Kinetic model of in vivo folding and inclusion body formation in recombinant Escherichia coli.

Aggregation of misfolded proteins can reduce the yield in recombinant protein production. The underlying complex processes are additionally influenced by cellular physiology. Nevertheless, a lumped-parameter model of kinetic competition between folding and aggregation was sufficient to track properly the specific concentration of a human protein produced in E. coli and its partitioning into soluble and insoluble cell fractions. Accurate estimation of the protein-specific parameters required informative experiments, which were designed using the Fisher information matrix. The model was employed to calculate the influence of the specific glucose uptake rate in high-cell-density cultivation of E. coli on accumulation and aggregation of the recombinant protein. Despite its simplicity, the model was flexible and unbiased concerning unidentified mechanisms. Assuming an exponentially decreasing production rate, the irreversible aggregation step was found to follow first order kinetics, while assuming a constant production rate with simultaneous degradation, the model predicted transient aggregation only. Implications for strain and process development are discussed.

[1]  M. Oliveberg,et al.  Transient aggregates in protein folding are easily mistaken for folding intermediates. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  U. Rinas,et al.  Simple fed-batch technique for high cell density cultivation of Escherichia coli. , 1995, Journal of biotechnology.

[3]  R. Jaenicke,et al.  Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. , 1979, Biochemistry.

[4]  P O Olins,et al.  Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli. , 1992, The Journal of biological chemistry.

[5]  Rody P. Cox,et al.  Chaperonins GroEL and GroES promote assembly of heterotetramers (α2β2) of mammalian mitochondrial branched-chain α-keto acid decarboxylase in Escherichia coli , 1992 .

[6]  F. Neidhardt,et al.  Transient rates of synthesis of individual polypeptides in E. coli following temperature shifts , 1978, Cell.

[7]  J. Corchero,et al.  Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria. , 1998, FEMS microbiology letters.

[8]  J. Corchero,et al.  Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms. , 1999, Biochimica et biophysica acta.

[9]  G. Lorimer,et al.  GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli , 1989, Nature.

[10]  C. Shin,et al.  Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli , 1998, Applied Microbiology and Biotechnology.

[11]  M. Kitagawa,et al.  Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, inEscherichia coli , 1998, Applied and Environmental Microbiology.

[12]  A. Fersht,et al.  Formation of short-lived protein aggregates directly from the coil in two-state folding. , 1999, Biochemistry.

[13]  Axel Munack,et al.  Design of Optimal Dynamical Experiments for Parameter Estimation , 1989, 1989 American Control Conference.

[14]  F. Baneyx,et al.  Protein Misfolding and Inclusion Body Formation in Recombinant Escherichia coli Cells Overexpressing Heat-shock Proteins (*) , 1996, The Journal of Biological Chemistry.

[15]  A. Goldberg,et al.  Production of abnormal proteins in E. coli stimulates transcription of ion and other heat shock genes , 1985, Cell.

[16]  J R Ghilardi,et al.  In vitro growth of Alzheimer's disease beta-amyloid plaques displays first-order kinetics. , 1996, Biochemistry.

[17]  Axel Munack,et al.  Chapter 8. Optimization of Sampling , 2001 .

[18]  A. Zvi,et al.  Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  U. Rinas,et al.  Kinetics of Heat‐Shock Response and Inclusion Body Formation During Temperature‐Induced Production of Basic Fibroblast Growth Factor in High‐Cell‐Density Cultures of Recombinant Escherichiacoli , 2000, Biotechnology progress.

[20]  B Fane,et al.  Global suppression of protein folding defects and inclusion body formation. , 1991, Science.

[21]  U. Rinas,et al.  Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli. , 1999, Journal of biotechnology.

[22]  A. Goldberg,et al.  Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES. , 1994, The Journal of biological chemistry.

[23]  J. King,et al.  Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. A model for inclusion body formation. , 1988, The Journal of biological chemistry.

[24]  F. Marston.  The purification of eukaryotic polypeptides synthesized in Escherichia coli. , 1986, The Biochemical journal.

[25]  A. Fersht,et al.  Conformational states bound by the molecular chaperones GroEL and secB: a hidden unfolding (annealing) activity. , 1996, Journal of molecular biology.

[26]  J. King,et al.  In vitro and ribosome-bound folding intermediates of P22 tailspike protein detected with monoclonal antibodies. , 1994, The Journal of biological chemistry.

[27]  E. Laskowska,et al.  Response of Escherichia coli cell membranes to induction of λc1857 prophage by heat shock , 1991, Molecular microbiology.

[28]  G. Winter,et al.  Improved oligonucleotide site-directed mutagenesis using M13 vectors. , 1985, Nucleic acids research.

[29]  J. King,et al.  Intracellular trapping of a cytoplasmic folding intermediate of the phage P22 tailspike using iodoacetamide. , 1994, The Journal of biological chemistry.

[30]  U. Rinas,et al.  Comparison of temperature- and isopropyl-β-d-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli , 1995 .

[31]  Johannes Buchner,et al.  Protein Aggregation in vitro and in vivo: A Quantitative Model of the Kinetic Competition between Folding and Aggregation , 1991, Bio/Technology.

[32]  J. Corchero,et al.  Limited in vivo proteolysis of aggregated proteins. , 1997, Biochemical and biophysical research communications.

[33]  M. Zółkiewski,et al.  ClpB Cooperates with DnaK, DnaJ, and GrpE in Suppressing Protein Aggregation , 1999, The Journal of Biological Chemistry.

[34]  A. Plückthun,et al.  Engineered turns of a recombinant antibody improve its in vivo folding. , 1995, Protein engineering.

[35]  P E Fraser,et al.  A kinetic model for amyloid formation in the prion diseases: importance of seeding. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  A. Goldberg,et al.  Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli. , 1992, The EMBO journal.

[37]  U. Rinas,et al.  Folding Kinetics of the All-β-sheet Protein Human Basic Fibroblast Growth Factor, a Structural Homolog of Interleukin-1β* , 1999, The Journal of Biological Chemistry.