Use of affinity tagged VMA1 intein for the production of recombinant pharmaceutical proteins.

AMARANTO, MARILLA; CORREA, EUGENIA; GARAY NOVILLO, JAVIER; BARRA, JOSÉ LUIS

Córdoba

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

We analyzed whether the affinity tagged Saccharomyces cerevisiae VMA1 intein, fused to achitin binding domain tag (CBD), can be used to produce recombinant pharmaceutical proteinswithout the N-Formilmethionine (fMet) terminal residue, using E. coli as protein factory. Wedesigned 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 codonoptimized and it was synthesized so as its first amino acid after the natural N-terminalmethionine, phenylalanine, is right after cleavage site of VMA1 intein. The CBD permitssubsequent protein affinity purification while VMA1 intein undergoes a self-cleavage reactionenabling the elution of HGH having now the phenylalanine residue as the first N-terminalresidue. Optimization of growth, induction, extraction and purification conditions allowed us toreproducibly produce the HGH recombinant protein in large quantities and with high purity.These results suggest that the CBD-VMA1 intein, having the coding sequence of the protein ofinterest cloned right after the intein cleavage site could be used for commercial production ofrecombinant proteins with impact in biopharmaceutical and/or biotechnological industries.