title: An automatic refolding apparatus for preparative-scale protein production.
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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai, China.
authors.forename: Yanye
authors.initials: Y
authors.lastname: Feng
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhang
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Ding
authors.forename: Yingping
authors.initials: Y
authors.lastname: Zhuang
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Zhong
authors.initials: Z
authors.lastname: Yang
mesh_terms: D000465:Algorithms; D000818:Animals; D057205:Automation, Laboratory; D002417:Cattle; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods; D014508:Urea / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D014508:Urea