Revealing biases inherent in recombination protocols

BackgroundThe recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.ResultsWe developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of β-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level).ConclusionBy comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling.

[1]  Narendra Maheshri,et al.  Computational and experimental analysis of DNA shuffling , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Frances H. Arnold,et al.  Molecular evolution by staggered extension process (StEP) in vitro recombination , 1998, Nature Biotechnology.

[3]  W. Stemmer,et al.  DNA shuffling of a family of genes from diverse species accelerates directed evolution , 1998, Nature.

[4]  D. Hoover,et al.  DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. , 2002, Nucleic acids research.

[5]  Gerd Folkers,et al.  Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling , 1999, Nature Biotechnology.

[6]  C D Maranas,et al.  Predicting crossover generation in DNA shuffling , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[7]  W. Stemmer,et al.  Optimized expression and specific activity of IL-12 by directed molecular evolution , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Shafikhani.  Factors affecting PCR-mediated recombination. , 2002, Environmental microbiology.

[9]  Wayne M Patrick,et al.  Novel methods for directed evolution of enzymes: quality, not quantity. , 2004, Current opinion in biotechnology.

[10]  R Y Tsien,et al.  Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Stephen J Benkovic,et al.  Evolution of highly active enzymes by homology-independent recombination. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Harayama,et al.  Novel family shuffling methods for the in vitro evolution of enzymes. , 1999, Gene.

[13]  J. Petrosino,et al.  Amino acid sequence determinants of beta-lactamase structure and activity. , 1996, Journal of molecular biology.

[14]  P. Bergquist,et al.  Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling. , 2001, Gene.

[15]  Donald Hilvert,et al.  Investigating and Engineering Enzymes by Genetic Selection. , 2001, Angewandte Chemie.

[16]  Marc Ostermeier,et al.  Finding Cinderella's slipper—proteins that fit , 1999, Nature Biotechnology.

[17]  F. Arnold,et al.  Functional and nonfunctional mutations distinguished by random recombination of homologous genes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Manfred T Reetz,et al.  Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Cameron Neylon,et al.  Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. , 2004, Nucleic acids research.

[20]  S. Lukyanov,et al.  GFP‐like chromoproteins as a source of far‐red fluorescent proteins , 2001, FEBS letters.

[21]  W. Stemmer,et al.  Evolution of a cytokine using DNA family shuffling , 1999, Nature Biotechnology.

[22]  Stan J. J. Brouns,et al.  DNA family shuffling of hyperthermostable beta-glycosidases. , 2002, The Biochemical journal.

[23]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Tsuneo Yamane,et al.  Chimeric Gene Library Construction by a Simple and Highly Versatile Method Using Recombination‐Dependent Exponential Amplification , 2003, Biotechnology progress.

[25]  Martin Zacharias,et al.  A statistical analysis of random mutagenesis methods used for directed protein evolution. , 2006, Journal of molecular biology.

[26]  W. Stemmer,et al.  Breeding of retroviruses by DNA shuffling for improved stability and processing yields , 2000, Nature Biotechnology.

[27]  W. Stemmer,et al.  Molecular evolution of an arsenate detoxification pathway by DNA shuffling , 1997, Nature Biotechnology.

[28]  Peter S. Shenkin,et al.  Amino Acid Sequence Determinants of β-Lactamase Structure and Activity , 1996 .

[29]  S. Benkovic,et al.  Incremental truncation as a strategy in the engineering of novel biocatalysts. , 1999, Bioorganic & medicinal chemistry.

[30]  U. Bornscheuer,et al.  Improved biocatalysts by directed evolution and rational protein design. , 2001, Current opinion in chemical biology.

[31]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[32]  S. Odelberg,et al.  Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. , 1995, Nucleic acids research.

[33]  Frances H. Arnold,et al.  Molecular breeding of carotenoid biosynthetic pathways , 2000, Nature Biotechnology.

[34]  Jon E. Ness,et al.  DNA shuffling of subgenomic sequences of subtilisin , 1999, Nature Biotechnology.

[35]  D. Pompon,et al.  High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. , 2000, Nucleic acids research.

[36]  W. Stemmer,et al.  Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling , 1996, Nature Biotechnology.

[37]  P. Bryan,et al.  Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains , 2005, Nucleic acids research.

[38]  F. Arnold,et al.  Optimization of DNA shuffling for high fidelity recombination. , 1997, Nucleic acids research.

[39]  Philip T. Pienkos,et al.  Growth factor engineering by degenerate homoduplex gene family recombination , 2002, Nature Biotechnology.

[40]  E. Chang.  Systemic Delivery of Tumor-Targeted p53 Gene Therapy Results in Chemo/Radiosensitization , 1999, Nature Biotechnology.

[41]  J. H. Shim,et al.  Combinatorial protein engineering by incremental truncation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[42]  F. Arnold,et al.  Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. , 1997, Journal of molecular biology.

[43]  Stephen J Benkovic,et al.  Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers. , 2003, Nucleic acids research.

[44]  T. Eggert,et al.  Enantioselective biocatalysis optimized by directed evolution. , 2004, Current opinion in biotechnology.

[45]  J. Seffernick,et al.  Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes. , 2001, Chemistry & biology.

[46]  Frances H Arnold,et al.  Analysis of shuffled gene libraries. , 2002, Journal of molecular biology.

[47]  J. Sorge,et al.  Creating seamless junctions independent of restriction sites in PCR cloning. , 1996, Gene.

[48]  Marc Ostermeier,et al.  A combinatorial approach to hybrid enzymes independent of DNA homology , 1999, Nature Biotechnology.

[49]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[50]  A. Meyerhans,et al.  DNA recombination during PCR. , 1990, Nucleic acids research.

[51]  Jon E. Ness,et al.  Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently , 2002, Nature Biotechnology.

[52]  DNA Shuffling Method for Generating Estrogen Receptor α and β Chimeras in Yeast , 2003 .

[53]  W. Stemmer,et al.  Molecular breeding of viruses , 2000, Nature Genetics.