Abstract
Overproduction of recombinant mecasermin was achieved by investigation of effect of three factors, temperature, inducer amount, and culture media, at three levels according to the Taguchi statistical design in Escherichia coli in a bench-scale bioreactor. In optimal conditions (induction temperature 28 °C, terrific broth with glucose (TB+G) medium, with 0.1 mM IPTG as inducer) 0.84 g/L mecasermin with expression levels of 38% of total protein and 4.13 g/L final dry cell biomass was produced, that is one of the highest values of recombinant protein has been reported in the batch system. The cell disruption was done by lysozyme pretreatment with sonication to the efficient purification of mecasermin. The isolated and washed inclusion bodies were solubilized in Gdn-HCl at pH 5.4 and folded with glutathione and purified with gel filtration. The purified rhIGF-1 (mecasermin) was formulated with arginine. Mecasermin protein remained t stable at 4 °C for up to 2 years. The quantitative and qualitative control indicated that mecasermin is expressed correctly (without the initial methionine by mass spectrometry), pure (without endotoxin and other protein impurities), correct folding (FTIR, RF-HPLC), monomer form (SEC-HPLC), and active (...Continue Reading
References
Feb 18, 1992·Biochemistry·S HoberB Nilsson
Jan 1, 1986·Methods in Enzymology·H Susi, D M Byler
May 18, 1993·Biochemistry·L O NarhiM A Weiss
Apr 18, 1998·Proceedings of the National Academy of Sciences of the United States of America·J C JolyJ R Swartz
Jul 27, 2002·Journal of Biotechnology·M M Carrió, A Villaverde
Jul 11, 2003·Health Physics·D L PrestonK Mabuchi
Sep 4, 2004·Microbial Cell Factories·Luis Felipe Vallejo, Ursula Rinas
Dec 21, 2004·Journal of Biotechnology·Hans Peter Sørensen, Kim Kusk Mortensen
Mar 23, 2005·Biochemistry·Brian M BaynesBernhardt L Trout
Jun 7, 2005·Cytokine & Growth Factor Reviews·Adam DenleyBriony E Forbes
Oct 3, 2006·Biotechnology and Bioengineering·Andrea VeraAntonio Villaverde
Feb 10, 2009·Advances in Therapy·Arlan L Rosenbloom
Apr 4, 2009·Biotechnology and Applied Biochemistry·S Yegane-SarkandyV Babaeipour
Jul 25, 2009·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Stephen F Kemp
Nov 7, 2009·Methods in Enzymology·Richard R Burgess
Apr 6, 2012·PloS One·Arun K UpadhyayAmulya K Panda
Jun 16, 2012·Growth Hormone & IGF Research : Official Journal of the Growth Hormone Research Society and the International IGF Research Society·Harinda RajapakshaBriony E Forbes
Jun 30, 2012·Nature Reviews. Drug Discovery·Steven A BerkowitzGraham B Jones
Jun 28, 2014·Biomolecules·Hiroshi Yamaguchi, Masaya Miyazaki
Apr 19, 2015·Microbial Cell Factories·Anupam SinghAmulya Kumar Panda
Nov 1, 2018·Biotechnology and Applied Biochemistry·Roghayyeh SeyfiFatemeh Abarghooi Kahaki