PRODUCTION IN BACTERIAL CELL A RECOMBINANT ANALOG OF PROINSULIN WITH THE PROPER DISULFIDE BONDS
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Authors
S. Ponomarenko
GlucoMetrix PVS GmbH, 14476, Potsdam, Am Mühlenberg, 11, BRD
Ch. Bouchaala
GlucoMetrix PVS GmbH, 14476, Potsdam, Am Mühlenberg, 11, BRD
Abstract
Mechanism of molecular folding of polypeptides synthesized in animal cells, is fundamentally different from the set found in prokaryotes and yeasts, which are traditionally used in the biopharmaceutical industry for expression of recombinant human proteins. The recombinant proteins expressed in a bacterial host system are chemically refolded in vitro to form their correct conformations. Arrangement of disulfide bridges is essential for the process of molecular folding of polypeptides into their three-dimensional structure and, eventually, for the formation of native protein conformation required for biological activity.
Here is described a new technology of protein expression in bacteria, exploiting prokaryotic system to fold of recombinant polypeptides with formation of correct disulfide bonds. According this technology, human insulin - important therapeutic protein - can be obtained without chemical refolding from recombinant analog of preproinsulin synthesized in bacteria. This is realized by creation in host cell of polypeptide structure with correct thiol bonds in cystine, which are analogous of disulfide bridges found in transit form of proinsulin.
According to the new technology, recombinant protein is expressed at a concentration sufficient for biosynthesis of the heterogeneous membrane protein in Escherichia coli cells. This semifolded proteopeptide is protected from proteolytic degradation, and it may be isolated as a complex similar to the proinsulin transit form. Transit forms of proinsulin are required for further transformation of hetero-dimer molecules into the native proteohormone structure. The new method without chemical refolding prevents accumulation of isoforms, significantly reduces time and material costs by production of recombinant human insulin in its native conformation.
Keywords
recombinant insulin, preproinsulin, refolding, membrane protein, ATP synthase
Article Details
References
Weiss M.A. Proinsulin and the Genetics of Diabetes Mellitus// J. Biol. Chem. -2009.- V. 284. N 29. – P. 19159–19163.
Ward C.W., Lawrence M.C. Landmarks in insulin research// Front.Endocrin. -2011.- V. 2. N 76. Crossref
Hoeg-Jensen T. Design of insulin variants for improved treatment of diabetes// In: Peptide and Protein Design for Biopharmaceutical Applications Editor Jensen K.J.-2009.- Chapter 7.- P. 249-286.
Kayser O. Grundwissen Pharmazeutische Biotechnologie// Verlag Teubner. Stuttgart. – 2002. 256 S.
Rudolph R., Lilie H., Schwarz E. In vitro Folding of Inclusion Body Proteins on an Industrial Scale.// In: Biothechnology. VCHVol. 5a Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes. Vol. Eds. Mountain A., Ney U.M., Schomburg D.- 2000.- P. 111-123.
Petrides D., Sapidou E., Calandranis J. Computer-aided process analysis and economic evaluation for biosynthetic human insulin production – A case study// Biotech.Bioengin.-1995.-V. 48. N 5.- P. 529-541.
Sambrook J., Fritsch E. F. Maniatis T. Molecular Cloning: a Laboratory Manual// 2nd edn.- Cold Spring Harbour Press, Cold Spring Harbour. NY.- 1989.
Jones P.C., Fillingame R.H. Genetic Fusions of Subunit c in the F0 Sector of H1-transporting ATP Synthase// J.Biol.Chem.-1998.- V. 273. N 45.- P. 29701–29705
Arechaga I., Butler P.G.G., Walker J.E. Self-assembly of ATP synthase subunit c rings// FEBS Letters.- 2002.- V.515.- P. 189-193
Qiao Z.-S., Min C.-Y., Hua Q.-X., Weiss M.A., Feng Y.-M. In Vitro Refolding of Human Proinsulin. Kinetic intermediates, putative disulfide-forming pathway, folding initiation site, and potential role of C-peptide in folding process// J.Biol.Chem. – 2003. - V. 278.-P. 17800-17809
Tramontano A. Protein Structure Prediction.// WILEY-VCH GmbH&Co KGaA Weinheim.- 2006. 208 p.
Girvin M.E., Rastogi V.K., Abildgaard F., Markley J.L., Fillingame R.H. Solution Structure of the Transmembrane H+-Transporting Subunit c of the F1Fo ATP Synthase// Biochemistry.- 1998.- V. 37.- N. 25.- P. 8817-9924
Schnick C., Forrest L.R., Sansom M.S., Groth G. Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis// Biochim Biophys Acta.-2000.- V.1459. N.1.-P.49-60..
Arechaga I., Miroux B., Karrasch S., Huijbregts R., de Kruijff B., Runswick M.J., Walker J.E. Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F1Fo ATPase// FEBS Letters.- 2000.- V. 482.- P. 215-219
Yuan Y., Wang Z.-H., Tang J.-G. Intra-A chain disulphide bond forms first during insulin precursor folding// Biochem. J. -1999.-V. 343.-P. 139-144.
Ponomarenko S.V. Biochemical characteristics of Escherichia coli ATP synthase with insulin peptide A fused to the globular part of the gamma-subunit. Biochemistry (Mosc). - 2006.-V.71.-P. 1006-1012
Hua Q.-X., Mayer J.P., Wenhua J., Zhang J., Weiss M.A. The Folding Nucleus of the Insulin Superfamily// J.Biol.Chem. – 2006.-V. 281. N. 38.- P. 28131-28142
Jones P.C., Jiang W., Fillingame R.H. Arrangement of the Multicopy H1-translocating Subunit c in the Membrane Sector of the Escherichia coli F1F0 ATP Synthase// J.Biol.Chem. –1998.- V. 273.- N. 27.-P. 17178–17185
Mitome N., Suzuki T., Hayashi S., Yoshida M. Thermophilic ATP synthase has a decamer c-ring: Indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling// PNAS.- 2004.- V. 101. N. 33.- P. 12159-12164
Hakulinen J.K., Klyszejko A.L., Hoffmann J., Eckhardt-Strelau L., Brutschy B., Vonck J., Meier T. Structural study on the architecture of the bacterial ATP synthase Fo motor// PNAS.- 2012. Crossref /10.1073/pnas.1203971109
Revington M., McLachlin D.T., Shaw G.S., and Dunn S.D. The Dimerization Domain of the b Subunit of the Escherichia coli F1F0-ATPase // J.Biol.Chem. – 1999.- V. 274.- N. 43.- P. 31094–31101
Suzuki T., Ozaki Y., Sone N., Feniouk B., Yoshida M. The product of uncI gene in F1Fo-ATP synthase operon plays a chaperone-like role to assist c-ring assembly// PNAS.- 2007.- V. 104.- N. 52.- P. 20776-20781
Saroussi S., Nelson, N. The little we know on the structure and machinery of V-ATPase// J. Experimental Biology.- 2009.- V. 212.-P. 1604-1610. Crossref
McCarty J.E.G._Expression of the unc Genes in Escherichia coli// J Bioenergetics Biomembranes.- 1988.- V. 20.- N. 1.- P. 19-39
Wang C.C., Tsou C.L. The insulin A and B chains contain sufficient structural information to form the native molecule.// Trends Biochem Sci.- 1991.- V. 16.- P. 279– 281.