Extracellular production of recombinant proteins using bacterial systems may enable the proteins concerned to retain important properties including structural authenticity and functional activity. By employing a secretory approach together with the Bacillus subtilis veg-expression-system, an alluring yield of human basic fibroblast growth factor (hbFGF), which had never been expressed as an authentic protein in recombinant approach, was successfully achieved by me as protein secreted in the culture supernatant.

However, despite the secretory nature of hbFGF, it has not been successfully expressed through excretion in Escherichia coli. With the development and application of a novel intein-mediated-cleavage-excretory system, I was able to demonstrate, for the first time, ex...[ Read more ]

Extracellular production of recombinant proteins using bacterial systems may enable the proteins concerned to retain important properties including structural authenticity and functional activity. By employing a secretory approach together with the Bacillus subtilis veg-expression-system, an alluring yield of human basic fibroblast growth factor (hbFGF), which had never been expressed as an authentic protein in recombinant approach, was successfully achieved by me as protein secreted in the culture supernatant.

However, despite the secretory nature of hbFGF, it has not been successfully expressed through excretion in Escherichia coli. With the development and application of a novel intein-mediated-cleavage-excretory system, I was able to demonstrate, for the first time, excretory production of both hbFGF and human epidermal growth factor (hEGF) in the same E. coli host. Both the growth factors, existing in their mature authentic structures, were detected in both the cell lysate and culture medium fractions. Furthermore, making use of the same intein-mediated-cleavage excretory system, high levels of endoglucanase A of Cellulomonas fimi and hEGF were co-produced in the E. coli host, supporting the conclusion that the system may serve as a versatile platform for use in the co-expression of proteins with widely different origins and properties.

Despite the merits of extracellular expression, hyper-expression of the secretory proteins often results in severe cell death. A novel strategy consisted of the use of an intein-mediated-cleavage system and “delayed” secretion of the precursor protein, was developed. By fusing the DnaB mini-intein at the N-terminus of the OmpA signal peptide of an exoglucanase (Exg), the cell death and plasmid curing caused by secretory Exg was decreased, leading to a significant improvement in the yield of excreted Exg.

The various novel expression strategies introduced in above and discussed in this dissertation may prove to be applicable for cost-effective production of a wide range of valuable proteins.