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Volumn 7, Issue 4, 1997, Pages 470-479

3D structural information as a guide to protein engineering using genetic selection

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN;

EID: 0030864556     PISSN: 0959440X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0959-440X(97)80109-1     Document Type: Article
Times cited : (34)

References (80)
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    • of special interest. Using a similar strategy as described in the previous reference [47], the importance of a negative charge in the active site of BsCM was examined. Combinatorial mutagenesis of codons 75 and 78 (encoding cysteine and glutamate in the wild type, respectively) showed that wild-type-like activity in vivo requires a carboxylic acid at one of the two positions. Some of the selected alternatives resemble the active site of the unrelated E. coli chorismate mutase.
    • Kast P, Hartgerink JD, Asif-Ullah M, Hilvert D. Electrostatic catalysis of the Claisen rearrangement: probing the role of Glu78 in Bacillus subtilis chorismate mutase by genetic selection. of special interest J Am Chem Soc. 118:1996;3069-3070 Using a similar strategy as described in the previous reference [47], the importance of a negative charge in the active site of BsCM was examined. Combinatorial mutagenesis of codons 75 and 78 (encoding cysteine and glutamate in the wild type, respectively) showed that wild-type-like activity in vivo requires a carboxylic acid at one of the two positions. Some of the selected alternatives resemble the active site of the unrelated E. coli chorismate mutase.
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    • Exploring the functional robustness of an enzyme by in vitro evolution
    • of outstanding interest. This excellent study used RNA hypermutagenesis with HIV reverse transcriptase and unbalanced dNTP ratios to iteratively mutagenize the gene for dihydrofolate reductase from plasmid R67. The gene for the 78-residue enzyme could be extensively mutagenized yielding up to 17 amino acid replacements without loosing its activity or tetramerization and substrate-binding properties. The role of the N-terminal amino acids, which are dispensable for activity but provide tolerance to the remaining 56 residues to substitution, still remains to be elucidated.
    • Martinez MA, Pezo V, Marlière P, Wain-Hobson S. Exploring the functional robustness of an enzyme by in vitro evolution. of outstanding interest EMBO J. 15:1996;1203-1210 This excellent study used RNA hypermutagenesis with HIV reverse transcriptase and unbalanced dNTP ratios to iteratively mutagenize the gene for dihydrofolate reductase from plasmid R67. The gene for the 78-residue enzyme could be extensively mutagenized yielding up to 17 amino acid replacements without loosing its activity or tetramerization and substrate-binding properties. The role of the N-terminal amino acids, which are dispensable for activity but provide tolerance to the remaining 56 residues to substitution, still remains to be elucidated.
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    • Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants
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    • Loop replacement and random mutagenesis of Ω-loop D, residues 70-84, in iso-1-cytochrome c
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    • Random mutagenesis of Thermus aquaticus DNA polymerase I: Concordance of immutable sites in vivo with the crystal structure
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    • In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities
    • of special interest. Five amino acids in the basic (α-helical DNA binding) region of a CCAAT/enhancer binding protein were randomized, and the corresponding genes were subjected to selection in vivo using a previously known transcriptional interference assay [79,80]. When the wild-type DNA recognition sequence was used in the selection, the original amino acids re-emerged. Novel binding specificities in vivo could be obtained with specifically altered DNA sites. This work, however, also shows some of the limitations of this selection system regarding the discrimination of signal over noise.
    • Sera T, Schultz PG. In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities. of special interest Proc Natl Acad Sci USA. 93:1996;2920-2925 Five amino acids in the basic (α-helical DNA binding) region of a CCAAT/enhancer binding protein were randomized, and the corresponding genes were subjected to selection in vivo using a previously known transcriptional interference assay [79,80]. When the wild-type DNA recognition sequence was used in the selection, the original amino acids re-emerged. Novel binding specificities in vivo could be obtained with specifically altered DNA sites. This work, however, also shows some of the limitations of this selection system regarding the discrimination of signal over noise.
