Cloning and expression of a recombinant human CBD (carbohydrate binding domain) : a comparison between two expression models

Abstract

Genetic engineering is a method that provides the production of recombinant proteins for different purposes. Nowadays there are several and commercially available expression systems aiming the production of these proteins. However, in same cases, the solubility and stability of the produced protein can be a problem, especially for eukaryotic proteins, which need post translational modifications to be biologically active. In the present work, different strategies were tested to increase the solubility of a human carbohydrate binding domain (CBD), present in laforin phosphatase. Namely, different expression systems, different hosts and fermentation conditions, the presence of additives and detergents during lyses, were used. The DNA coding sequence was cloned by PCR into three prokaryotic expression systems: pET 29a, pET 25b; and two eukaryotic systems: pGAPZaC and pPICZaC. The CBD was expressed at high level in pET system. In pET29a the CBD protein was obtained in inclusion bodies. In pET25b, a small amount of soluble protein was obtained in the presence of arginine and CHAPS, in the lyses buffer. Although a soluble recombinant protein was obtained, it was not stable in solution, aggregating easily. On the other hand, the utilisation of two expression systems of Pichia pastoris led to the production of soluble and stable CBD in extra cellular medium; however, this CBD was obtained at low expression level and its activity was not confirmed.