[Modification, expression and bioactivity analysis of hK-Fc fusion protein].
To prolong serum half-life of human kallikrein (hK) and enhance its secretion rate, we modified hK gene and constructed a new form of recombinant hK protein (hK'-Fc). We amplified hK gene and Fc sequence, replaced the signal peptide of hK gene with murine signal peptide, constructed native expression plasmid of pcDNA-hK-Fc and modified expression plasmid of pcDNA-hK'-Fc, then transfected to CHO cells respectively. After the stable cell lines were screened, we compared the secretion rate between native fusion protein and modified fusion protein, purified fusion protein through Protein A+G affinity chromatography column and investigated the bioactivity of fusion protein. The results showed that recombinant vectors encoding fusion protein hK-Fc and hK'-Fc were constructed successfully; CHO cell lines stably secreting fusion protein were obtained, the yield is higher than 11 mg/L; Secretion rate was enhanced by 5-10 times after the signal peptide of fusion protein was modified; Fusion protein has enzymatic activity in vitro. The above results could promote the following researches on serum half-life of the fusion protein and develop a new stroke medicine with better clinical efficacy.
Zhou
Tong
T
State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China.
Tao
Jianjun
J
Li
Linguo
L
Hou
Yongmin
Y
Yu
Long
L
D000818:Animals; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D015815:Cell Adhesion Molecules; C106607:FCGBP protein, human; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D020840:Tissue Kallikreins