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recR基因是大腸桿菌細胞中與DNA重組有關的基因之一，其基因產物則為參與RecF重組途徑之作用成員之一。recR基因之發現雖然早在1989年，其選殖與定序亦於稍後陸續完成，但是關於Recf蛋白生化功能之研究與探討卻至今仍無明顯之進展。
本篇論文所探討的主題是recR基因之表現，目的是希望能大量表現RecR蛋白以利其純化及將來生化功能之探討。另外，本文也分析RecR蛋白質大量表現的情況下對於細胞生理功能之影響。為了達成上述目的，在我們的研究中選用兩種具有不同啟動子的表現載體：分別是具有Ptac啟動子的pKK388-1以及具有T7ψ10啟動子的PET-3d。並利用分子選殖技術將recR基因序列之結構部份接於兩種表現載體之上。
經由IPTG誘導後以SDS-PAGE分析細胞中RecR蛋白之表現，我們發現具有T7ψ10啟動子之表現載體可大量表現RecR，而其有Ptac啟動子的載體則僅微量表現RecR。另在誘導RecR蛋白表現的情況下，我們發現細胞之DNA修復作用及細胞存活性之變化與基礎表現狀態下比較並無明顯之差異。此結果顯示RecR之大量表現對於細胞之正常生理功能並無明顯之影響。
為進一步了解RecR蛋白之生化功能，我們從經IPTG誘導之細胞萃取液純化RecR。在經由Pheny1 sepharose CL-4B column以及PBE94 column加以純化之後，我們已可得到純度約80%的RecR蛋白質，此純化之RecR蛋白在體外DNA重組作用扮演之角色將於日後探討。 The recR gene was identified in 1989 as one of the RecF path-way recombination genes in Escherichia coli. Although the recR gene has been cloned and its product identified, the biochemical functions of RecR protein and its role in homologous recombination remain unknown.
In this Study, the Structural portion of the recR gene was Synthesized by polymerase chain reaction (PCR) and cloned onto expression vectors which contain a Nco I fusion cloning site. Follow- ing IPTG induction, the expression of RecR was analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the recombinant plasmids carrying a T7 promoter (pVW62 and pVW63) overexpressed RecR in large quantity while the recombinant plasmid carrying a Ptac (pVW61) expressed RecR poorly. The effects of RecR overexpression on cell viability and U.V. Sensitivity were alSo examined. It was observed that the overexpression of RecR did not lead to cell death, nor did it affect the U.V. Sensitivity of uvrA recBC sbcBC recR cells.
Finally, aS a prelude to understand the biochemical functions of RecR protein, we have purified RecR protein from cells which overexpressed RecR. After chromatography with Phenyl SepharoSe CL -4B and PBE 94, RecR with about 80% purity was obtained. Further works on the biochemical functions of RecR will be pursued.
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