Recombinant production of the anti-HIV protein actinohivin of actinomycete origin in Escherichia coli and analysis of essential region of actinohivin using its mutants

放线菌素（Actinohivin， AH）是一种从放线菌K97-0003培养液中分离得到的新型抗hiv蛋白，由114个氨基酸残基组成。本课题旨在阐明AH的构效关系，为制备新的低分子量抗hiv化合物提供基础依据。将产AH菌株克隆的AH基因导入大肠杆菌表达载体pet30xa /LIC中，构建AH表达质粒。携带该质粒的大肠杆菌BL21 plysS （DE3）产生了约20 mg/L的重组AH融合蛋白。融合蛋白经镍螯合柱和反相硅胶柱层析纯化，蛋白酶因子Xa酶切得到重组AH。重组AH表现出与放线菌产生的天然AH相当的合胞体形成抑制活性。AH由三个分子间同源片段组成。因此，我们构建了6种由一段或两段AH组成的分枝变异，并对其合胞体形成的抑制活性进行了测试。认为AH的三个片段对合胞体形成抑制活性至关重要。由于突变体AH在2段的两个半胱氨酸残基被丝氨酸取代，AH也具有与AH相似的抑制活性，因此认为半胱氨酸残基不影响AH的活性，其结构特征表明AH是一种属于碳水化合物结合模块家族13的蛋白质。由于研究表明AH识别HIV表面蛋白gp120的高甘露糖型寡糖部分（未发表的数据），因此有望鉴定参与AH和gp120相互作用的氨基酸残基，并阐明两者的结合机制。

Actinohivin (AH) which is a novel anti-HIV protein isolated from the culture broth of the actinomycete strain K97-0003, consists of 114 amino acid residues. The purposes of this project were to clarify the structure-activity relationship of AH and to obtain the basic evidence for the creation of new low moleculer weight anti-HIV compounds.AH gene coloned from AH producing strain was introduced into E. coli expression vector pET30 Xa/LIC to construct the AH expression plasmid. E. coli BL21 plysS (DE3) carrying this plasmid produced about 20 mg/L of the recombinant AH fusion protein. The fusion protein was purified by nickel chelate column and reversed phase silica gel column chromatography, followed by protease factor Xa digestion to obtain the recombinant AH. The recombinant AH showed a syncytium formation inhibitory activity equivalent to that of the natural AH produced by the actinomycete.AH consists of three intermolecular homologous segments. So, six kinds of the trancate variations which consist of one or two segments of AH were constructed and tested for the syncytium formation inhibitory activity. It was considered that three segments of AH are essential for the syncytium formation inhibitory activity. Since the mutant AH in which two cysteine residues in a segment 2 were replaced by serine also has a similar inhivitory activity to AH, it was thought that cysteine residues does not affect the activity of AH the feature of the structure revealed that AH is a protein which belongs to carbohydrate binding module family 13. Because it is shown that AH recognizes the high mannose-type oligosaccharide moieties of the HIV surface protein gp 120 (unpublished data), it is expected to be identify the amino acid residues which participate in the interaction of AH and gp120 and to clarify the binding mechanism of both molecules.