Rhyum S B, Jin B R, Park H R, Hong H J
Protein Engineering Research Group, Genetic Engineering Research Institute, KIST, Taejon, South Korea.
J Biotechnol. 1994 Aug 31;36(3):221-30. doi: 10.1016/0168-1656(94)90153-8.
PreS1 region gene fragment encoding the N-terminal 56 amino acid (aa) of hepatitis B virus (HBV, adr subtype), which encodes B- and T-cell epitopes and an hepatocyte receptor binding site, was synthesized by PCR and fused to the 3'-end of MalE gene encoding maltose-binding protein (MBP) to yield expression plasmid pMalpreS1-56. The plasmid was introduced into Escherichia coli DH5 alpha and expressed at 37 degrees C under the control of inducible tac promoter. The resulting fusion protein was highly expressed in a soluble form, about 40% of total cellular proteins, but it bound only partially to an amylose column. Therefore, the soluble preS1 fusion protein was purified to near homogeneity by two passages of anion-exchange chromatography followed by gel filtration. The yield of the fusion protein was 70 mg per 1 culture that had been induced by IPTG for 6 h. The purified fusion protein was specifically cleaved by a Factor Xa digestion to release the preS1 peptide, which was then purified by gel filtration to homogeneity. The purity, integrity, antigenicity and immunogenicity of the purified preS1 peptide was confirmed by glycerol-SDS-PAGE, Western analysis, N-terminal amino acid sequencing and an indirect ELISA.
合成了编码乙型肝炎病毒（HBV，adr亚型）N端56个氨基酸（aa）的前S1区基因片段，该片段编码B细胞和T细胞表位以及一个肝细胞受体结合位点，通过聚合酶链反应（PCR）将其与编码麦芽糖结合蛋白（MBP）的MalE基因的3'端融合，构建表达质粒pMalpreS1-56。将该质粒导入大肠杆菌DH5α，并在可诱导的tac启动子控制下于37℃表达。所产生的融合蛋白以可溶性形式高表达，约占细胞总蛋白的40%，但仅部分结合到直链淀粉柱上。因此，通过两次阴离子交换层析继以凝胶过滤，将可溶性前S1融合蛋白纯化至近乎均一。融合蛋白的产量为每1升经异丙基-β-D-硫代半乳糖苷（IPTG）诱导6小时的培养物70毫克。纯化的融合蛋白经凝血因子Xa酶切特异性裂解，释放出前S1肽，然后通过凝胶过滤纯化至均一。通过甘油-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（甘油-SDS-PAGE）、蛋白质免疫印迹分析、N端氨基酸测序和间接酶联免疫吸附测定（ELISA）证实了纯化的前S1肽的纯度、完整性、抗原性和免疫原性。
