u g h early days in the attempts to turn a protease into a peptide ligase, this is an example of what is really m e a n t by protein engineering.
MJ Zoller, ARIAD Pharmaceuticals, 26 Lansdowne Street, Cambridge, MA 02139, USA.
Introduction The use of recombinant DNA technology in the analysis of protein structure and function is n o w well established as I described in m y review in this issue last year [1]. Proteins present in nature at exceedingly small levels can n o w be expressed and purified with ease using molecular biology techniques. In addition, this technology has allowed the production of a vast array of variants, such as proteins that differ from the natural molecule by only a single amino acid, chimeric proteins, and proteins designed d e novo. All of this b e c a m e possible through advances in oligonucleotide synthesis technology, the isolation of enzymes that operate on DNA, research into the principles of g e n e expression, and recently, the d e v e l o p m e n t of the polymerase chain reaction (PCR). The techniques to produce recombinant proteins have b e c o m e more accessible with time. This is evident from the explosion in techniques books, courses that teach recombinant DNA methods, and kits for mutagenesis, PCR, and expression. On another front, sophisticated r a n d o m mutagenesis methodologies are being developed to generate libraries of antibodies, cytokines, and peptides. This review focuses on current strategies for gene construction, methods for generating random mutants, and applications of coupled in vitro transcription-translation.
Methods for gene synthesis and mutagenesis Putting genes together by template-assisted assembly Protein coding sequences can be obtained from cloned genomic DNA (introns m a y be a problem), from a
cloned cDNA, or by construction and cloning of a synthetic gene. A standard m e t h o d for gene synthesis (Fig. 1) is to anneal a set of overlapping oligonucleotides, then enzymatically link t h e m together using DNA ligase [2]. The synthetic oligonucleotides encode both the top and bottom strands of the gene. Thus, a gene encoding a 300-amino-acid protein would be 900 base pairs in length and would require a set of oligonucleotides comprised of 1800 nucleotides. In cases where a g e n e of related sequence exists, a method termed 'templateassisted assembly' can be used to reduce the n u m b e r of oligonucleotides b y half (Fig. 2). By this method, a series of oligonucleotides is synthesized from only o n e strand of the gene of interest. The oligonucleotides are annealed to a single-stranded DNA template containing the complementary strand of a related gene. Once annealed, the oligonucleotides lie head-to-tail along the template. The ends are ligated using DNA ligase, and the resulting long oligonucleotide is first isolated and then used in a standard oligonucleotide-directed m u tagenesis reaction. By this approach, Carter et al. [3"] used six oli,gonucleotides to construct a gene encoding the constant domain of a ~visotype light chain using a 1( light-chain template. These two genes share only 70% sequence identi W at the nucleotide level. The only limitations to this approach are the synthetic capabili W of current DNA synthesizers (they can only reliably synthesize oligomers up to 100 nucleotides) and the fact that the oligomers must b e designed so their ends are perfectly complementary to the template DNA. Daugherty et al. [4"] used PCR-based template assembly to construct a gene encoding a humanized monoclonal antibody. Alternatively, a set of mutagenic oligonucleotides can be annealed to a single-stranded template DNA and extended via standard primer-extension
Abbreviations cDNA--cytoplasmic DNA; HIV--human immunodeficiencyvirus; PCR--polymerasechain reaction.
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© Current Biology Ltd ISSN 0958-1669
New recombinant DNA methodologyfor protein engineeringZoller 349 oligonucleotide mutagenesis [1]. However, a n u m b e r of clones usually need to b e sequenced to find one that has incorporated all of the oligonucleotides.
Coding sequence of a related protein
14
Oligonucleotides~_./~/
Oligonucleotides
\ ~
~
Anneal
Anneal
(
"1
Single-stranded template DNA
1, DNA ligase
DNA ligase
(
/x
A
/x
)
DNA polymerase DNA ugase
Clone into expression vector /x
A
A
Fig. 1. Assembly of a synthetic gene by ligation of complementary overlapping oligonucleotides [2]. See text for details.
