Dataset Information

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.

ABSTRACT: The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.

SUBMITTER: Yang Z

PROVIDER: S-EPMC3146519 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.

Yang Zhong Z Zhang Linlin L Zhang Yan Y Zhang Ting T Feng Yanye Y Lu Xiuxiu X Lan Wenxian W Wang Jufang J Wu Houming H Cao Chunyang C Wang Xiaoning X

PloS one 20110729 7

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventi ...[more]

PMID: 21829569

Similar Datasets

Project description:BackgroundSpider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process.ResultsHere, we present a mild solubilization strategy-one-step heating method to solubilize spidroins from IBs, with combining spidroins' high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70-90 °C for 20 min, pH 7.0-10, urea concentration 2-4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology.ConclusionsThe one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.

Dataset Information

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.

Publications

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets