Production of hepatopoietin Cn in high-cell-density cultures of recombinant Escherichia coli and detection of its antioxygen activity.
Hepatopoietin Cn (HPPCn), a member of the leucine-rich acidic nuclear protein (LANP) family, is a novel human hepatic growth factor that stimulates DNA synthesis and hepatocyte proliferation in vivo and in vitro by activating several signaling pathways. We performed pilot-scale production of recombinant human HPPCn (rhHPPCn) protein to elucidate its role in liver protection. An Escherichia coli BL21 (DE3) strain containing a pET-24a-HPPCn expression vector was cultivated in a 40-l bioreactor. High-cell-density fed-batch fermentation using this strain yielded a high concentration of rhHPPCn protein in the form of inclusion bodies. After washing and solubilization of the protein, rhHPPCn refolding was achieved by dilution of the denaturant. Then, the renatured protein was purified chromatographically. Assessment of the biological activity of the obtained protein showed that rhHPPCn could attenuate the oxidative stress in ethanol-induced hepatocyte injury. We successfully achieved large-scale production of biologically active rhHPPCn protein and obtained yields greater than 40 mg rhHPPCn per gram dry cell weight by applying the above-mentioned renaturation and purification procedures. The obtained volumetric yield corresponds to 640 mg of biologically active rhHPPCn per liter of culture broth. Additionally, the recombinant HPPCn protein was confirmed to protect hepatocytes against ethanol-induced oxidative stress.
Liu
Yang
Y
Department of Experimental Hematology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, People's Republic of China. john_liuyang@hotmail.com
Li
Xiao-Li
XL
Zhang
Dong-Dong
DD
Wang
Qiang
Q
Liu
Yong
Y
Liu
Dong-Mei
DM
Wu
Chu-Tse
CT
Cui
Chun-Ping
CP
D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000975:Antioxidants; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C553972:hepatopoietin Cn, human; D017228:Hepatocyte Growth Factor