PMID: 8612642Mar 1, 1996Paper

Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide

European Journal of Biochemistry
P JonassonM Uhlén

Abstract

The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ-proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse-phase chromatography, mass spectrometry and N-terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin-sensitive sites in a single step. The implications for production and recovery of various reco...Continue Reading

References

Aug 1, 1992·Current Opinion in Biotechnology·M UhlénB Nilsson
Feb 1, 1987·Protein Engineering·B NilssonM Uhlén
Aug 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·S H Shen
Nov 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·J Brosius, A Holy
May 1, 1994·Trends in Biotechnology·P A NygrenM Uhlén
Jul 29, 1994·Journal of Biotechnology·Y Kang, J W Yoon

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Citations

Jul 31, 1996·Journal of Biotechnology·J NilssonM Uhlén
Apr 6, 2004·Journal of Biotechnology·F J M MergulhãoG A Monteiro
Apr 9, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Cyril DianDarcy Birse
Mar 13, 2008·PloS One·Chee Gee LiewPeter W Andrews
Dec 21, 2010·Biotechnology Journal·Ronny FalkSophia Hober
Jan 29, 2020·Journal of Cellular Physiology·Ayelén Melisa BlancoSuraj Unniappan
Dec 24, 2004·Diabetes·Alokesh DuttaroyOlivier Blondel
Apr 7, 2021·Applied Microbiology and Biotechnology·Partha HazraMutyalasetty Kusumanchi
Nov 5, 1997·Protein Expression and Purification·J NilssonP A Nygren
Aug 24, 2017·International Journal of Biological Macromolecules·Jashandeep KaurJagdeep Kaur
Oct 28, 2004·Journal of the American Chemical Society·Masaaki SatoShin-Ichiro Nishimura

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