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Efficient process development for high‐level production, purification, formulation, and characterization of recombinant mecasermin in Escherichia coli
Author(s) -
Mofid Mohammad Reza,
Babaeipour Valiollah,
Jafari Sevda,
Haddad Leila,
Moghim Sharareh,
Ghanavi Jalaledin
Publication year - 2021
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1990
Subject(s) - escherichia coli , recombinant dna , chromatography , lysozyme , inducer , bioreactor , inclusion bodies , chemistry , lac operon , size exclusion chromatography , sonication , yeast extract , yeast , biochemistry , enzyme , fermentation , organic chemistry , gene
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 (bioactivity test). Also, the purification results revealed that expression at low temperature results in the efficient purification of the overproduced mecasermin with high quantity and quality.