Molecular evolution by staggered extension process (StEP) in vitro recombination

Nature Biotechnology
H ZhaoF H Arnold

Abstract

We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.

References

Aug 1, 1992·PCR Methods and Applications·R C Cadwell, G F Joyce
Feb 1, 1987·Scientific American·F W Stahl
May 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·S J ScharfH A Erlich
Oct 25, 1994·Proceedings of the National Academy of Sciences of the United States of America·W P Stemmer
Mar 15, 1997·Nucleic Acids Research·H Zhao, F H Arnold
May 1, 1997·Molecular Biology and Evolution·R D Bradley, D M Hillis
Jul 22, 1997·Proceedings of the National Academy of Sciences of the United States of America·H Zhao, F H Arnold
Jan 7, 1998·Trends in Biotechnology·O Kuchner, F H Arnold
Feb 28, 1998·Nucleic Acids Research·Z ShaoF H Arnold

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Citations

Jul 9, 1999·Biotechnology and Bioengineering·F Brühlmann, W Chen
Oct 10, 2001·Angewandte Chemie·Sean V. TaylorDonald Hilvert
Oct 25, 2001·Archives of Biochemistry and Biophysics·K NakamuraF P Guengerich
May 15, 2007·Analytical and Bioanalytical Chemistry·V MöhrleG Eberz
Jul 12, 2005·Journal of Molecular Evolution·Niles Lehman, Peter J Unrau
Dec 4, 2004·Applied Microbiology and Biotechnology·Paolo GiudiciStefano Cassanelli
May 23, 2012·Applied Microbiology and Biotechnology·Xue-Fei WangXuan-Wei Zhou
Mar 10, 2010·Current Microbiology·Weihua ZhuYi Cao
Jul 28, 2012·Biodegradation·Smriti RayuBrajesh K Singh
Feb 14, 2009·Molecular Biotechnology·André O S LimaDouglas E Eveleigh
Apr 29, 2005·Biomolecular Engineering·Edward G HibbertPaul A Dalby
Apr 29, 2005·Biomolecular Engineering·Birte Höcker
Apr 29, 2005·Biomolecular Engineering·Ting WangTianhong Wang
Apr 29, 2005·Biomolecular Engineering·Linda G Otten, Wim J Quax
Mar 31, 2004·Bioorganic & Medicinal Chemistry·Eduardo García-JuncedaAlfonso Fernández-Mayoralas
Jan 29, 2000·Journal of Immunological Methods·C SchaffitzelA Plückthun
Sep 11, 2002·Journal of Molecular Biology·Paul E O'MailleMing-Daw Tsai
Jun 26, 2003·Journal of Molecular Biology·Kaori Hiraga, Frances H Arnold
Jan 11, 2001·Biochimica Et Biophysica Acta·P N Bryan
Jan 11, 2001·Biochimica Et Biophysica Acta·P P SheridanJ E Brenchley
Jan 11, 2001·Biochimica Et Biophysica Acta·M LehmannM Wyss
Sep 22, 2001·Biochimica Et Biophysica Acta·P L WintrodeF H Arnold
Feb 24, 2001·Trends in Biotechnology·G Carrea, G Colombo
Jul 27, 2002·Journal of Biotechnology·Raymond M D VerhaertWim J Quax
Oct 11, 2003·Biotechnology Advances·Sajja Hari Krishna
Feb 7, 2001·Current Opinion in Biotechnology·M ChartrainB C Buckland
Jun 14, 2000·Current Opinion in Biotechnology·J R Cherry
Sep 7, 2000·Current Opinion in Biotechnology·S Lutz, S J Benkovic
Sep 7, 2000·Current Opinion in Biotechnology·I P Petrounia, F H Arnold
Sep 7, 2000·Current Opinion in Biotechnology·M OlsenG Georgiou
Aug 28, 1998·Current Opinion in Biotechnology·P J Hudson
Apr 21, 1999·Current Opinion in Biotechnology·W ChenA Mulchandani
Aug 17, 1999·Current Opinion in Biotechnology·M E HimmelC E Wyman
Sep 2, 2003·Current Opinion in Structural Biology·Paul A Dalby
Aug 17, 1999·Current Opinion in Structural Biology·R W Roberts, W W Ja
Feb 13, 2001·Trends in Biochemical Sciences·F H ArnoldA Gershenson
Aug 6, 1998·Current Opinion in Chemical Biology·L Giver, F H Arnold
Feb 19, 2000·Current Opinion in Chemical Biology·K E Jaeger, M T Reetz

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