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    • Random mutagenesis targeted to the active site of the EcoRV restriction endonuclease
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    • Affinity selective isolation of ligands from peptide libraries through display on a lac repressor 'headpiece dimer'
    • of special interest. As an alternative to phage display, the strategy of displaying random peptide libraries on DNA-binding proteins such as Lacl was further improved. By using in vitro affinity selection of peptide - Lacl fragment-encoding plasmid complexes starting from a random linker library, a stable covalently linked dimeric variant of the Lacl head piece was obtained. Although this novel and potentially monovalent system proved to be effective in affinity selection of peptide ligands to a monoclonal antibody, the mode of plasmid DNA binding appears (unexpectedly) to be nonspecific and requires further investigation. This work is yet another example for 'you get what you select for' in that the outcome of the selection experiment did not correspond to the design, but still fulfilled the selection criterion.
    • Gates CM, Stemmer WPC, Kaptein R, Schatz PJ. Affinity selective isolation of ligands from peptide libraries through display on a lac repressor 'headpiece dimer'. of special interest J Mol Biol. 255:1996;373-386 As an alternative to phage display, the strategy of displaying random peptide libraries on DNA-binding proteins such as Lacl was further improved. By using in vitro affinity selection of peptide - Lacl fragment-encoding plasmid complexes starting from a random linker library, a stable covalently linked dimeric variant of the Lacl head piece was obtained. Although this novel and potentially monovalent system proved to be effective in affinity selection of peptide ligands to a monoclonal antibody, the mode of plasmid DNA binding appears (unexpectedly) to be nonspecific and requires further investigation. This work is yet another example for 'you get what you select for' in that the outcome of the selection experiment did not correspond to the design, but still fulfilled the selection criterion.
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    • Chemical selection for catalysis in combinatorial antibody libraries
    • of special interest. An enriched antibody library of 100 transition-state analog binders was subjected to a novel catalysis-based in vitro selection strategy. The advantage of the selection is unclear, however, as the catalytic antibody fragment had to be identified after an in vivo screening procedure in E. coli cells.
    • Janda KD, Lo L-C, Lo C-HL, Sim M-M, Wang R, Wong C-H, Lerner RA. Chemical selection for catalysis in combinatorial antibody libraries. of special interest Science. 275:1997;945-948 An enriched antibody library of 100 transition-state analog binders was subjected to a novel catalysis-based in vitro selection strategy. The advantage of the selection is unclear, however, as the catalytic antibody fragment had to be identified after an in vivo screening procedure in E. coli cells.
    • (1997) Science , vol.275 , pp. 945-948
    • Janda, K.D.1    Lo, L.-C.2    Lo, C.-H.L.3    Sim, M.-M.4    Wang, R.5    Wong, C.-H.6    Lerner, R.A.7
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    • Cell-free synthesis of peptide libraries displayed on polysomes
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    • In vitro selection and evolution of functional proteins by using ribosome display
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    • Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli
    • of special interest. An in vivo selection system for ribozyme activity is proposed that is based on the acquired trimethoprim resistance of E. coli cells which occurs if an active ribozyme cleaves the primary transcript of an engineered dihydrofolate reductase gene to make the initiation region accessible for translation. Unfortunately, the signal-to-background ratio in the current configuration is too low to make this system useful for genuine evolutionary experiments.
    • Fujita S, Koguma T, Ohkawa J, Mori K, Kohda T, Kise H, Nishikawa S, Iwakura M, Taira K. Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli. of special interest Proc Natl Acad Sci USA. 94:1997;391-396 An in vivo selection system for ribozyme activity is proposed that is based on the acquired trimethoprim resistance of E. coli cells which occurs if an active ribozyme cleaves the primary transcript of an engineered dihydrofolate reductase gene to make the initiation region accessible for translation. Unfortunately, the signal-to-background ratio in the current configuration is too low to make this system useful for genuine evolutionary experiments.