Putting genestogether by overlap extension Overlap extension PCR [5] can be used to synthesize a gene without template DNA. This approach was recently used to humanize a monoclonal antibody [4"] and to construct a synthetic gene encoding the human immunodeficiency virus (HIV)-2 Rev protein [6]. The gene is synthesized in two steps (Fig. 3). First, a mixture of the overlapping oligonucleotides is amplified for 7-25 cycles, then an aliquot of this reaction mixture is reamplified in the presence of two flanking primers. The ends of the internal segments overlap b y 18-20 base pairs. The authors note that a n u m b e r of cloned genes have to b e sequenced to identify one that is free of Taq polymerase misincorporation errors (discussed below). Dillon and Rosen [6] indicate that it is necessary to perform two rounds of PCR in order to obtain a PCR product of the desired size. Amplification with all of the primers in a single reaction results in a large n u m b e r of the DNA fragments that are shorter than the expected length.
PCR facilitates construction of chimeric genes
An important strategy to identify gene function is b y the analysis of chimeric proteins. Among the several ways to construct genes encoding chimeras are ligation of segments via c o m m o n restriction sites [7], loop-out deletion mutagenesis using oligonucleotides [8], and PCR amplification. While all three methods are fairly straightforward for construction of simple chimeras,
Introduce into
E. coli
Fig. 2. Creating chimeric genes via template-assembled oligonucleotides [3",4"]. See text for details. such as a gene encoding the N-terminal part of one protein and the C-terminal part of a second protein, the construction of a complex chimera, in which an internal segment of a protein is replaced with the analogous section from another protein, is more difficult. Here, PCR methods are particularly useful. In m y review last year [1] I described one such PCR method, termed 'sticky-feet directed mutagenesis' b y Clackson and Winter [9]. The segment to b e inserted is first p r e p a r e d by PCR amplification. Next, one strand of the PCR product is used in a standard primer extensionmutagenesis reaction using the other protein coding sequence as template. An alternative method, described more recently by Clackson et al. [10"] in an experiment to produce a library of single-chain antibodies, uses only PCR. This is a variation of overlap extension described above. First, the variable regions from the heavy and light chains were amplified b y PCR, then the two segments were joined b y a third PCR fragment that e n c o d e d a flexible linker. The overlap between the end of the linker fragment and the ends of the variable segments was 24 nucleotides.
350
Protein engineering
A residue) to the 3" end of the amplified fragment [14]. This extra nucleotide can b e removed by treatment of the amplified DNA with T4 DNA polymerase. Kuipers et al. [15] suggested the use of PCR primers that start one nucleotide 3' of a T in the target DNA. In this way, if the extra A residue is added it will not cause a mutation since the T-A base pair formed is found in the template. This method m a y not always be feasible and the additional nucleotide is not always added.
Oligonucleotides
18 bp overlap
IlllH
IIIlll
IIIIII a
III ~-__
m
llitll
_llllll P
IIIlll m
'
1 b
Fig. 3. Gene construction by overlap extension using multiple oligonucleotides [4",5,6]. (a). First round of PCR and (b) the second round of PCR. See text for details.
