GODINO AGUSTINA
Congresos y reuniones científicas
Título:
Use of Ssp DnaB mini-intein for the purification of recombinant pharmaceutical proteins
Autor/es:
PAULA VACCRELLO; AMARANTO MARILLA; BARRA JOSÉ LUIS; AGUSTINA GODINO
Lugar:
Paraná, Entre Ríos, Argentina
Reunión:
Congreso; LIV Reunión anual SAIB; 2018
Resumen:
Inteins are self-splicing polypeptides with ability to excise themselves from flanking protein regions with remarkable precision. The aim of thiswork was to implement a purification methodology using the Synechocystissp. DnaB mini-intein (SspDnaB) for the production of recombinanthuman growth hormone (rhGH) in Escherichia coli. We designed an expression vector to produce rhGH N-terminal fused to the CBD-SspDnaBchimeric protein. The CBD (Chitin Binding Domain) tag allows purification of proteins by affinity chromatography while SspDnaB mini-inteinundergoes a self-cleavage reaction enabling the elution of rhGH without the affinity tag. Two rhGH variants were studied, the natural hGHwhose first amino acid is phenylalanine (Phe-hGH) and a variant with an additional methionine at its N-terminal end (Met-hGH). The hGHcoding sequences were E. coli codon optimized and synthesized with the first aminoacid (Met or Phe according to the rhGH variant) right aftercleavage site of SspDnaB mini-intein. Optimization of growth and induction conditions allowed the expression of large quantities of both rhGHvariants. Then, the recombinant proteins were extracted from E. coli cells and purified by affinity chromatography. Both rhGH variants wereefficiently bound to the chitin column by the CBD. However, Phe-hGH could not be recovered suggesting that Phe is not a recommendedaminoacid at the N-terminal end of the target protein for the self-cleavage of the SspDnaB mini-intein. On the other hand, Met-hGH wasefficiently recovered with high purity obtaining a yield of approximately 12 g/L.