USE OF Ssp DnaB MINI-INTEIN FOR THE PURIFICATION OF RECOMBINANT PHARMACEUTICAL PROTEINS

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

Entre Rios

Congreso; LIV Reunión anual SAIB; 2018

SAIB

Inteins are self-splicing polypeptides with the ability to excise themselves from flanking protein regions with remarkable precision. The aim of this work was to implement a purification methodology using the Synechocystis sp. DnaB mini-intein (Ssp DnaB) for the production of recombinant human growth hormone (rhGH) in Escherichia coli. We designed an expression vector to produce rhGH N-terminal fused to the CBD-Ssp DnaB chimeric protein. The CBD (Chitin Binding Domain) tag allows purification of proteins by affinity chromatography while Ssp DnaB mini-intein undergoes a self-cleavage reaction enabling the elution of rhGH without the affinity tag. Two rhGH variants were studied, the natural hGH whose first amino acid is phenylalanine (Phe-hGH) and a variant with an additional methionine at its N-terminal end (Met-hGH). The hGH coding sequences were E. coli codon optimized and synthesized with the first amino acid (Met or Phe according to the rhGH variant) right after cleavage site of Ssp DnaB mini-intein. Optimization of growth and induction conditions allowed the expression of large quantities of both rhGH variants. Then, the recombinant proteins were extracted from E. coli cells and purified by affinity chromatography. Both rhGH variants were efficiently bound to the chitin column by the CBD. However, Phe-hGH could not be recovered suggesting that Phe is not a recommended amino acid at the N-terminal end of the target protein for the self-cleavage of the Ssp DnaB mini-intein. On the other hand, Met-hGH was efficiently recovered with high purity obtaining a yield of approximately 12 g/L.