USE OF AFFINITY TAGGED VMA1 INTEIN FOR THE PRODUCTION OF RECOMBINANT PHARMACEUTICAL PROTEINS.

MARILLA AMARANTO; MARIA EUGENIA CORREA; JAVIER N. GARAY NOVILLO; JOSÉ LUIS BARRA

Córdoba

Congreso; LII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

We analyzed whether the affinity tagged Saccharomyces cerevisiae VMA1 intein, fused to a chitin binding domain tag (CBD), can be used to produce recombinant pharmaceutical proteins without the N-Formilmethionine (fMet) terminal residue, using E. coli as protein factory. We designed and constructed an expression vector to produce the human growth hormone (HGH) C -terminal fused to the CBD-intein chimeric protein. HGH coding sequence was E. coli codon optimized and it was synthesized so as its first amino acid after the natural N-terminal methionine, phenylalanine, is right after cleavage site of VMA1 intein. The CBD permits subsequent protein affinity purification while VMA1 intein undergoes a self-cleavage reaction enabling the elution of HGH having now the phenylalanine residue as the first N-terminal residue. Optimization of growth, induction, extraction and purification conditions allowed us to reproducibly produce the HGH recombinant protein in large quantities and with high purity. These results suggest that the CBDVMA1 intein, having the coding sequence of the protein of interest cloned right after the intein cleavage site could be used for commercial production of recombinant proteins with impact in biopharmaceutical and/or biotechnological industries.