Expression of a recombinant elastin‐like protein in pichia pastoris

The translation of highly repetitive gene sequences is often associated with reduced levels of protein expression and may be prone to mutational events. In this report, we describe a modified concatemerization strategy to construct a gene with enhanced sequence diversity that encodes a highly repetitive elastin‐like protein polymer for expression in Pichia pastoris. Specifically, degenerate oligonucleotides were used to create a monomer library, which after concatemerization yielded a genetically nonrepetitive DNA sequence that encoded identical pentapeptide repeat sequences. By limiting genetic repetition, the risk of genetic deletions, rearrangements, or premature termination errors during protein synthesis is minimized. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

[1]  Y. Jigami,et al.  Production of humanized glycoproteins in bacteria and yeasts. , 2007, Current opinion in chemical biology.

[2]  J. Scheller,et al.  Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling. , 2006, The Biochemical journal.

[3]  Jack Hoopes,et al.  Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins , 2006, Science.

[4]  J. Scheller,et al.  Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides. , 2006, Plant biotechnology journal.

[5]  E. Chaikof,et al.  Viscoelastic and mechanical behavior of recombinant protein elastomers. , 2005, Biomaterials.

[6]  M. Hearn,et al.  Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production , 2005, Journal of molecular recognition : JMR.

[7]  Patrick S Daugherty,et al.  Evolutionary optimization of fluorescent proteins for intracellular FRET , 2005, Nature Biotechnology.

[8]  J. Scheller,et al.  Purification of Spider Silk-elastin from Transgenic Plants and Application for Human Chondrocyte Proliferation , 2004, Transgenic Research.

[9]  Teresa Mitchell,et al.  Production of Complex Human Glycoproteins in Yeast , 2003, Science.

[10]  Ashutosh Chilkoti,et al.  Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides. , 2003, Biomacromolecules.

[11]  S. Lovett,et al.  Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[12]  T. Gerngross,et al.  A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha , 2002, Applied and Environmental Microbiology.

[13]  David L Kaplan,et al.  Genetic engineering of fibrous proteins: spider dragline silk and collagen. , 2002, Advanced drug delivery reviews.

[14]  V. Conticello,et al.  Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences. , 2002, Advanced drug delivery reviews.

[15]  Y. Ogihara,et al.  Molecular analysis of the complete set of length mutations found in the plastomes of Triticum-Aegilops species. , 2002, Genome.

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

[17]  Ying Liu,et al.  Endotoxin removal in some medicines and human serum albumin solution by affinity membranes. , 2002, Se pu = Chinese journal of chromatography.

[18]  Elizabeth R. Wright,et al.  Thermoplastic elastomer hydrogels via self-assembly of an elastin-mimetic triblock polypeptide , 2002 .

[19]  Ashutosh Chilkoti,et al.  Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. , 2002, Biomacromolecules.

[20]  C. Wandrey,et al.  Purification of Polymeric Biomaterials , 2001, Annals of the New York Academy of Sciences.

[21]  J W Larrick,et al.  Producing proteins in transgenic plants and animals. , 2001, Current opinion in biotechnology.

[22]  S. Lovett,et al.  Instability of repetitive DNA sequences: The role of replication in multiple mechanisms , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. Chisholm,et al.  Production of Human Type I Collagen in Yeast Reveals Unexpected New Insights into the Molecular Assembly of Collagen Trimers* , 2001, The Journal of Biological Chemistry.

[24]  J. Scheller,et al.  Production of spider silk proteins in tobacco and potato , 2001, Nature Biotechnology.

[25]  A. Weiss,et al.  Expression of Recombinant Human Tropoelastin in Saccharomyces cerevisiae Containing a Synthetic Gene with a High Codon Adaptation Index Coupled to the SUC2 Invertase Signal Sequence , 2001 .

[26]  S. Streatfield,et al.  Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants , 2001, Trends in Plant Science.

[27]  K. Vandermeid,et al.  Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations. , 2001, Journal of chromatography. B, Biomedical sciences and applications.

[28]  C. J. Kim,et al.  Immunologic reactivity of a lipopolysaccharide-protein complex of type A Pasteurella multocida in mice. , 2000, Journal of veterinary science.

