Efficient expression and secretion of recombinant hirudin III in E. coli using the L-asparaginase II signal sequence.
One of the hirudin variants HV3 was efficiently expressed in Escherichia coli using the L-asparaginase II signal sequence and the product was secreted into the culture medium. For the secretory manufacture of HV3, the L-asparaginase II signal sequence containing a single NheI restriction site at its 3' end was designed using the degenerate codons and PCR-amplified from E. coli chromosomal DNA. The synthetic HV3 coding sequence was fused to the signal sequence in-frame by its 5' NheI restriction site. The above signal-HV3 fusion gene was inserted into an expression vector pTA, which was derived from pkk223-3 such that its expression was under the control of the tac promotor. The resulting HV3 secretion expression vector pTASH thus constructed was introduced into an E. coli host cell AS1.357 with high L-asparaginase II producing level. After inducing with IPTG, the expression product was efficiently secreted into the culture medium and shake-flask culturing gave a yield of approximately 5 x 10(5)ATU/L (approximately 60mg/L). The secreted HV3 was easily purified from culture supernatant using ultrafiltration, ion-exchange column chromatography, and FPLC reverse-phase chromatography. The purified rHV3 from the culture supernatant had the expected N-terminal amino sequence and strong antithrombin activity, suggesting that the signal sequence was completely removed and the product was processed accurately during the secretion process.
School of Biopharmaceutics, China Pharmaceutical University, 24 Tongjia Street, Nanjing 210009, Jiangsu Province, People's Republic of China.
Shuhua
S
Tan
Wutong
W
Wu
Jingjing
J
Liu
Yi
Y
Kong
Yinghui
Y
Pu
Riying
R
Yuan
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005343:Fibrinolytic Agents / Q000302:isolation & purification; D005818:Genetic Engineering; D005822:Genetic Vectors; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein
D005343:Fibrinolytic Agents; D006629:Hirudins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C072300:hirudin HV3; D001215:Asparaginase