Versatile format of minichaperone-based protein fusion system

Abstract

Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

Novel His-tag Variants for Insertion Inside Polypeptide Chain

“…Two proteins to which the idea of newly designed internal His-tags was applied are both used as fusion partners for different purposes. GrAD207, a permutated apical domain of T. thermophilus GroEL, was designed for stabilization in solution of proteins prone to aggregation, and allowed obtaining two initially insoluble proteins in stable soluble constructs. , The opportunity to purify these and other possible fusions with GrAD207 under mild native conditions should be a precious addition to its features. GrAD207 belongs to α and β structural classes, three-layer ββα fold (3.50 CATCH index).…”

Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability

“…6F ), in contrast to the heat-inactivated mPex. The other fusion tag has been previously applied for increasing renaturation efficiency, for example, the small molecular chaperone [ 29 ]. So, the M6 variant is an ideal alternate for tag removal in the refolding mixture under oxidative environment.…”

Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study

“…Thus, the development of plasmid vectors to co-express metalloproteins from haloarchaea and the genes coding for chaperons or enzymes related to the biosynthesis of cofactors (such as [Fe-S] or MoCo) is one of the main targets to be achieved in the future. In this context, the minichaperone-based protein fusion system recently described could be beneficial to achieve the overexpression of haloarchaeal proteins [84,85] Finally, because each protein is unique and due to the complex interactions among the reagents in experiments, it is mandatory to set up reaction conditions that would be optimal for each specific process to get recombinant proteins. Therefore, methods for the optimization of experimental conditions based on a one-factor-at-a-time approach should be replaced by a carefully selected small set of experiments characterized by their low cost and low time requirements.…”