期刊
JOURNAL OF BIOTECHNOLOGY
卷 332, 期 -, 页码 126-134出版社
ELSEVIER
DOI: 10.1016/j.jbiotec.2021.04.003
关键词
hGH; Intein; DnaX; Disulfide bond; Periplasmic; Escherichia coli
资金
- Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCYT) grant PICT [2016-1051, 2018-0139]
- Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCYT) grant PICT Start-Up [2018-00811]
- Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCYT) grant PICT-II-B-2018 Argentina Innovadora -Jovenes [2018-01519]
- Secretaria de Ciencia y Tecnologia (SECYT-UNC) grant PRIMAR-TP [248/2018, 32520170100233CB]
- SECyT-UNC (2018-2020) [411/18, 455/18]
Several intein-based self-cleaving affinity tags were evaluated for expression and purification of recombinant proteins in Escherichia coli. Different tags resulted in different outcomes, with some leading to complete cleavage of the protein while others were unable to release the target protein.
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 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-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.
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