2001 Fiscal Year Final Research Report Summary
Recombinant production of the anti-HIV protein actinohivin of actinomycete origin in Escherichia coli and analysis of essential region of actinohivin using its mutants
| Project/Area Number |
12672117
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
INOKOSHI Junji Kitasato University, School of Pharmaceutical Sciences, Assistant Professor, 薬学部, 講師 (30151640)
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| Co-Investigator(Kenkyū-buntansha) |
CHIBA Harumi Kitasato University, School of Pharmaceutical Sciences, Research associate, 薬学部, 助手 (90276163)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Actinohivin / anti-HIV / recombinant protein / syncytium formation / production / actinomycete / Eschelichia coli / inhibitor |
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| Research Abstract |
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.
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Research Products
(6 results)
