Seyyedeh Masumeh Mirnurollahi1, Azam Bolhassani2, Shiva Irani1, Noushin Davoudi3. 1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2. Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran. 3. Biotechnology Research Center, Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran.
Abstract
BACKGROUND: Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. OBJECTIVES: In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. MATERIALS AND METHODS: The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. RESULTS: The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. CONCLUSIONS: This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.
BACKGROUND:Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. OBJECTIVES: In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. MATERIALS AND METHODS: The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. RESULTS: The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. CONCLUSIONS: This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.
Authors: Kazumoto Murata; Martin Lechmann; Ming Qiao; Toshiaki Gunji; Harvey J Alter; T Jake Liang Journal: Proc Natl Acad Sci U S A Date: 2003-05-14 Impact factor: 11.205