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    • Fujita, S.1    Koguma, T.2    Ohkawa, J.3    Mori, K.4    Kohda, T.5    Kise, H.6    Nishikawa, S.7    Iwakura, M.8    Taira, K.9
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    • Genetic and biochemical probes for protein - protein interactions
    • of special interest. Recent developments in the application of the two-hybrid system and its derivatives are discussed in this useful short review, and a comprehensive list of relevant references is provided.
    • McNabb DS, Guarente L. Genetic and biochemical probes for protein - protein interactions. of special interest Curr Opin Biotechnol. 7:1996;554-559 Recent developments in the application of the two-hybrid system and its derivatives are discussed in this useful short review, and a comprehensive list of relevant references is provided.
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    • McNabb, D.S.1    Guarente, L.2
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    • Determinants of helix-loop-helix dimerization affinity. Random mutational analysis of SCL/tal
    • of special interest. The basic helix-loop-helix domain of the hematopoietic transcription factor SCL/tal was examined for its dimerization specificity with another transcription factor, E2-2, using the yeast two-hybrid system. Randomizing mutagenesis using error-prone PCR, coupled with selection for transcriptional activation of LEU2 (restoring leucine prototrophy), resulted in the identification of variants with higher affinity for E2-2. The structural consequences of one of the mutations were analyzed by computational modeling using the crystallographic coordinates of a related protein.
    • Goldfarb AN, Lewandowska K, Shoham M. Determinants of helix-loop-helix dimerization affinity. Random mutational analysis of SCL/tal. of special interest J Biol Chem. 271:1996;2683-2688 The basic helix-loop-helix domain of the hematopoietic transcription factor SCL/tal was examined for its dimerization specificity with another transcription factor, E2-2, using the yeast two-hybrid system. Randomizing mutagenesis using error-prone PCR, coupled with selection for transcriptional activation of LEU2 (restoring leucine prototrophy), resulted in the identification of variants with higher affinity for E2-2. The structural consequences of one of the mutations were analyzed by computational modeling using the crystallographic coordinates of a related protein.
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    • Goldfarb, A.N.1    Lewandowska, K.2    Shoham, M.3
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    • Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2
    • of outstanding interest. A combinatorial library of 20-residue peptides displayed in the active-site loop of E. coli thioredoxin was subjected to selection in vivo using the yeast two-hybrid system. Individual recombinant proteins containing the constrained peptides were shown to bind (and inhibit) the target protein, human cyclin-dependent kinase 2. Specific in vivo selected protein aptamers that have inhibitory function inside cells could be used to dissect complex networks of protein interactions. To test whether individual free (unconstrained) peptides have inhibitory activity would be interesting.
    • Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. of outstanding interest Nature. 380:1996;548-550 A combinatorial library of 20-residue peptides displayed in the active-site loop of E. coli thioredoxin was subjected to selection in vivo using the yeast two-hybrid system. Individual recombinant proteins containing the constrained peptides were shown to bind (and inhibit) the target protein, human cyclin-dependent kinase 2. Specific in vivo selected protein aptamers that have inhibitory function inside cells could be used to dissect complex networks of protein interactions. To test whether individual free (unconstrained) peptides have inhibitory activity would be interesting.
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    • Colas, P.1    Cohen, B.2    Jessen, T.3    Grishina, I.4    McCoy, J.5    Brent, R.6
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    • A three-hybrid system for detecting small ligand-protein receptor interactions
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    • A tri-hybrid system for the analysis and detection of RNa-protein interactions
    • Putz U, Skehel P, Kuhl D. A tri-hybrid system for the analysis and detection of RNa-protein interactions. Nucleic Acids Res. 24:1996;4838-4840.
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    • Reverse two-hybrid and one-hybrid systems to detect dissociation of protein - protein and DNA - protein interactions
    • of outstanding interest. A novel method termed 'reverse two (one)-hybrid system' is introduced which allows the direct selection of proteins or peptides that have impaired protein - protein interactions. The system is based on the yeast URA3 gene, whose expression by a transcriptional activator (which binds to its promoter-proximal DNA sequence) leads to cytotoxicity in the presence of 5-fluoroorotic acid. Disruption of DNA-binding and transcriptional activation in the context of a two (one)-hybrid system allows cell survival under those conditions. This novel technology could be used to directly select for peptides and proteins that induce specific protein - protein dissociation. Such tools may be useful as therapeutic agents to reverse aberrant protein interactions associated with disease.
