Separation of recombinant human vasoactive intestinal peptide–humanin fusion protein via three different approaches

Both vasoactive intestinal peptide (VIP) and humanin (HN) can provide neuroprotection against β-amyloid toxicity and are believed to be beneficial in the treatment of Alzheimer's disease. For the sake of tandem co-expression of recombinant human VIP and HN(rhVIP-HN) fusion protein, new VIP-HN gene is constructed and cloned into the plasmid pET28a(+) and is expressed in Escherichia coli BL21(DE3). Since the fusion protein was present as inclusion bodies, three different preparation approaches are employed to obtain the hexa-histidine tagged rhVIP-HN (His6-rhVIP-HN) fusion protein by Ni2+ chelating resin chromatography. The comparison of the results obtained from the three approaches reveals that renaturation during the separation process (Approach III) is the most efficient for large-scale preparation of His6-rhVIP-HN. Ultimately, 46 mg of the target protein was obtained from one gram of the inclusion body with purity up to 90%. Since there is not separated step in renaturation procedure, Approach III could be more time-saving, buffer-saving and easy to operate than the other two approaches. These results can be useful for preparation of rhVIP-HN in large scale.