Artículo
Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli
Amaranto, Marilla
; Vaccarello, Paula; Correa, Elisa María Eugenia
; Barra, Jose Luis
; Godino, Agustina
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Fecha de publicación:
05/2021
Editorial:
Elsevier Science
Revista:
Journal of Biotechnology
ISSN:
0168-1656
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
We 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 self-cleavage 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-terminus of the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acid of the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-based self-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bonds and free of extra amino acids.
Palabras clave:
DISULFIDE BOND
,
DNAX
,
ESCHERICHIA COLI
,
HGH
,
INTEIN
,
PERIPLASMIC
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Articulos(CIQUIBIC)
Articulos de CENTRO DE INVEST.EN QCA.BIOL.DE CORDOBA (P)
Articulos de CENTRO DE INVEST.EN QCA.BIOL.DE CORDOBA (P)
Citación
Amaranto, Marilla; Vaccarello, Paula; Correa, Elisa María Eugenia; Barra, Jose Luis; Godino, Agustina; Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli; Elsevier Science; Journal of Biotechnology; 332; 5-2021; 126-134
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