Abstract
Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production.
Highlights
An explosion in the field of structural genomics and protein expression has greatly increased our knowledge of how to manipulate proteins [1,2]
Different methods were carried out to evaluate the performance of this refolding apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin-human artemin fusion protein (Trx-ARTN); dilution refolding for thioredoxin-human insulin-like growth factor I fusion protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA)
The widely used simple dilution, dialysis and on-column refolding methods are to test with several proteins: a combination of dilution and dialysis for SDF-1/CXCL12 and Trx-ARTN; dilution refolding for Trx-IGF1 and EGFP; and on-column refolding for BSA
Summary
An explosion in the field of structural genomics and protein expression has greatly increased our knowledge of how to manipulate proteins [1,2]. One of the most attractive means of producing recombinant proteins utilizes genetically modified E. coli, due to their low cost, high productivity, and ease of use [3]. A substantial hindrance to these efforts is the formation of insoluble inclusion bodies. It is possible to denature and refold the insoluble proteins, with an increasing number of refolding approaches [4]. The procedures for refolding proteins from inclusion bodies consisted of 1) solubilizing inclusion bodies with denaturing agents, and 2) refolding proteins through the removal of denaturants. The refolding of proteins from inclusion bodies must still be largely performed with a series of trial-and-error renaturation experiments
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