Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli

Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein

“…The in vivo reconstituted relative fluorescence (RF) was measured in dual expression constructs with the left-out peptide fused to a carrier gene, intein. 15 Intein is a single-turnover enzyme whose activity is to splice the N and C-terminal ''extein'' sequences together to make a single polypeptide chain. In this case, only the N-terminal extein is present, so upon completion of translation and folding the intein cleaves, leaving a free peptide to bind to the LOO protein.…”

“…To create the construct for expressing the missing peptides, the sequence of the segment left-out from the LOO-GFP was fused to a carrier protein, Ssp-DnaB mini-intein. 15 The intein gene was amplified from pTWIN1 vector (New England Biolabs, Ipswich, MA) and cloned into pCDFDuet-1 vector via BglII/EcoRV sites. DNA encoding the missing peptide was synthesized by annealing overlapping oligos and inserted into pCDFDuet-1 vector carrying the intein gene via AgeI/EcoRV sites.…”

Expression and Simple Purification Strategy for the Generation of Antimicrobial Active Enterocin P from Enterococcus faecium Expressed in Escherichia coli ER2566

“…Although the current approach resulted in an insoluble and inactive form of EntP fusion protein, this may actually be advantageous due to: (i) increased stability of the fusion protein by reducing the susceptibility to intracellular proteases; (ii) reduced toxicity caused by active EntP, thus the fusion proteins can be synthesized and accumulated at high concentration in the cells; (iii) avoiding premature cleavage in vivo since a correct fold conformation is required for the self-splicing activity of intein (22). For instance, in a previous study by Gutiérrez et al, the low level of EntP when expressed in its active form in E. coli was believed to indicate toxicity to the host cells (9).…”

Purification of Recombinant Protein for Industrial Use

“…For example, the intein-based IMPACT system uses a protein fusion consisting e.g., of an N-terminal chitin-binding domain (affinity tag), a central intein and a C-terminal target protein [29]. Binding to a chitin matrix is followed by on-column cleavage using either a thiol reagent or pH and temperature shift to yield intein cleavage and elution of the target protein [30,31].…”