CHARACTERIZATION AND BINDING STUDIES OF A RECOMBINANT PRES1 PEPTIDE

如上一份年度报告所述，我们无法净化 重组前S1肽或含有它的融合蛋白主要 因为它的表达水平很低。我们已经准备了一个新的 大量表达融合蛋白的构建体（ca. 80，000道尔顿） 在大肠杆菌包涵体中。在用尿素和 用1，1，2-三氯三氟乙烷除去磷脂， 用人Xa因子处理融合蛋白溶液， preS序列。然而，由蛋白质组成的10，000道尔顿肽 分离并纯化preS1的氨基末端91个氨基酸（通过Mono-Q 色谱法）。这是主要产品，而不是预期的16，000 由插入的preS1-preS2序列组成的道尔顿肽。因子xa 切割涉及精氨酸羧基的肽键，而不是 不仅在插入的四肽位点-Ile-Glu-Gly-Arg-，而且在 - Gly-Arg-存在于preS1的90和91位。这 用I-125标记重组前S1肽 Bolton-Hunter（BH）试剂。标记的BH-preS1肽表现为 免疫学上与未标记的preS1相同， 商业单克隆preS抗体或我们自己的特异性多克隆兔 抗合成preS1肽（21 - 47）。人肝细胞膜 由尸检的肝脏标本制备（由NIH提供 临床中心和DC综合医院）。标记的preSl可以结合至 人肝细胞膜和结合可以被抑制， 未标记的配体或抗preS1单克隆抗体。两只兔子被 用这种肽免疫，但几次后抗preS1滴度仍然很低 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.