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从枯草芽孢杆菌中克隆hbs基因并在大肠杆菌中表达HBsu蛋白。

Cloning the hbs gene from Bacillus subtilis and expression of the HBsu protein in Escherichia coli.

作者信息

Ghodsi S, Gharavi S, Ghadam P

机构信息

Biology Department, Faculty of Sciences, Alzahra University, Vanak, Tehran, IR Iran.

出版信息

Iran J Microbiol. 2010 Sep;2(3):152-6.

PMID:22347565
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3279786/
Abstract

BACKGROUND AND OBJECTIVES

Bacillus subtilis HBsu is a 10 kD heat-stable protein shown to be involved in binding to DNA and is encoded by the hbs gene. Large-scale production for biochemical analysis is achieved through cloning and expression of the recombinant protein.

MATERIALS AND METHODS

This gene was amplified from B. subtilis ATCC 6633 using PCR and cloned into pET28a (+) expression vector. The construct was used to transform Escherichia coli BL21 (DE3). The expression of the protein was induced by the addition of 1mM IPTG. To confirm the expression of the cloned gene, SDS-PAGE was carried out and production of an approximately 11 KD recombinant tagged protein was confirmed for the cloned hbs gene.

RESULTS AND CONCLUSION

The identity of the recombinant HBsu was verified and characterized by SDS-PAGE which can then be utilized for further applications.

摘要

背景与目的

枯草芽孢杆菌HBsu是一种10 kD的热稳定蛋白，已证明其参与与DNA的结合，由hbs基因编码。通过重组蛋白的克隆和表达实现了用于生化分析的大规模生产。

材料与方法

使用PCR从枯草芽孢杆菌ATCC 6633中扩增该基因，并将其克隆到pET28a(+)表达载体中。该构建体用于转化大肠杆菌BL21(DE3)。通过添加1mM IPTG诱导蛋白表达。为了确认克隆基因的表达，进行了SDS-PAGE，并确认克隆的hbs基因产生了约11 KD的重组标签蛋白。

结果与结论

通过SDS-PAGE验证并表征了重组HBsu的身份，随后可将其用于进一步的应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/893d44fbb90f/IJM-2-152-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/003931d0225b/IJM-2-152-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/0c243e560db7/IJM-2-152-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/a0ae33995ee9/IJM-2-152-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/789e8e48ce7f/IJM-2-152-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/893d44fbb90f/IJM-2-152-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/003931d0225b/IJM-2-152-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/0c243e560db7/IJM-2-152-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/a0ae33995ee9/IJM-2-152-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/789e8e48ce7f/IJM-2-152-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f32e/3279786/893d44fbb90f/IJM-2-152-g005.jpg

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本文引用的文献

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2
Surface salt bridges modulate the DNA site size of bacterial histone-like HU proteins.表面盐桥调节细菌组蛋白样HU蛋白的DNA结合位点大小。
Biochem J. 2005 Aug 15;390(Pt 1):49-55. doi: 10.1042/BJ20050274.
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IHF and HU stimulate assembly of pre-replication complexes at Escherichia coli oriC by two different mechanisms.整合宿主因子（IHF）和组蛋白样蛋白（HU）通过两种不同机制刺激大肠杆菌oriC处前复制复合物的组装。
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Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB.枯草芽孢杆菌两种DNA结合蛋白HBsu和AbrB的结构-功能关系及调控
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The histone-like protein HU binds specifically to DNA recombination and repair intermediates.类组蛋白HU特异性结合DNA重组和修复中间体。
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The Bacillus subtilis HBsu protein modifies the effects of alpha/beta-type, small acid-soluble spore proteins on DNA.枯草芽孢杆菌HBsu蛋白可改变α/β型小酸溶性芽孢蛋白对DNA的影响。
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