D N A polymerases differ in their fidelity
Once Taq DNA polymerase b e c a m e widely used in cloning experiments, researchers found that DNA synthesis was not error free. This is not a problem where the aggregate PCR product will b e analyzed by sequencing or by restriction digestion. On the other hand, experiments in which a particular DNA molecule is cloned from a PCR reaction m a y give rise to an undesirable mutation, for example o n e that changes a codon. The p r o b l e m of errors in PCR has b e e n extensively investigated by Eckert and Kunkel [11"] and by K e o h a v o n g and Thilly [12]. In surveying the literature, Eckert and Kunkel found that DNA amplified by Taq polymerase contained on average one mistake per 10000 nucleotides per cycle. Experiments performed by Eckert and Kunkel [13"] s h o w e d that a combination of low concentrations of deoxyribonucleotides and magnesium, a buffer with a p H of 6, high levels of target DNA, and a low number of cycles, yielded the highest fidelity of DNA synthesis w h e n using Taq. It was noted, however, that these conditions may not b e optimal for the amount of DNA produced by PCR. The Taq polymerase has another problem: it adds a non-template encoding nucleotide (usually an
New thermostable D N A polymerases improve fidelity
Biochemical studies of Taq polymerase s h o w e d that the major source of errors during DNA synthesis is the lack of proof-reading activity. Unlike the DNA polymerases from Escherichia coli or p h a g e T4, the Taq DNA polymerase lacks the 3'-5' exonuclease activity found in these more accurate polymerases. Although E. coli DNA polymerase can be used in PCR, the use of thermostable DNA polymerases is simpler and resuits in higher yields of product. To improve the fidelity of DNA synthesis in PCR, researchers b e g a n to search for thermostable DNA polymerases that exhibit proof-reading activity. The fidelity of synthesis by the DNA polymerase from Thermococcus litoralis (sold commercially as Vent polymerase) w a s s h o w n to b e approximately 3-5 times higher than that with Taq polymerase [16,17]. Recently the thermostable DNA polymerase from Pyrococcus furiosus has b e c o m e available and has b e e n reported to exhibit a 12-fold higher fidelity c o m p a r e d with Taq [18]. Care should b e taken to follow the suggested buffer and nucleotide conditions for these other polymerases as they m a y differ from those optimized for Taq polymerase. As noted by Eckert and Kunkel [11.'], using a more accurate polymerase will reduce but not eliminate the chance of synthesis errors. Other factors, such as unbalanced concentration of nucleotides and DNA damage at high temperature, can contribute to undesired mutations in the amplified DNA. It is clear from polymerase fidelity studies that the PCR conditions and choice of polymerase can greatly affect the resultant fidelity of synthesis and the yield of DNA. The presence of an u n w a n t e d codon change can have drastic effects on the property of the encoded protein. Thus, as reco m m e n d e d b y Eckert and Kunkel Ill-q, conditions that provide a low error rate yet result in a workable yield of DNA for cloning should be chosen. The question naturally arises: should the cloned PCR amplified fragment be sequenced? Experience indicates that u n d e r optimal conditions - - low magnesium and nucleotide concentration, pH 6-7, a large amount (over 50 ng) of template DNA, and only 10-15 amplification cycles - - it w o u l d be unlikely for a Taq polymerase-generated PCR fragment of about 500 base pairs in length to contain a mutation. But to be absolutely sure, sequencing is the safest option. Some researchers take the approach of assaying two clones of each construct generated in separate PCRs. This same advice is applicable in experiments that use mutant genes generated by primer extension mutagenesis. However, as the er-
New recombinant DNA methodology for protein engineering Zoller 351 ror rates of T7 or T4 DNA polymerases are 10-50 times lower than thermostable polymerases, sequencing the entire coding sequence is not as necessary.
Methods to generate amino acid diversity Mutant libraries are prepared using degenerate oligonucleotides There are several mutational strategies used in the analysis of protein structure and function [19]. Site-directed mutagenesis, in which a specific substitution is made in a coding sequence, is used to ascertain the role of a particular residue. In contrast, r a n d o m mutagenesis is used to identify functionally important regions or residues. Historically, this has been done in vitro by chemical modification, enzymatic misincorporation, insertion of linkers or enzymatic deletion. Often used in conjunction with a genetic selection or simple screen, random mutagenesis can help to narrow d o w n the number of residues for further analysis or to identify a variant with a desired property. As I described previously [1], in the absence of genetic selection, systematic site-directed approaches, such as homolog-scanning or charged-to-alanine-scanning mutagenesis, have proved to be efficient methods to identify a region to study further. Once a region of interest has been identified, functionally important residues can be found by conducting an alanine scan. The key residues can then be analyzed individually in more detail by testing the effects of all 19 