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来自敏捷硫杆菌的细胞色素c550：克隆、在大肠杆菌中的表达及异源全蛋白的纯化。

Cytochrome c550 from Thiobacillus versutus: cloning, expression in Escherichia coli, and purification of the heterologous holoprotein.

作者信息

Ubbink M, Van Beeumen J, Canters G W

机构信息

Gorlaeus Laboratories, Department of Chemistry, Leiden University, The Netherlands.

出版信息

J Bacteriol. 1992 Jun;174(11):3707-14. doi: 10.1128/jb.174.11.3707-3714.1992.

DOI:10.1128/jb.174.11.3707-3714.1992
PMID:1339423
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC206060/
Abstract

The gene coding for cytochrome c550 from Thiobacillus versutus, cycA, has been cloned and sequenced. It codes for a protein of 134 amino acids plus a 19-amino-acid-long signal peptide. Both coding and noncoding DNA sequences of the clone are homologous to the Paracoccus denitrificans DNA sequence. An expression vector was constructed by cloning the cycA gene directly behind the lac promoter of pUC. The cycA gene was expressed in Escherichia coli under semianaerobic conditions, and mature holo-cytochrome c550 was isolated with the periplasmic soluble protein fraction. Under both aerobic and anaerobic conditions, significantly less cytochrome c550 was produced. The heterologously expressed cytochrome c550 was isolated and purified to better than 95% purity and was compared with cytochrome c550 isolated and purified from T. versutus. No structural differences could be detected by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis UV-visible light spectroscopy, and 1H nuclear magnetic resonance spectroscopy, indicating that E. coli produces the cytochrome and attaches the heme correctly.

摘要

已克隆并测序了来自氧化亚铁硫杆菌的细胞色素c550编码基因cycA。它编码一个由134个氨基酸组成的蛋白质以及一个19个氨基酸长的信号肽。该克隆的编码和非编码DNA序列均与反硝化副球菌的DNA序列同源。通过将cycA基因直接克隆到pUC的lac启动子后面构建了一个表达载体。cycA基因在半厌氧条件下在大肠杆菌中表达，成熟的全细胞色素c550从周质可溶性蛋白组分中分离出来。在有氧和厌氧条件下，细胞色素c550的产量均显著降低。对异源表达的细胞色素c550进行了分离和纯化，纯度优于95%，并与从氧化亚铁硫杆菌中分离和纯化的细胞色素c550进行了比较。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、紫外-可见光谱和1H核磁共振光谱未检测到结构差异，表明大肠杆菌产生了细胞色素并正确地连接了血红素。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/206060/42bd9191fe7f/jbacter00077-0303-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/206060/b8aa99f952da/jbacter00077-0301-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/206060/42bd9191fe7f/jbacter00077-0303-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/206060/b8aa99f952da/jbacter00077-0301-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/206060/42bd9191fe7f/jbacter00077-0303-a.jpg

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