Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference.
To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.
Qian
Bingjun
B
SJTU-SIBS-PSU Joint Center for Life Sciences, School of Life Science and Biotechnology, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, PR China.
Shen
Huifeng
H
Xiong
Jingjing
J
Chen
Li
L
Zhang
Lu
L
Jia
Junwei
J
Wang
Yuan
Y
Zhang
Zhuchuan
Z
Yuan
Zheng
Z
Cao
Kaiming
K
Zhang
Dabing
D
D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D003062:Codon; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C050806:presurface protein 1, hepatitis B surface antigen