USE OF A MINI-INTEIN FOR THE AFFINITY PURIFICATION OF RECOMBINANT THERAPEUTIC PROTEINS FROM ESCHERICHIA COLI PERIPLASM

GODINO, A.; AMARANTO, M.; BARRA, J. L.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB

from Escherichia coli periplasm. This development combines the delivery of proteins to the periplasmic space, together with a self-cleavable affinity tag for the purification process. The expression of recombinant proteins directed by a periplasmic signal peptide allows the expression of proteins with any N-terminal amino acid (except Pro), and the periplasmic environment facilitates disulfide bridge formation. We use the recombinant human growth hormone (rhGH) as a model protein. The therapeutic rhGH starts with a Phe residue, it has two disulfide bridges and cannot have affinity tags. We designed an expression vector to produce the rhGH N-terminal fused to a periplasmic signal peptide and the C-terminal fused to a mini-intein / Chitin Binding Domain (CBD) chimeric affinity tag. The CBD allows purification of recombinant proteins by chitin affinity chromatography, while the mini-intein undergoes a self-cleavage reaction enabling the elution of rhGH without any extra amino acid. Optimization of the upstream conditions allowed us the expression of six times more recombinant protein in the periplasm than that obtained under non-optimized conditions. The mini-intein/CBD polypeptide allowed the purification of the recombinant protein by affinity chromatography using a chitin column. Elution of the rhGH without any extra amino acid was achieved after induction of the self-cleavage of the mini-intein by decreasing the pH of the buffer and increasing the temperature of the affinity column. The rhGH without any tag was efficiently recovered with high purity under these experimental conditions. These results suggest that this expression and purification system could be used for the production of therapeutic and research proteins with any N-terminal amino acid and without affinity tag.