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)是从放线菌K97-0003菌株的发酵液中分离到的一种新型抗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，为新型低分子抗HIV药物的开发提供基础依据。将AH产生菌的AH基因克隆到大肠杆菌表达载体pET30 Xa/LIC中，构建AH表达载体。在大肠杆菌BL21plysS(DE3)中表达约20 mg/L的重组AH融合蛋白。融合蛋白经镍螯合柱层析和反相硅胶柱层析纯化，再经蛋白酶Xa消化，获得重组AH。重组AH具有与放线菌产生的天然AH相当的合胞形成抑制活性，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组成的反式突变体，并测试了它们对合胞体形成的抑制活性。认为AH的三个片段是合胞体形成抑制活性所必需的。由于一个片段2上的两个半胱氨酸残基被丝氨酸取代的突变体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。结构特征表明AH是一个属于糖结合模块家族13的蛋白质。由于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与gp120相互作用的氨基酸残基，并阐明这两个分子的结合机制。

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it is shown that AH recognizes the high mannose-type oligosaccharide moieties of the HIV surface protein gp 120 (unpublished data), it is expected to be identify the amino acid residues which participate in the interaction of AH and gp120 and to clarify the binding mechanism of both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.