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从未培养的环境微生物中筛选和纯化新型细胞色素b5

Screening and purification for novel cytochrome b5 from uncultured environmental micro-organisms.

作者信息

Roh C, Villatte F, Kim B-G, Schmid R D

机构信息

School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.

出版信息

Lett Appl Microbiol. 2007 May;44(5):475-80. doi: 10.1111/j.1472-765X.2007.02118.x.

Abstract

AIMS

We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b(5) (cyt b(5)) gene directly from metagenomic DNA is described.

METHODS AND RESULTS

Using the degenerate primer set, a cyt b(5) gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b(5) from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b(5) was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized.

CONCLUSIONS

Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA.

SIGNIFICANCE AND IMPACT OF THE STUDY

Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes.

摘要

目的

我们描述了一种基于序列的聚合酶链反应(PCR)方法,该方法适用于直接从宏基因组DNA中分离细胞色素b5(cyt b5)基因的新型可溶性血红素结合结构域。

方法与结果

使用简并引物组,直接从宏基因组DNA中分离出cyt b5基因。基于基于序列的PCR方法,沼泽红假单胞菌菌株中cyt b5的相似保守基序构成了新的靶基因。使用pET表达系统将编码cyt b5的基因克隆到大肠杆菌BL21(DE3)中并进行表达。通过镍-亚氨基三乙酸亲和色谱法纯化表达的重组酶并对其进行表征。

结论

基于序列的策略是从宏基因组DNA中应用新基因的有效方法。

研究的意义和影响

对宏基因组中的新基因进行研究,由于大多数微生物物种尚未得到充分开发,可能是生物过程中一个有趣且有用的资源库。

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