Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 204, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2022.106229
Keywords
Repifermin; Keratinocyte growth factor 2; Escherichia coli; Recombinant protein purification; Optimization of purification
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In this study, high-level production of bioactive recombinant human keratinocyte growth factor 2 (KGF-2) was achieved for large-scale production. 6HFh8-KGF-2 was purified using nickel affinity chromatography. The results showed that the high-level expression of 6HFh8-KGF-2 in Escherichia coli could effectively induce MCF-7 cell proliferation.
Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.
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