High‐throughput automated refolding screening of inclusion bodies

Abstract

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…While this assay measures solubility and not necessarily folding, it has been shown previously that it can be used for estimation of protein folding with high confidence. 28 However, to distinguish between some falsepositive measurements obtained by this approach (Supporting Information Table S1, in gray) (see also Materials and Methods section), we visually re-analyzed each well and found 17 (of 95) visually clear wells, which indicates proper folding of a2N under these conditions (Supporting Information Table S1, in green). Figure 3 shows a summary of these experiments.…”

“…27 Thus, correct refolding should be validated by either: (i) circular dichroism spectroscopy, (ii) dynamic light scattering, (iii) SEC, or (iv) functional biochemical and interaction assays. 28 Since first two methods are not compatible with high concentrations of NDSB-256 reagent present in our first preparation, we validated its binding properties after denaturation using surface-plasmon resonance protein-protein interaction assay. In these ''BIAcore TM '' experiments, the purified and refolded a2N was immobilized on a CM4 chip by amine-coupling and the affinity of its interaction with ARNO was studied.…”

Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins

“…IBs usually consist of almost pure, aggregated proteins which are typically misfolded and thus biologically inactive [19]. In spite of the numerous known reasons for the IB formation, to date there has been no universal technique for efficient folding of the aggregation-prone recombinant proteins [20,21]. Although there are few examples where formation of IBs is highly desirable, such as their use for oral vaccination as an alternate route for the delivery of recombinant antigens for immunization [22], in most cases obtaining a recombinant protein in the soluble form is absolutely crucial.…”

Improved Refolding Efficacy of Recombinant Human Interferon α-2b via pH Modulation

“…In fact, increasing pH above 8.0 dramatically decreases C cf value. Thus, the previous data obtained from refolding efficacy revealed that solubilization cannot be a true approach to finding the optimum correct protein refolding conditions [16,17]. Data from this study showed that "mixed refolding efficacy", correct folding of interferon, as determined by potency assay were increased simultaneously and was predictable till pH 8.0.…”

“…All refolding processes were performed batchwise at 4 °C and the refolding efficiency was determined as the ratio of solubilized protein concentration to the total denatured protein [16,17] at pH 7.0, 7.5, 8.0 and 8.5. The refolding buffer (50 mM Tris-HCl and 50 mM NaCl) was prepared at various pHs (pH 7.0, 7.5, 8.0 and 8.5).…”