共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Krishna Kumar Singh Ruchi Jain Harini Ramanan Deepak Kumar Saini 《Molecular biotechnology》2014,56(12):1121-1132
Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol. 相似文献
16.
17.
18.
Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators. Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions. Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity. In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells. The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction. Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1. The purified protein yielded a single, 43kDa protein band upon SDS-polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources. Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions. Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation. 相似文献
19.
Hiromura M Choi CH Sabourin NA Jones H Bachvarov D Usheva A 《The Journal of biological chemistry》2003,278(16):14046-14052