Development of gene expression systems and high cell density cultivation for the production of bioadhesive precursor protein

Abstract

In nature when materials need to be joined in most cases the only option is via adhesion, often promoted by adhesives. Since bioadhesives of marine organisms (such as mussels, barnacles and oysters) are applied and cured in moist environments, they are potentially useful for many medical and dental applications. Mussel adhesive protein is comprised mainly of about 75 repeats of the decapeptide sequence Ala-Lys-Pro-Ser-Tyr-HydroxyPro-HydroxyPro-Thr-Dopa-Lys. Biological synthesis system of bioadhesive protein analog in Escherichia coli was developed, because isolation of the uncured adhesive from mussels for commercial use is not practical. For the expression of bioadhesive precursor protein in a tandem repeats, 4 expression vectors using inducible T7 expression system were developed and successfully expressed the bioadhesive precursor protein with various molecular weight. Orientation of the repeats was suppressed by the complementary nature of nonpalindromic ends generated in BlpI. High cell density culture and high level expression are essential for the mass production of bioadhesive precursor protein. In order to achieve these goals, the effects of T7 lysozyme and plasmid size on the cell growth and production of a bioadhesive precursor protein were studied in fed-batch culture. Cell growth and bioadhesive precursor protein expression level were not significantly different between BLR(DE3)[pKYC0.8] and BLR(DE3)[pLysS] [pKYC0.8] in batch culture. In fed-batch culture, however, BLR(DE3) [pLysS][pKYC0.8] showed growth retardation and much lower level of bioadhesive precursor protein expression than that obtained with BLR(DE3)[pKYC0.8]. This result suggests that the presence of pLysS in the T7 expression system strongly affects the cell growth and expression of BP protein in high cell density cultivation. Therefore, if the target protein is not toxic to the cell, higher expression level of foreign protein can be obtained with BLR(DE3) strain without pLysS in hi...