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| Expression of hK1-L-Fc Fusion Protein in CHO Cells and Analysis of its Bioactivity |
| DENG Chun-ping1, TAO Jian-jun2, HOU Yong-min2, ZHONG Ling1 |
1. School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China;
2. Techpool Bio-pharma Co. Ltd., Guangzhou 510520, China |
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Abstract To further modify the recombinant human kallikrein 1(hK1) for higher activity, new form of recombinant human kallikrein 1(hK1-L-Fc) was constructed by fusion of the human kallikrein1 gene and the coding sequences for Fc fragment of human IgG1 with a flexible linker peptide. The fusion gene hK1-L-Fc was constructed by PCR, then inserted into expression vector pcDNA3.1, and expressed in Chinese Hamster Ovary cells. The chimeric protein was purified by Protein A affinity chromatography, and further analyzed by SDS-PAGE、Western blotting、MALDI-TOF-MS and HPLC. The bioactivity of fusion protein in vitro was determined by the reaction with its substrate. The results showed that recombinant expression vector pcDNA3.1-hK1-L-Fc was constructed successfully and the cell lines stably secreting fusion protein were obtained; The expression output of the fusion protein was higher than 0.7 mg/L in batch culture; The purity of the protein with about 60 kDa molecular weight was higher than 95%, and the biological activity of the fusion protein was more than 9.2 U/mg, which increased more than 18% than that of hK1-Fc fusion protein.
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Received: 13 October 2010
Published: 01 April 2011
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