Use of Ssp DnaB mini-intein for the purification of recombinant pharmaceutical proteins.

Congreso; LIV Reunión anual SAIB,; 2018

Inteins are self-splicing polypeptides with ability to excise themselves from flankingprotein regions with remarkable precision. The aim of this work was to implement apurification methodology using the Synechocystis sp. DnaB mini-intein (Ssp DnaB) for theproduction of recombinant human growth hormone (rhGH) in Escherichia coli. Wedesigned an expression vector to produce rhGH N-terminal fused to the CBD-Ssp DnaBchimeric protein. The CBD (Chitin Binding Domain) tag allows purification of proteins byaffinity chromatography while Ssp DnaB mini-intein undergoes a self-cleavage reactionenabling the elution of rhGH without the affinity tag. Two rhGH variants were studied, thenatural hGH whose first amino acid is phenylalanine (Phe-hGH) and a variant with anadditional methionine at its N-terminal end (Met-hGH). The hGH coding sequences wereE. coli codon optimized and synthesized with the first amino acid (Met or Phe according tothe rhGH variant) right after cleavage site of Ssp DnaB mini-intein. Optimization of growthand 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 affinitychromatography. Both rhGH variants were efficiently bound to the chitin column by theCBD. However, Phe-hGH could not be recovered suggesting that Phe is not arecommended amino acid at the N-terminal end of the target protein for the self-cleavage ofthe Ssp DnaB mini-intein. On the other hand, Met-hGH was efficiently recovered with highpurity obtaining a yield of approximately 12 g/L.