Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography.
Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing.
Yang
Qing
Q
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.
Liang
Zhen
Z
Zhang
Lihua
L
Zhang
Xiaodan
X
Zhao
Jiuyang
J
Zhang
Weibing
W
Zhang
Yukui
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D004345:Drug Industry; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007313:Insecta; D007610:Kallikreins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods*; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods*; D013997:Time Factors
D004792:Enzyme Precursors; D007610:Kallikreins; D012697:Serine Endopeptidases; C052910:prokallikrein, tissue