amino acids substituted at a site. Wells and coworkers [20] used cassette mutagenesis with degenerate oligonucleotides in their pioneering experiments on the enzyme subtilisin. In this case, an oligonucleotide cassette was synthesized with NNN at the c o d o n of interest, where N is derived from an equimolar, mixture of A,C,G,T. The degenerate cassette was ligated into the subtilisin coding sequence and clones encoding the 19 substitution mutants were isolated and used to direct synthesis of the various mutant enzymes (NNG/C can be used instead of NNN to reduce the frequency of stop codons to 1 in 32). When more than one c o d o n is mutated using this approach the number of encoded sequences increases exponentially. Thus, two randomized codons encode 400 (202) different combinations, three encode 8000 (203), and so on. While isolation of clones that encode the twenty different codons might take a bit of effort, isolation of clones that would encode every combination of two randomized codons would take a long time. Thus, for practical reasons, large mutant libraries are screened using a genetic strategy. Sauer and coworkers [21] used this approach to probe functionally and structurally important amino acids in a bacteriophage repressor. Recently, Pielak and coworkers [22"1 applied this approach to yeast cytochrome c. As I described last year [1], the display of random peptides on phage uses libraries of mutants generated by cloning random cassettes into M13 phage. Wells and coworkers [23"q have randomized a segment of the growth hormone cod-
ing sequence involved in receptor binding and have screened the library for tighter binding human growth h o r m o n e variants using the phage display selection technique. This approach yielded a variant that exhibited about 10-times tighter binding than the wild type. The ability to transform E. coli limits the size of these libraries to about 108-109 transformants per microgram of DNA. Therefore with NNG/C at each codon, it is practical to mutate only six codons and still be able to sample the entire library (206 = 6.4x 107 coding sequences within 326 = i x 109 DNA molecules). Degenerate oligonucleotides prepared using NNG/C encode all 20 amino acids. Arkin and Youvan [24°'] have described a set of nucleotide mixtures that encode a subset of amino acids. For example, NTG/C encodes the hydrophobic amino acids phenylalanine, isoleucine, leucine, methionine and valine. Other mixtures can be designed that encode primarily hydrophilic, small amphipathic or aromatic amino acids. These mixtures could be used to prepare degenerate oligonucleotides to mutate a defined segment of a protein coding region. Knowledge of the chemical nature of the amino acids that form the region of interest would be used to tailor a particular nucleotide mixture for each amino acid to be mutated (or to prevent the substitution of an undesired amino acid). The advantage of this approach over the NNG/C method is that it reduces the degeneracy in a w a y that w o u l d be functionally acceptable. For example, substitution of a lysine for a buried phenylalanine w o u l d most likely have a catastrophic effect on protein structure and/or stability. By substituting only hydrophobic amino acids, there is a greater chance of producing an active protein that is not denatured. Reducing the degeneracy of the library allows for more than six codons to be mutated given current transformation frequencies. Roberts et al. [25] used oligonucleotides synthesized with limited nucleotide mixtures to prepare a library of bovine pancreatic trypsin inhibitor mutants in a phage display vector. In a second example, Garrard and coworkers [3"] mutated the complementarity-determining regions of an antibody using degenerate oligonucleotides biased at certain positions to encode only a subset of amino acids.
Suppressor strains simplify testing multiple substitutions Last year Normanly et al. [26] described a set of E. coli strains containing amber suppressor tRNAs that insert different amino acids. These strains could be used to test the effects of a variety of amino acids at one position of a protein. An amber c o d o n is introduced into the desired position of the protein coding sequence cloned in an expression vector. This vector is introduced into each of the different strains and the protein is expressed and isolated from each one. In this way, each strain produces a protein with a different amino acid substitution. In an exhaustive example of this approach [27"q, variants of bacteriophage T4 lysozyme (164 amino acids) were expressed in amber suppressor strains of Salmonella t y p h i m u r i u m . An amber c o d o n
352 Proteinengineering was placed at every position of the coding sequence except the initiator methionine. The 163 amber-encoding mutants were introduced into 13 suppressor strains allowing analysis of o v e r 2000 single amino acid substitution variants. From the analysis of plaque formation, which is d e p e n d e n t o n the activity of T4 lysozyme, the tolerance to substitution could be correlated with structure and activity. Although the intepretation of the data was complex, one simple conclusion is that lysine is the most frequently unacceptable substitution and alanine the least deleterious. Such an approach m a y be used as an initial screen to identify interesting variants to study further.
