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 - 126

0168-1656

e evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatography purification 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-terminal thiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with selfcleavage inducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tag resulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in a cytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed using an N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion protein was properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-ter