Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli

AMARANTO, MARILLA; VACCARELLO, PAULA; CORREA, ELISA M.E.; BARRA, JOSÉ L.; GODINO, AGUSTINA

JOURNAL OF BIOTECHNOLOGY

ELSEVIER SCIENCE BV

Año: 2021 vol. 332 p. 126 - 134

0168-1656

We evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatographypurification of recombinant proteins produced in Escherichia coli. We used human growth hormone (hGH)as target protein that contains two internal disulfide bridges and an N-terminal phenylalanine. Use of N-terminalthiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with selfcleavageinducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tagresulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in acytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed usingan N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion proteinwas properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-terminusof the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acidof the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-basedself-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bondsand free of extra amino acids.