Abstract
A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).
Petrounia, I.P. & Arnold, F.H. Designed evolution of enzymatic properties. Curr. Opin. Biotechnol. 11, 325–330 (2000).
Roberts, R.W. & Szostak, J.W. RNA–peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94, 12297–12302 (1997).
Lehtovaara, P.M., Koivula, A.K., Bamford, J. & Knowles, J.K. A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro. Protein Eng. 2, 63–68 (1988).
Cadwell, R.C. & Joyce, G.F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).
Fabret, C. et al. Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains. Nucleic Acids Res. 28, EE95 (2000).
Wells, J.A., Vasser, M. & Powers, D.B. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene 34, 315–323 (1985).
Kegler-Ebo, D.M., Docktor, C.M. & DiMaio, D. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes. Nucleic Acids Res. 22, 1593–1599 (1994).
Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).
Higuchi, R., Krummel, B. & Saiki, R.K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367 (1988).
Dalbadie-McFarland, G. et al. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc. Natl. Acad. Sci. USA 79, 6409–6413 (1982).
Glaser, S.M., Yelton, D.E. & Huse, W.D. Antibody engineering by codon-based mutagenesis in a filamentous phage vector system. J. Immunol. 149, 3903–3913 (1992).
Neuner, P., Cortese, R. & Monaci, P. Codon-based mutagenesis using dimer-phosphoramidites. Nucleic Acids Res. 26, 1223–1227 (1998).
Sondek, J. & Shortle, D. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc. Natl. Acad. Sci. USA 89, 3581–3585 (1992).
Gaytan, P., Yanez, J., Sanchez, F., Mackie, H. & Soberon, X. Combination of DMT–mononucleotide and Fmoc–trinucleotide phosphoramidites in oligonucleotide synthesis affords an automatable codon-level mutagenesis method. Chem. Biol. 5, 519–527 (1998).
Ormo, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Ito, Y., Suzuki, M. & Husimi, Y. A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation. Biochem. Biophys. Res. Commun. 264, 556–560 (1999).
Heim, R., Cubitt, A.B. & Tsien, R.Y. Improved green fluorescence. Nature 373, 663–664 (1995).
Cubitt, A.B. et al. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).
Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).
Sumaoka, J., Igawa, T., Furuki, K. & Komiyama, M. Homogeneous Ce(IV) complexes for efficient hydrolysis of plasmid DNA. Chem. Lett. 56–57 (2000).
Berger, S.L. Expanding the potential of restriction endonucleases: use of hepaxoterministic enzymes. Anal. Biochem. 222, 1–8 (1994).
Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
Wiehler, J., von Hummel, J. & Steipe, B. Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487, 384–389 (2001).
Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989 (2000).
Fradkov, A.F. et al. Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett. 479, 127–130 (2000).
Miyazaki, K. & Arnold, F.H. Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function. J. Mol. Evol. 49, 716–720 (1999).
Cunningham, B.C. & Wells, J.A. High-resolution epitope mapping of hGH–receptor interactions by alanine-scanning mutagenesis. Science 244, 1081–1085 (1989).
Hohsaka, T., Ashizuka, Y., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into streptavidin through in vitro frameshift suppression. J. Am. Chem. Soc. 118, 9778–9779 (1996).
Hohsaka, T., Ashizuka, Y., Sasaki, H., Murakami, H. & Sisido, M. Incorporation of two different nonnatural amino acids independently into a single protein through extension of the genetic code. J. Am. Chem. Soc. 121, 12194–12195 (1999).
Hohsaka, T., Kajihara, D., Ashizuka, Y., Murakami, H. & Sisido, M. Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems. J. Am. Chem. Soc. 121, 34–40 (1999).
Murakami, H., Hohsaka, T., Ashizuka, Y., Hashimoto, K. & Sisido, M. Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin. Biomacromolecules 1, 118–125 (2000).
Murakami, H., Hohsaka, T., Ashizuka, Y. & Sisido, M. Site-directed incorporation of p-nitrophenylalanine into streptavidin and site-to-site photoinduced electron transfer from a pyrenyl group to a nitrophenyl group on the protein framework. J. Am. Chem. Soc. 120, 7520–7529 (1998).
Hohsaka, T., Ashizuka, Y., Taira, H., Murakami, H. & Sisido, M. Incorporation of nonnatural amino acids into proteins by using various four-base codons in the E. coli in vitro translation system. Biochemistry 40, 11060–11064 (2001).
Baptiste, J., Edwards, D.M., Delort, J. & Mallet, J. Oligodeoxy-ribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5′-ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Res. 19, 5227–5232 (1991).
Shibata, Y. et al. Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method. Biotechniques 30, 1250–1254 (2001).
Pheiffer, B.H. & Zimmerman, S.B. Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligo-nucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853–7871 (1983).
Lund, A.H., Duch, M. & Pedersen, F.S. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24, 800–801 (1996).
Acknowledgements
This work was supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No.11102003). The authors thank K. Takakura and. K. Inoguchi of Okayama University of Science for allowing us to use the Hitachi FM-BIO II instrument. H.M. received a Research Fellowship from the Japan Society for Promotion of Science for Young Scientists.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Murakami, H., Hohsaka, T. & Sisido, M. Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs. Nat Biotechnol 20, 76–81 (2002). https://doi.org/10.1038/nbt0102-76
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0102-76
This article is cited by
-
A novel framework for engineering protein loops exploring length and compositional variation
Scientific Reports (2021)
-
Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis
Nature Communications (2020)
-
Tailoring Proteins to Re-Evolve Nature: A Short Review
Molecular Biotechnology (2018)
-
Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations
BMC Biochemistry (2015)
-
Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process
Biotechnology Letters (2008)