Journal of Microbiology

Chang Soo Kang

3 Articles

Expression, Purification, and Characterization of Recombinant Fibulin-5 in a Prokaryote Expression System: Myoung Seok Jeong , Chang Soo Kang , Yeon Soo Han , In Seok Bang; J. Microbiol. 2010;48(5):695-700. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0320-6

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Fibulin-5 is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. To investigate anti-angiogenesis activities and context-specific activities on responsive cells of recombinant fibulin-5 (rfibulin-5) expressed in Escherichia coli, the cDNA of fibulin-5 cloned from a human placenta cDNA library was inserted into the pET32a (+) vector to allow fibulin-5 expression as a Trx fusion protein. The fusion protein Trx-fibulin-5, expressed as insoluble inclusion bodies, was solubilized and its resulting expression level reached to 15% of the total cell protein. The Trxfibulin-5 was purified effectively by N2+-chelating chromatography and then identified by Western blotting analysis with an anti-His tag antibody. The purified Trx-fibulin-5 was refolded by dialysis against redox reagents, and the rfibulin-5 released from the fusion protein by enterokinase cleavage was purified using a RESOURCE RPC column. The final purified rfibulin-5 effectively inhibited angiogenesis in chicken embryos in a dose-dependent manner through a chorioallantoic membrane (CAM) assay. Additionally, rfibulin-5 potently suppressed in vitro proliferation of human umbilical vein endothelial cells, but stimulated that of human dermal fibroblasts. The expression and in vitro refolding of rfibulin-5 resulted in production of an active molecule with a yield of 2.1 mg/L.

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Animal Reproduction Science.2015; 163: 30. CrossRef
Production and on-column re-folding of human vascular endothelial growth factor 165 in Escherichia coli
Sun Kwon Bang, Young Sik Kim, Byung Soo Chang, Cheol Beom Park, In Seok Bang
Biotechnology and Bioprocess Engineering.2013; 18(5): 835. CrossRef

Expression of Recombinant Hybrid Peptide Hinnavin II/α-Melanocyte-Stimulating Hormone in Escherichia coli: Purification and Characterization: Son Kwon Bang , Chang Soo Kang , Man-Deuk Han , In Seok Bang; J. Microbiol. 2010;48(1):24-29. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0317-1

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The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/α-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni2+-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the combinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.

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Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds
Yunqi Ma, Chang-Joo Lee, Jang-Su Park
Antibiotics.2020; 9(9): 541. CrossRef
Design and high-level expression of a hybrid antimicrobial peptide LF15-CA8 in Escherichia coli
Xing-Jun Feng, Li-Wei Xing, Di Liu, Xue-Ying Song, Chun-Long Liu, Jing Li, Wen-Shan Xu, Zhong-Qiu Li
Journal of Industrial Microbiology and Biotechnology.2014; 41(3): 527. CrossRef
Expression systems for heterologous production of antimicrobial peptides
Nádia Skorupa Parachin, Kelly Cristina Mulder, Antônio Américo Barbosa Viana, Simoni Campos Dias, Octávio Luiz Franco
Peptides.2012; 38(2): 446. CrossRef

Biologically Active and C-Amidated HinnavinII-38-Asn Produced from a Trx Fusion Construct in Escherichia coli: Chang Soo Kang , Seung-Yeol Son , In Seok Bang; J. Microbiol. 2008;46(6):656-661. Published online December 24, 2008; DOI: https://doi.org/10.1007/s12275-008-0214-z

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The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15~20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.

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International Journal of Peptide Research and Therapeutics.2013; 19(2): 99. CrossRef
Expression systems for heterologous production of antimicrobial peptides
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Functional Divergence among Silkworm Antimicrobial Peptide Paralogs by the Activities of Recombinant Proteins and the Induced Expression Profiles
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PLoS ONE.2011; 6(3): e18109. CrossRef

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