請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/20891
標題: Streptomyces regensis cytochrome P-450 monooxygenase基因的選殖及表現
Cloning and expression of cytochrome P-450 monooxygenase from Streptomyces regensis
作者: Chung, Chun-Yu
張純毓
關鍵字: Cloning gene
基因選殖
P-450 monooxygenase
出版社: 分子生物學研究所
摘要: Abstract Hypercholesterolemia is one of the major causes of arteriosclerosis and coronary heart disease. Cholesterol is supplied by absorption from diet and biosynthesis, and is excreted mainly as bile acid into feces. To reduce body cholesterol, three major strategies can be considered: (a) inhibition of cholesterol absorption, (b) inhibition of bile acid re absorption, (c) inhibition of cholesterol biosynthesis. Cholesterol is synthesized from acetyl-coenzyme A (Co A) in a process that includes more than 20 enzymatic steps. The rate-limiting enzyme of this pathway is 3-hydroxy- 3-methylgultaryl (HMG)-Co A reductase. When the enzyme is inhibited, the cholesterol level is decrease. Lovastatin and pravastatin have been used as clinical drugs. Pravastatin has stronger and more tissue-selective inhibition of cholesterol synthesis. Besides, it has lower side effect. In 1980, Sankyo Co. has found that compactin can be converted to pravastatin by cytochrome P-450 (Cyt P-450) monooxygenase system of Streptomyces carbophilus. Up to now, only Cyt P-450 monooxygenase gene of S. carbophilus was cloned and approved the convertible ability of compactin to pravastatin. Three primers were designed from the conserved sequences of P450 monooxygenase genes. A DNA fragment was amplified from Streptomyces regensis CCRC 11890 chromosomal DNA using Nested polymerase chain reaction. It was a partial fragment of P450 monooxygenase gene. Using the DNA fragment as probe, two Escherichia coli XLOLR (pBK-CMV-3 and pBK-CMV-15) clones that exhibited P450 monooxygenase activity was isolated from the S. regensis genomic library using plaque hybridization. Plasmid pBK-CMV-3 contains a 4.5 kb insert, and plasmid pBK-CMV-15 contains a 5.5kb insert. DNA sequencing of two insert DNAs revealed three closely spaced open reading frames (ORF), which were predicted to encode P450 monooxygenase. These three genes were designated as P450sre-1、P450sre-2 and P450sre-3, which transcribed in the same orientation. Identity of amino acid sequences among P450sre-1、P450sre-2、P450sre-3 gene and S. carbophilus P450 monooxygenase gene in order as 30﹪、45﹪and 45﹪, respectively. The sequences of P450sre-1、P450sre-2 and P450sre-3 gene in pBK-CMV-3 and pBK-CMV-15 were identical, but the catalytic site of P450sre-3 gene in pBK-CMV-15 was incomplete. The ORF of P450sre-1 gene consisted of 1368 bp and was predicted to encode 419 amino acids, with a calculated molecular mass of 51 kDa. The ORFs of P450sre-2 and P450sre-3 genes consisted of 1236 bp and were predicted to encode 412 amino acids, with a calculated molecular mass of 45.3 kDa. The GC contents of P450sre-1、P450sre-2 and P450sre-3 gene was 75.18﹪、70.15﹪ and 60.09﹪, respectively. The amino acid sequences showed 30﹪~ 65﹪identity with other P450 monooxygenase genes. Three P450 monooxygenase genes were cloned into pQE-30 expression vector respectively and expressed in E. coli Nova Blue. The enzyme were purified by metal affinity chromatography. In vivo analysis showed that E. coli Nova Blue harboring P450sre gene didn’t have the convertible ability, but in vitro analysis showed that E. coli Nova Blue containing P450sre-3 gene could convert of Na-compactin to pravastatin. Three P450 monooxygenase genes were also cloned into pIJ702 expression vector and expressed in S. lividans 66. In vivo or in vitro analysis, P450sre-1 and P450sre-2 both could convert Na-compactin to pravastatin, whereas the P450sre-3 gene only showed the convertible ability in vitro analysis. Perhaps lacks of promoter, P450sre-3 gene doesn''t have the convertible ability in S. lividans 66.
