Jiro Kohda

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins.

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

Preparation of Thermus thermophilus holo-chaperonin-immobilized microspheres with high ability to facilitate protein refolding.

Carboxylated poly(styrene/acrylamide) (P(St/AAm)-H) microspheres with different acrylamide contents were prepared by emulsifier-free emulsion polymerization. Thermus thermophilus holo-chaperonin (cpn) was covalently immobilized onto these microspheres with high yield. The T. thermophilus holo-cpn-immobilized microspheres were used for refolding of guanidine hydrochloride (Gdn-HCl)-denatured enzymes and showed sufficiently high ability to facilitate refolding of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD) and pig heart lactate dehydrogenase (LDH) at 30 degrees C and Bacillus stearothermophilus LDH at 60 degrees C. The specific ability of T. thermophilus holo-cpn-immobilized microspheres increased with increasing immobilization amount and reached plateau at around 10-15 mg/m(2). When the immobilization amounts of T. thermophilus holo-cpn were approximately 10 mg/m(2), the microspheres retained sufficiently high ability to facilitate protein refolding during repeated use. Therefore, the P(St/AAm)-H microspheres on which approximately 10 mg/m(2) of T. thermophilus holo-cpn is immobilized are very effective for refolding of various (Gdn-HCl)-denatured enzymes over a wide temperature range.

Affinity Purification of Fusion Chaperonin Cpn60-(His)6 from Thermophilic Bacterium Bacillus Strain MS and Its Use in Facilitating Protein Refolding and Preventing Heat Denaturation

The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned. Cpn60 with a hexahistidine affinity tag (His)6 fused to its C‐terminus (cpn60‐(His)6) was overproduced in Escherichia coli. Cpn60‐(His)6 was expressed in a soluble form in E. coli. and purified to homogeneity in a single step by nickel chelate affinity chromatography. Cpn60‐(His)6 formed a tetradecamer and had ATPase activity. Cpn60‐(His)6 mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 °C, respectively, in an ATP‐dependent manner. In addition, cpn60‐(His)6 prevented heat denaturation of pig heart MDH and T. flavus MDH at 30 and 80 °C, respectively, in an ATP‐dependent manner. Therefore, cpn60‐(His)6 facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.

Protein refolding system using holo-chaperonin from the thermophilic bacterium Thermus thermophilus

Abstract Thermus thermophilus holo-chaperonin (holo-cpn), a cpn60 14-mer-cpn10 7-mer complex, which was overproduced in Escherichia coli , was easily purified from a crude cell extract by heat treatment. The refolding of guanidine hydrochloride (GdnHCl) unfolded enzymes in the absence and presence of T. thermophilus holo-cpn was studied using Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD) and bovine deoxyribonuclease I (DNase I) as model enzymes. T. thermophilus holo-cpn significantly enhanced the recovery of the enzyme activities at around 30–40°C, which is the same temperature range within which the enzymes spontaneously recovered their highest activity. Since the complex formed by T. thermophilus cpn60 and cpn10 was stable, it could be separated from G6PD and DNase I in the refolding mixture by ultrafiltration using membranes with molecular weight cut-offs of 300 kDa and 100 kDa, respectively, and successfully reused. To increase the final concentration of renatured enzyme, step-wise addition of unfolded enzyme into the refolding mixture containing T. thermophilus holo-cpn was found to be effective. A combination of step-wise addition of unfolded enzyme and recycling of T. thermophilus holo-cpn using the ultrafiltration system significantly increased both the recovery of enzyme activity and the final concentration of renatured enzyme.

Development of Efficient Protein Refolding Systems Using Chaperonins

Application of chaperonins to in vitro protein refolding was investigated. E. coli chaperonin GroEL/ES and Thermus thermophilus holo-chaperonin (T. thermophilus holo-cpn) were used for construction of protein refolding systems The systems based on immobilized chaperonin, fusion chaperonin and chaperonin in combination with ultrafiltration were investigated. Fusion chaperonin with the hexa-histidine affinity tag (GroEL-(His)6) was efficiently purified by affinity column. The refolding activity of GroEL-(His)6 was proved to be similar to that of native one. GroEL/ES was immobilized by covalent bond. In addition, GroEL-(His)6 was immobilized on metal chelate resin by affinity interaction. These immobilized chaperonins retained sufficiently high refolding activities and were reused efficiently. Especially, immobilized GroEL-(His)6 showed a high refolding activity. On the other hand, in ultrafiltration system, T. thermophilus holo-cpn, which forms a stable complex, was efficiently separated from refolded proteins and repeatedly used for protein refolding. The selection of the system is mainly dependent on the type of chaperonin most effective for the refolding of target proteins.