Streptomyces longwoodensis로부터 Autoregulator Receptor Protein 유전자의클로닝 및 특성
Characterization and Cloning of the Gene Encoding Autoregulator Receptor Protein from Streptomyceslongwoodensis.
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공시균인 S. longwoodensis IFO 14251의 autoregulators및 receptor gene 탐색의 일환으로 기존의 Streptomyces 속receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였다. 예상되는 100 bp 크기의 단편을 pUC19 vector에ligation하여 E. coli DH5α에 transformation한 후, plasmid를분리하여 BamHI을 처리하여 2% agarose gel에 전기영동한결과, pUC19 외에 receptor gene PCR product가 100 bp위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로PCR을 수행한 후, 염기배열을 결정하여 분석한 결과,Streptomyces sp. 유래의 receptor gene의 일부분임이 확인되었다. 따라서 S. longwoodensis IFO 14251에는 lysocellin생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 100 bp의PCR product를 probe로 이용하여 Southern 및 colonyhybridization을 통하여 4.4 kb의 SphI 단편을 가지는plasmid(pSLT)를 제작하였고 이를 sequencing한 결과,Streptomyces 속 유래의 autoregulator receptor 단백질을 코드하는 유전자와 3개의 open reading frame(651 bp)을 확인하여 sltR이라 명명하였다. 유전자 해석 결과, 기존의 autoregulatorreceptor proteins과 비교시 35~46%의 homology를 나타내었다. 재조합 단백질의 발현을 위해, sltR/pET-17bplasmid를 제작하고 E. coli BL21(DE3)/pLysS을 host cell로 이용하여 재조합 단백질을 발현시켰다. SltR 재조합 단백질의 정제는 DEAE-Sephacel column chromatography와DEAE-5PW chromatography(HPLC)를 통해 수행하였다.Gel filtration chromatography(HPLC, 55 kDa)와 SDSPAGE(28 kDa)를 통해 분자량을 확인한 결과, 재조합 단백질은 dimer형태로 존재하는 것으로 확인되었다. 또한, 결합활성에 있어 A-factor type의 autoregulator에 대해 가장 강한 결합활성을 나타내었다.
Abstract
For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli DH5α. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on 2% gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame (ORF) were isolated protein showing moderate homology (35~46%) with the γ-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR. The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.
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