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
