Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Abstract

We have recently employed an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), in conjunction with efficient expression and secretory functions formed between the ompA leader sequence and the human epidermal growth factor (EGF) gene (fused at the 5' end of VMA), and the human basic fibroblast growth factor (bFGF) gene (fused at the 3' end of VMA), to engineer an efficient intein-based Escherichia coli system for high-level co-expression of EGF and bFGF as authentic mature products. Both products were found not only excreted to the culture medium but also located, surprisingly, in the cytoplasm (Kwong and Wong 2013). In this study, we employed two structurally varied inteins, VMA and Mycobacterium xenopi GyraseA (GyrA), and further demonstrated that despite acting alone, both VMA and GyrA were able to mediate successful co-expression of two widely different proteins, EGF and an endoglucanase (Eng) in E. coli. Although EGF and Eng were initially expressed as large precursors/intermediates, they were soluble and auto-cleavable to finally yield the desired products in both the cytoplasm and culture media. The results further substantiate our postulation that the aforementioned intein/E. coli approach might lead to the development of cost-effective and versatile host systems, wherein all culture fractions are involved in producing the target proteins.

Intein mediated hyper-production of authentic human basic fibroblast growth factor in Escherichia coli

“…Both EGF 23 24 25 and bFGF were identified as precisely processed products detached from their fusion intein partner, VMA, in both the culture medium and the cytoplasm 9 . The same approach has also been employed for co-expression and auto-cleavages of fusion products formed between different inteins and widely dissimilar proteins 11 .…”

“…Our laboratory has been involved in the engineering of various recombinant host systems for efficient expression of valuable proteins 9 10 11 12 13 14 15 16 17 . Recently, with Bacillus subtilis as the host, we have been successful in achieving secretory expression of fully bioactive bFGF 10 .…”

“…Mediated by the coding sequence for an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), we have engineered an efficient protein expression construct, pWK3R, to successfully co-express bFGF and human epidermal growth factor (EGF) as bioactive and authentic products 9 . The same expression approach has also been employed to efficiently co-express widely dissimilar recombinant proteins 11 . Since the target proteins are present in both the cytoplasm and growth media of the expression systems concerned, the utilization of all compartments of the culture for storage may effectively enhance the overall yields of the desired proteins 9 11 .…”

“…The same expression approach has also been employed to efficiently co-express widely dissimilar recombinant proteins 11 . Since the target proteins are present in both the cytoplasm and growth media of the expression systems concerned, the utilization of all compartments of the culture for storage may effectively enhance the overall yields of the desired proteins 9 11 .…”

Efficient Generation of Hydrazides in Proteins by RadA Split Intein

“…In this case, the transient thioester created during the N→S shift of inteins is resolved by a strong nucleophile, such as hydrazine (hydrazinolysis, Figure A). This method was reported for the Mycobacterium xenopi GyrA (Mxe GyrA; 198 residues; 28 kDa) intein, which yielded 65 % of the desired hydrazide . Because intein N cleavage was inefficient, long incubations with hydrazine alone led to protein degradation.…”