    • Vidal M, Brachmann RK, Fattaey A, Harlow E, Boeke JD. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein - protein and DNA - protein interactions. of outstanding interest Proc Natl Acad Sci USA. 93:1996;10315-10320 A novel method termed 'reverse two (one)-hybrid system' is introduced which allows the direct selection of proteins or peptides that have impaired protein - protein interactions. The system is based on the yeast URA3 gene, whose expression by a transcriptional activator (which binds to its promoter-proximal DNA sequence) leads to cytotoxicity in the presence of 5-fluoroorotic acid. Disruption of DNA-binding and transcriptional activation in the context of a two (one)-hybrid system allows cell survival under those conditions. This novel technology could be used to directly select for peptides and proteins that induce specific protein - protein dissociation. Such tools may be useful as therapeutic agents to reverse aberrant protein interactions associated with disease.
    • (1996) Proc Natl Acad Sci USA , vol.93 , pp. 10315-10320
    • Vidal, M.1    Brachmann, R.K.2    Fattaey, A.3    Harlow, E.4    Boeke, J.D.5
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    • Genetic characterization of a mammalian protein - protein interaction domain by using a yeast reverse two-hybrid system
    • of special interest. The feasibility of the 'reverse two-hybrid system' is demonstrated by selecting variants from a partially mutagenized gene library encoding the C-terminal domain of transcription factor E2F1 that are unable to interact with protein DP1 (selection step 1) but are still of full length (selection step 2). Before selecting for full-length clones from those that did not bind DP1, however, plasmid curing and yeast mating was necessary.
    • Vidal M, Braun P, Chen E, Boeke JD, Harlow E. Genetic characterization of a mammalian protein - protein interaction domain by using a yeast reverse two-hybrid system. of special interest Proc Natl Acad Sci USA. 93:1996;10321-10326 The feasibility of the 'reverse two-hybrid system' is demonstrated by selecting variants from a partially mutagenized gene library encoding the C-terminal domain of transcription factor E2F1 that are unable to interact with protein DP1 (selection step 1) but are still of full length (selection step 2). Before selecting for full-length clones from those that did not bind DP1, however, plasmid curing and yeast mating was necessary.
    • (1996) Proc Natl Acad Sci USA , vol.93 , pp. 10321-10326
    • Vidal, M.1    Braun, P.2    Chen, E.3    Boeke, J.D.4    Harlow, E.5
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    • The yeast two-hybrid system: Forward and reverse
    • of special interest. This commentary on the reports by Vidal and coworkers [74,75] suggests a modification of the 'reverse two-hybrid system' to allow the simultaneous selection of binders to one protein and nonbinders to another. The modification would exploit genetic selection for two different auxotrophic markers, his and ura, and circumvent an involved screening option in the original system.
    • White MA. The yeast two-hybrid system: forward and reverse. of special interest Proc Natl Acad Sci USA. 93:1996;10001-10003 This commentary on the reports by Vidal and coworkers [74,75] suggests a modification of the 'reverse two-hybrid system' to allow the simultaneous selection of binders to one protein and nonbinders to another. The modification would exploit genetic selection for two different auxotrophic markers, his and ura, and circumvent an involved screening option in the original system.
    • (1996) Proc Natl Acad Sci USA , vol.93 , pp. 10001-10003
    • White, M.A.1
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    • Chook YM, Ke H, Lipscomb WN. Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog. Proc Natl Acad Sci USA. 90:1993;8600-8603.
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    • Chook YM, Gray JV, Ke H, Lipscomb WN. The monofunctional chorismate mutase from Bacillus subtilis: structure determination of chorismate mutase and its complexes with a transition state analog and prephenate, and implications for the mechanism of the enzymatic reaction. J Mol Biol. 240:1994;476-500.
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