摘要 高膽固醇血症為動脈硬化及冠狀動脈疾病最重要危險因素之一。人體內的膽固醇來自於食物及生合成,再以bile acids的方式分泌排到體外,因此降低體內的膽固醇可採用下述策略:(a) 抑制膽固醇的吸收,(b) 抑制bile acid的再吸收,(c) 抑制膽固醇的生合成。膽固醇的生合成,由 acetyl - Co A 算起,超過20個步驟。其中,3-hydroxy - 3 - methylgultaryl (HMG) - Co A reductase 是生合成膽固醇速率的限制酵素,當此酵素的活性被抑制時,人體內膽固醇的含量也隨之下降。 目前已開發成為臨床用處方用藥的有:lovastatin及pravastatin,其中以pravastatin最具組織選擇性,藥效強且副作用小。1980年,日本 Sankyo Co. 首先發現Streptomyces carbophilus能將compactin很有效率的轉換成pravastatin,是利用菌體本身的Cytochrome P-450 ( Cyt P-450 ) monooxygenase system,至目前為止,具有此種轉換能力的菌株,只有S. carbophilus的Cyt P-450 monooxygenase基因被選殖出來,並且被證明有轉換compactin為pravastatin的能力。 比對已知的P450 monooxygenase基因的高度保留區域,根據其胺基酸序列設計退化性引子 ( degenerated primer ),以 Streptomyces regensis CCRC 11890 的染色體DNA作為模版,利用二次聚合酉每鏈鎖反應 (nested PCR),可以合成一 DNA 片段。上網比對確定此片段為P450 monooxygenase基因的一部份。以此片段作為探針 (probe),由S. regensis的染色體基因庫 (genomic library),以溶菌斑雜交法 (plaque hybridization) 篩選出兩個在Streptomyces lividans 66菌體內可表現P450 monooxygenase活性的質體 pBK-CMV-3 和pBK-CMV-15。經由 DNA 序列分析,顯示 pBK-CMV-3 含有 4.5 kb 的嵌入片段,而pBK-CMV-15 則含有 5.5 kb 的嵌入片段。此二個DNA片段皆含有三個位置相鄰的 open reading frame (ORF),均為P450 monooxygenase,此三個P450 monooxygenase基因的轉錄方向皆相同,此三個基因分別命名為P450sre-1、P450sre-2和P450sre-3。將此三個基因的胺基酸序列與S. carbophilus比對,其相似度分別為30%、45%及45% 。pBK-CMV-3 和pBK-CMV-15質體上的P450sre-1、P450sre-2和P450sre-3 基因胺基酸序列是相同的,但是pBK-CMV-15上的P450sre-3 基因的酵素活性部位是不完整。 P450sre-1基因全長 1,257 bp,此段 ORF 可以轉譯出 419 個胺基酸,預估分子量為 46 kDa;P450sre-2 基因全長 1,236 bp,此段 ORF 可以轉譯出 412 個胺基酸,預估分子量為 45.3 kDa;P450sre-3 基因全長 1,236 bp,此段 ORF 可以轉譯出 412 個胺基酸,預估分子量為 45.3 kDa。其 G+C 含量依次為75.18﹪、70.15﹪和69.09﹪,P450sre-1、P450sre-2和P450sre-3 基因與其他來源的P450 monooxygenase基因的胺基酸序列之相似度介於30%至65% 之間。將 P450sre-1、P450sre-2和P450sre-3 基因分別選殖入表現載體 pQE-30 中,並使其在 E. coli 中大量表現,再利用帶有金屬的親和性管柱來回收P450 monooxygenases。在菌體內的分析結果指出,三種 P450 基因的E. coli轉形株無法將Na - compactin轉換成為pravastatin,若將E. coli轉形株打破,則發現攜帶P450sre-3 基因之E. coli轉形株具有轉換的能力。再將P450sre-1、P450sre-2和P450sre-3 基因分別選殖入S. lividans 66表現載體 pIJ702 中,無論是在菌體內或者是試管實驗都顯示出 P450sre-1 和P450sre-2 基因之S. lividans 66的轉形株,均有能力將Na - compactin轉換成pravastatin;P450sre-3 基因之S. lividans 66轉形株則只在試管實驗中展現出轉換能力,其原因可能在於缺乏 promoter,以致於無法在S. lividans 66菌體內展現轉換能力。
URI: http://hdl.handle.net/11455/20891
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