Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

BackgroundWith the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript.ResultsIn this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias.ConclusionThe results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

[1]  Ursula Egner,et al.  Identifying protein construct variants with increased crystallization propensity––A case study , 2006, Protein science : a publication of the Protein Society.

[2]  J. Lutkenhaus,et al.  Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Bulmer.  The selection-mutation-drift theory of synonymous codon usage. , 1991, Genetics.

[4]  Jan Löwe,et al.  Structural insights into the conformational variability of FtsZ. , 2007, Journal of molecular biology.

[5]  S. Freeland,et al.  Optimal encoding rules for synthetic genes: the need for a community effort , 2007, Molecular systems biology.

[6]  H. Erickson,et al.  Atomic structures of tubulin and FtsZ. , 1998, Trends in cell biology.

[7]  Alan Villalobos,et al.  Gene Designer: a synthetic biology tool for constructing artificial DNA segments , 2006, BMC Bioinformatics.

[8]  L. Isaksson,et al.  Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy , 1986, Journal of bacteriology.

[9]  Paul M. Sharp,et al.  Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes , 1986, Nucleic Acids Res..

[10]  T. Ikemura.  Codon usage and tRNA content in unicellular and multicellular organisms. , 1985, Molecular biology and evolution.

[11]  Tomio Ogasawara,et al.  A bilayer cell‐free protein synthesis system for high‐throughput screening of gene products , 2002, FEBS letters.

[12]  S Falkow,et al.  Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. , 1997, Microbiology.

[13]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  G. Björk,et al.  Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency , 2004, Molecular and General Genetics MGG.

[15]  M Nayal,et al.  Valence screening of water in protein crystals reveals potential Na+ binding sites. , 1996, Journal of molecular biology.

[16]  V S Lamzin,et al.  Automated refinement for protein crystallography. , 1997, Methods in enzymology.

[17]  D. Ardell,et al.  Influences on gene expression in vivo by a Shine–Dalgarno sequence , 2006, Molecular microbiology.

[18]  Jan Löwe,et al.  Structural insights into FtsZ protofilament formation , 2004, Nature Structural &Molecular Biology.

[19]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[20]  M. Harding,et al.  Geometry of metal-ligand interactions in proteins. , 2001, Acta crystallographica. Section D, Biological crystallography.

[21]  L. Amos,et al.  Crystal structure of the bacterial cell-division protein FtsZ , 1998, Nature.

[22]  John Walchli,et al.  Gene Composer: database software for protein construct design, codon engineering, and gene synthesis , 2009, BMC biotechnology.

[23]  J. Kane,et al.  Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. , 1995, Current opinion in biotechnology.

[24]  P. Sharp,et al.  Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do 'prefer' optimal codons. , 1989, Nucleic acids research.

[25]  J. Bennetzen,et al.  Codon selection in yeast. , 1982, The Journal of biological chemistry.

[26]  Y Endo,et al.  A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Marjorie M Harding.  Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. , 2002, Acta crystallographica. Section D, Biological crystallography.

[28]  J. van Duin,et al.  Control of prokaryotic translational initiation by mRNA secondary structure. , 1990, Progress in nucleic acid research and molecular biology.

[29]  Ivan Ivanov,et al.  Missing Codon Pairs in the Genome of Escherichia Coli , 2002, Bioinform..

[30]  E. Robinson,et al.  Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[32]  G. W. Hatfield,et al.  Nonrandom utilization of codon pairs in Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[33]  T. Creighton.  Methods in Enzymology , 1968, The Yale Journal of Biology and Medicine.

[34]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[35]  Jan van Duin,et al.  Control of prokaryotic translational initiation by mRNA secondary structure , 1990 .

[36]  I. Ivanov,et al.  Effect of 3′ Terminal Codon Pairs with Different Frequency of Occurrence on the Expression of cat Gene in Escherichia coli , 2004, Current Microbiology.

[37]  H. Margalit,et al.  Hierarchy of sequence-dependent features associated with prokaryotic translation. , 2003, Genome research.

[38]  L. Duret,et al.  tRNA gene number and codon usage in the C. elegans genome are co-adapted for optimal translation of highly expressed genes. , 2000, Trends in genetics : TIG.

[39]  W. Kwiatkowski,et al.  Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli , 2007, Journal of Structural and Functional Genomics.

[40]  Etsuko N. Moriyama,et al.  Codon Usage Bias and tRNA Abundance in Drosophila , 1997, Journal of Molecular Evolution.

[41]  S. Govindarajan,et al.  Codon bias and heterologous protein expression. , 2004, Trends in biotechnology.

[42]  R. Hale,et al.  Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. , 1998, Protein expression and purification.

[43]  Shōzō Ōsawa,et al.  Evolution of the genetic code , 1995 .

[44]  S. Choe,et al.  Characterization of the family of Mistic homologues , 2006, BMC Structural Biology.

[45]  S. Karlin,et al.  Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria , 2001, Journal of bacteriology.

[46]  Gang Wu,et al.  SGDB: a database of synthetic genes re-designed for optimizing protein over-expression , 2006, Nucleic Acids Res..

[47]  K. Collins,et al.  The reverse transcriptase component of the Tetrahymena telomerase ribonucleoprotein complex. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  P. Nordlund,et al.  The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins. , 2008, Protein expression and purification.

[49]  G. Björk,et al.  Transfer RNA modification: influence on translational frameshifting and metabolism , 1999, FEBS letters.

[50]  Udo Oppermann,et al.  Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. , 2008, Protein expression and purification.

[51]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.