CHARACTERIZATION AND BINDING STUDIES OF A RECOMBINANT PRES1 PEPTIDE

正如在前一份年度报告中所描述的那样，我们无法净化 重组前S1多肽或含有其主要成分的融合蛋白 因为它的表达水平很低。自那以后，我们准备了一份新的 大量表达融合蛋白的结构(约80,000道尔顿) 在大肠杆菌中的包涵体中。用尿素增溶后， 粗品1，1，2-三氯三氟乙烷脱除磷脂 融合蛋白溶液经人凝血因子Xa处理后释放 PRES序列。然而，一个10,000道尔顿的多肽由 用Mono-Q法分离纯化了前S1的91个氨基酸 层析)。这是主要的产品，而不是预期的16,000 由插入的preS1-preS2序列组成的道尔顿多肽。因子Xa 裂解涉及精氨酸羧基的多肽键，而不是 仅在插入的四肽位点-Ile-Glu-Gly-Arg-，也在 -Gly-Arg-存在于preS1的第90和91位。这 I-125偶联标记重组PreS1多肽 Bolton-Hunter(BH)试剂。标记的BH-preS1多肽似乎表现出 当使用以下任一种方法进行RIA时，免疫学上均为未标记的PreS1 商品化的PreS单抗或我们自己的特异性多克隆兔 抗合成的PreS1多肽(21-47)。人肝细胞膜 由尸检的肝脏标本制成(由美国国立卫生研究院提供 临床中心和哥伦比亚特区总医院)。标记的PRESL可以结合到 人肝细胞膜及其结合可被抑制 未标记的配体或抗PreS1的单抗。已经有两只兔子 用该多肽免疫，但在几次免疫后，抗PreS1效价仍然很低 加强剂量。工作仍在进行中。

As described in the previous annual report, we had been unable to purify the recombinant preS1 peptide or a fusion protein containing it mainly because of its low level of expression. We have since prepared a new construct which abundantly expresses a fusion protein (ca. 80,000 daltons) in inclusion bodies in Escherichia coli. After solubilization with urea and removal of phospholipids with 1,1,2trichlorotrifluoroethane, the crude fusion protein solution was treated with human Factor Xa to release the preS sequence. However, a 10,000 dalton peptide consisting of the amino-terminal 91 amino acids of preS1 was isolated and purified (by Mono-Q chromatography). This was the main product, rather than the expected 16,000 dalton peptide consisting of the inserted preS1-preS2 sequence. Factor Xa cleaves the peptide bond that involves the carboxyl group of arginine, not only at the inserted tetrapeptide site, -Ile-Glu-Gly-Arg-, but also at -Gly-Arg- which is present at positions 90 and 91 of preS1. This recombinant preS1 peptide was labeled by conjugating with I-125 Bolton-Hunter (BH) reagent. The labeled BH-preS1 peptide appeared to behave immunologically as unlabeled preS1 when RIAs were performed with either commercial monoclonal preS antibodies or our own specific polyclonal rabbit anti-synthetic preS1 peptide (21-47). Human hepatocyte membranes were prepared from autopsied liver specimens (kindly provided by the NIH Clinical Center and D.C. General Hospital). The labeled preSl can bind to human hepatocyte membranes and the binding can be inhibited by either the unlabeled ligand or anti-preS1 monoclonal antibodies. Two rabbits have been immunized with this peptide, but anti-preS1 titers remain low after several booster doses. Work is still in progress.