Journal
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
Volume 68, Issue 4, Pages 776-788Publisher
WILEY
DOI: 10.1002/bab.1990
Keywords
characterization; fermentation; Mecasermin; purification; rhIGF-1
Funding
- Iranian Council of Stem Cell Technology [190008]
- Isfahan University of Medical Sciences
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High overproduction of recombinant mecasermin was achieved in a bench-scale bioreactor by optimizing three factors, resulting in one of the highest values reported in batch systems. Purification at low temperature was found to efficiently purify the protein with high quantity and quality.
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 inEscherichia coliin a bench-scale bioreactor. In optimal conditions (induction temperature 28 degrees 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 degrees 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.
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