[29]  K. Kivirikko,et al.  Expression of recombinant human type I-III collagens in the yeast pichia pastoris. , 2000, Biochemical Society transactions.

[30]  Yong-Jin Choi,et al.  Random Changes of Amino Acid Residues with Expected Frequency by Saturated Point Mutagenesis , 2000, Molecules and cells.

[31]  S. Lee,et al.  Removal of Endotoxin during Purification of Poly(3-Hydroxybutyrate) from Gram-Negative Bacteria , 1999, Applied and Environmental Microbiology.

[32]  S. Lovett,et al.  Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions. , 1999, Journal of molecular biology.

[33]  V. Conticello,et al.  Rapid assembly of synthetic genes encoding protein polymers , 1999 .

[34]  S. Lee,et al.  Efficient and economical recovery of poly(3-hydroxybutyrate) from recombinant Escherichia coli by simple digestion with chemicals. , 1999, Biotechnology and bioengineering.

[35]  I. Frazer,et al.  Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer‐associated human papillomavirus type 16 , 1999, Clinical and experimental immunology.

[36]  H. Waterham,et al.  Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. , 1997, Gene.

[37]  P. Monsan,et al.  A nondegradative route for the removal of endotoxin from exopolysaccharides. , 1995, Analytical biochemistry.

[38]  C. Scorer,et al.  The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. , 1993, Gene.

[39]  N. Wainwright,et al.  Lipopolysaccharide detoxification by endotoxin neutralizing protein. , 1993, The Journal of surgical research.

[40]  William C. Raschke,et al.  Recent Advances in the Expression of Foreign Genes in Pichia pastoris , 1993, Bio/Technology.

[41]  C. Scorer,et al.  Foreign gene expression in yeast: a review , 1992, Yeast.

[42]  Karen N. Allen,et al.  On the deletion of inverted repeated DNA in Escherichia coli: effects of length, thermal stability, and cruciform formation in vivo. , 1991, Genetics.

[43]  C. Goochee,et al.  The Oligosaccharides of Glycoproteins: Bioprocess Factors Affecting Oligosaccharide Structure and their Effect on Glycoprotein Properties , 1991, Bio/Technology.

[44]  M. Radman,et al.  Control of large chromosomal duplications in Escherichia coli by the mismatch repair system. , 1991, Genetics.

[45]  J. Mandel,et al.  Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome , 1991, Science.

[46]  M. Romanos,et al.  High-Level Expression of Tetanus Toxin Fragment C in Pichia Pastoris Strains Containing Multiple Tandem Integrations of the Gene , 1991, Bio/Technology.

[47]  M. Goodman,et al.  Depsipeptide analogues of elastin repeating sequences: Conformational analysis , 1990, Biopolymers.

[48]  M. Pabst,et al.  Removal of endotoxin from protein solutions by phase separation using Triton X-114. , 1990, Journal of immunological methods.

[49]  M. Seidman,et al.  Use of supF, an Escherichia coli tyrosine suppressor tRNA gene, as a mutagenic target in shuttle-vector plasmids. , 1989, Mutation research.

[50]  J. Cregg,et al.  Pichia pastoris as a host system for transformations , 1985, Molecular and cellular biology.

[51]  A. Albertini,et al.  On the formation of spontaneous deletions: The importance of short sequence homologies in the generation of large deletions , 1982, Cell.

[52]  J. Hoeffler,et al.  Gene Expression Systems: Using Nature For The Art Of Expression , 2006 .

[53]  E. Chaikof,et al.  Protein-Based Thermoplastic Elastomers , 2005 .

[54]  Teresa Mitchell,et al.  Production of complex human glycoproteins in yeast. , 2003, Science.

[55]  J. Arrand,et al.  High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin. , 2002, Cytokine.

[56]  S. Fahnestock,et al.  Synthetic spider dragline silk proteins and their production in Escherichia coli , 1997, Applied Microbiology and Biotechnology.

[57]  L. Bedzyk,et al.  Production of synthetic spider dragline silk protein in Pichia pastoris , 1997, Applied Microbiology and Biotechnology.

[58]  H. Daniell,et al.  Hyperexpression of a synthetic protein-based polymer gene. , 1997, Methods in molecular biology.