Expression of protein variants in vitro Expression of recombinant proteins is not limited to the standard systems of bacteria, yeast or mammalian ceils [28]. Insect ceils are an excellent system for the expression of a n u m b e r of proteins such as eukaryotic tyrosine kinases a n d viral oncogenes [29]. Channel proteins have b e e n studied b y the microinjection of channel mRNA into frog oocytes [30"]. Once inside the cell, the RNA is translated and a functional channel forms at the oocyte plasma membrane. Channel cDNA cloned into a plasmid containing a p r o m o t e r for an RNA polymerase from a bacteriophage such as SP6, T3 or T7 provides a convenient source of the RNA [31]. Protein variants can be p r o d u c e d by making the desired mutations in the DNA template. For s o m e applications, there is n o need to use any cellular system and instead protein synthesis can b e accomplished i n vitro. RNA can be synthesized i n v i t r o then added to an i n v i t r o translation system [32] or DNA templates can be a d d e d to a coupled transcription-translation extract [33]. The protein p r o d u c e d can be assayed directly or purified before enzymatic or structural analysis. Lesley e t al. [34] have recently described a clever modification of this system in w h i c h protein is synthesized i n v i t r o from PCR generated DNA templates. The promoter for bacteriophage T7 is positioned in front of the coding sequence of interest b y PCR with a primer that contains the T7 promoter, then the PCR fragment is added to an E. c o l i S-30 extract [33]. Noren e t al. [35] have exploited i n v i t r o transcriptiontranslation to incorporate unnatural amino acids into proteins. An a m b e r mutation is introduced into the coding sequence at the desired position. This plasmid is added to the transcription-translation extract, s u p p l e m e n t e d with a suppressor tRNA to which the desired unnatural amino acid has b e e n chemically coupled. This system has b e e n used to prepare site-specific variants of ~qactamase and recently, a light-activated lysozyme [36"']. The ability to incorporate amino acids other than the standard 20 greatly expands the repertoire of engineered proteins. However, this system has b e e n criticized for the rather low yield of protein. Yields tend to vary depending on the particular pro-
tein of interest, but lysozyme was obtained at levels b e t w e e n 40-50 I.tg m l - 1
Conclusion There is n o w a plethora of methods at hand to synthesize or mutate a coding sequence, many of which came about as a result of the application of the PCR. The low fidelity of DNA replication in a PCR is being i m p r o v e d by the use of n e w thermostable DNA polymerases. As protein engineers find applications for the n e w random mutagenesis methods, it is expected that sophisticated DNA synthesis machines will b e developed. The study of i n v i t r o synthesized proteins could b e greatly e x p a n d e d b y i m p r o v e m e n t in protein yield. Important technologies that I did not cover in this rev i e w are the use of fusion proteins [37], epitope tags [38] and metal-binding peptides [39"] to facilitate protein purification. T w o emerging areas to watch with interest are engineering n e w proteases with unique specificities [40] and the d e v e l o p m e n t of peptide ligases for the generation of n e w protein variants [41"].
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest
1.
ZOttERM: N e w Molecular Biology Methods for P r o t e i n E n g i n e e r i n g . Curt Opin Btotechnol 1991, 2:526-531; Curr Opin Struc Biol 1991, 1:605-610.
2.
FERRETIL, KARNIK SS, KHORANAHG, NASSALN, OPRIAN DD: T o t a l Synthesis o f a G e n e f o r B o v i n e R h o d o p s i n . Proc Natl A c a d Sci U S A 1986, 83:599-603.
CARTERP, GARRARD L, HENNER D: Antibody Engineering U s i n g V e r y Long Template-assembled OHgonucleotides. Methods 1992, in press. A ~Llight chain is constructed from a ~c light chain template using six oligonucleotides by template assembly (also k n o w n as the 'six-pack experiment'). In a second experiment, a library of mutant antibodies is generated using degenerate oligonucleotides and PCR-based g e n e synthesis, f 3.
