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.
Highlights
The over-expression of recombinant proteins in Escherichia coli (E. coli) is widely used to produce proteins in large amounts, due to noticeable advantages such as growth on inexpensive carbon sources, rapid biomass accumulation, amenability to high celldensity fermentation and relative ease for increasing production scale [1,2,3,4]
According to the statistics in the Center for Eukaryotic Structure Genomics (CESG), among 8048 cloned targets in E. coli, only about 30% of them were expressed in soluble forms, whereas the others either were degraded or formed insoluble aggregates, known as inclusion bodies
The conformation of the refolded proteins were monitored by acquiring circular dichroism (CD) or NMR two-dimensional 1H-15N HSQC spectrum; the aggregationstates of the refolded target proteins were analyzed by either running dynamic light scattering (DLS) experiment, or running nondenaturing PAGE gel, or using size exclusion chromatography
Summary
The over-expression of recombinant proteins in Escherichia coli (E. coli) is widely used to produce proteins in large amounts, due to noticeable advantages such as growth on inexpensive carbon sources, rapid biomass accumulation, amenability to high celldensity fermentation and relative ease for increasing production scale [1,2,3,4]. To obtain soluble active proteins from inclusion bodies, the insoluble inclusion bodies need to be first solubilized in denaturant, and followed by a step of refolding process (for comparison, we refer to it here as ‘‘the one-step-denaturing and refolding’’ method) [5]. This procedure has been used over 20 years and works quite well for many inclusion body proteins, with approximately 40% being refolded to soluble and biologically active forms [5,6,7]. In most cases, there is a significant amount of precipitation when refolding the proteins, resulting in a great loss of overall yield of the target proteins
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