4.
DAUGHERTYB, DEMARTINO J, LAW M-F, KAWKA D, SINGER I, MARK G: Polytnerase Chain Reaction Facilitates the C l o n i n g , CDR-grafting, and Rapid Expression o f a M u r i n e M o n o c l o n a l A n t i b o d y D i r e c t e d a g a i n s t the
CD18 Component of Leukocyte Integrins. Nucleic Acids Res 1991, 19:2471-2476. T w o m e t h o d s of engineering an antibody are presented. O n e m e t h o d is a PCR-based template assembly a n d the other m e t h o d u s e s overlap extension PCR. While the methodology is useful, the authors failed (on purpose?) to include the s e q u e n c e of the oligonucleotides encoding the complementarity-determining regions.
5.
HORTONRM, HUNT liD, HO SN, PULLENJK, PEASE LR: Engin e e r i n g Hybrid Genes without the Use o f Restriction Enzymes: Gene s p l i c i n g b y Overlap E x t e n s i o n . Gene
1989, 77:61-68. 6.
DILLONPJ, ROSEN CA: A Rapid Method for the Construction o f Synthetic Genes Using the Polymerase C h a i n Reaction. Biotechniques 1990, 9:298-300.
New recombinant DNA methodology for protein engineering Zoller 353 7.
LAMMERSR, ULLR2CHA: C o n s t r u ~ . i o n a n d E x p r e s s i o n o f C h i m e r i c Cell Surface Receptors. Methods Enzymol 1991, 198:225-232.
8.
CHAN VL, SMITHM: In Vitro G e n e r a t i o n o f Specific Delet i o n s i n DNA Cloned i n M13 Vectors U s i n g Synthetic O l i g o d e o x y r i b o n u c l e o t i d e s : Mutants i n t h e 5'-flanking R e g i o n o f t h e Yeast Alcohol D e h y d r o g e n a s e II Gene. Nucleic Acids Res 1984, 12:2407-2419.
9.
CLACKSONT, WINTERG: 'Sticky Feet'-directed M u t a g e n e s i s a n d Its Application to S w a p p i n g A n t i b o d y D o m a i n s . Nucleic Acids Res 1989, 17:10163-10170.
10.
CLACKSONT, HOOGENBOOM FIR, GRIFFITHS AD, WINTER G: M a k i n g A n t i b o d y F r a g m e n t s Using Phage Display Libraries. Nature 1991, 352: 624-628. Heavy and light chain antibod3; segments from mouse eDNA are prepared and ligated together by overlap extension PCR via a linking fragment that encodes a flexible peptide chain. 11. •.
ECKERTKA, KUNKEL TA: DNA P o l y m e r a s e Fidelity a n d t h e P o l y m e r a s e C h a i n Reaction. PCR: Methods and Applications 1991, 1:17-24. An excellent review of the use of thermostable DNA polymerases to amplify DNA. 12
ECKERTKA, KUNKELTA: H i g h Fidelity DNA S y n t h e s i s b y t h e T h e r m u s a q u a t i c u s DNA P o l y m e r a s e . Nucleic Acids Res 1990, 18:3739-3744. Conditions are described that optimize the fidelity of DNA synthesis by Taq polymerase. Optimal conditions for fidelity will be sub-optimal for the yield of DNA produced. A compromise is suggested. 14.
CLARKJ: Novel N o n - t e m p l a t e d Nucleotide A d d i t i o n Rea c t i o n s Catalyzed b y Procaryotie a n d Eucaryotic DNA P o l y m e r a s e s . Nucleic Acids Res 1988, 16:9677-9686.
15.
KUIPERSOP, BOOT HJ, DEVOS W'M: I l n p r o v e d Site-directed M u t a g e n e s i s M e t h o d U s i n g PCR. Nucleic Acids Res 1991, 19:4558.
16.
CARIELLON, SWENBERGJ, SKOPEKT: Fidelity o f T h e r m o c o c c u s litoralis DNA Polyinerase (VentTM) i n PCR Det e r m i n e d b y D e n a t u r i n g G r a d i e n t Gel E l e c t r o p h o r e s i s . Nucleic Acids Res 1991, 19:4193-4198.
18.
24. •.
ARKINA, YOUVAN D: O p t i m i z i n g Nucleotide Mixtures to Encode Specific Subsets o f A m i n o Acids f o r Semi-rand o m Mutagenesis. Biotechnology 1992, 10:296-300. A unique investigation of the codon chart. Degenerate oligonucleotides that encode subsets of the twenty amino acids can be synthesized by adjusting the composition of nucleotide precursor pools. Using oligonucleotides composed of limited pools may allow a greater number of codons to be mutated and may improve the proportion of functional proteins expressed. 25.
ROBERTS B, MARK!~ND W, LEY A, KENT R, WHITE D, GUTERMAN S, LADNER RC: Directed E v o l u t i o n o f a ProteRn: Selection o f P o t e n t N e u t r o p h i l Elastase I n h i b i t o r s Displayed o n M13 F u s i o n Phage. Proc Natl Acad Sci U S A 1992, 89:2429-2433.
26.
NORMANLYJ, KLEINALG, MASSONJ-M, ABELSONJ, MILLERJH: C o n s t r u c t i o n o f E s c h e r i c h i a coli A m b e r S u p p r e s s o r tRNA G e n e s Ilia D e t e r m i n a t i o n o f tRNA Specificity. J Mol Biol 1990, 213:719-726.
KEOHAVONGP, THILLY WG: Fidelity o f DNA P o l y m e r a s e s i n DNA Amplification. Proc Natl Acad Sci U S A 1989, 86:9253-9257.
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BOWIEJU, REIDHAAR-OLSENJF, LIM WA, SALTERRT: Decip h e r i n g t h e Message i n P r o t e i n S e q u e n c e T o l e r a n c e to Arnitto Acid Substitutions. Science 1990, 247:1306-1310.
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27. •.
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LESLEY S, BROW M, BURGESS R: Use o f I n Vitro Protern S y n t h e s i s f r o m P o l y m e r a s e C h a i n Reaction-generated T e m p l a t e s to Study I n t e r a c t i o n o f E s c h e r i c h i a coli T r a n s c r i p t i o n Factors w i t h Core RNA P o l y m e r a s e a n d f o r Epitope M a p p i n g o f M o n o c l o n a l Antibodies. J Biol Chem 1991, 266:2632-2638.
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37.
SMITH DB, JOHNSON KS: S i n g l e - s t e p P u r i f i c a t i o n o f Polypeptides Expressed i n E s c h e r i c b i a c o l i a s Fusions w i t h G l u t a t h i o n e - S - t r a n s f e r a s e . Gene 1988, 6 7 : 3 1 4 0 .
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FIELD J, NIKAWA J, BROEK D, MACDONALD R, RODGERS L, WILSON IA, LERNER RA, WIGLER M: P u r i f i c a t i o n o f a RAS-responsive Adenylyl Cyclase C o m p l e x f r o m Saccharomyces cerevisiae b y Use o f a n Epitope Addition Method. Mol Cell Biol 1988, 8:2159-2165.
39.
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CARTER I°~ ABRAI-LMSEN L, WELLS JA: P r o b i n g the Mecha n i s m a n d I m p r o v i n g the Rate o f Substrate-assisted Catalysis i n S u b t i l i s i n BPN'. Biochemistry 1991, 30:614245148.
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ABRAHMSEN L, TOM J, BURNIER J, BUTCHER KA, KOSSIAKOFF A, WELLS JA: E n g i n e e r i n g S u b t i l i s i n and Its Substrates for Efficient L i g a t i o n o f Peptide Bonds in Aqueous Solution. Biochemistry 1991, 30:4151~i159. T h o u g h early days in the attempts to turn a protease into a peptide ligase, this is an example of what is really m e a n t by protein engineering.
MJ Zoller, ARIAD Pharmaceuticals, 26 Lansdowne Street, Cambridge, MA 02139, USA.
