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We investigated the expression and stability of enhanced green fluorescent protein (eGFP) under extreme conditions using two types of high-copy-number vectors and two types of anchoring motifs (CotB and C-terminally truncated ∆CotB spore coat proteins) for the development of a spore surface display system in Bacillus subtilis. The fused cotB-gfp and ΔcotB-gfp DNA fragments were cloned into the pUB19 (pUB110-derived) and pHY300PLK vectors. Four types of expression vectors were transformed into B. subtilis 168. The expression level of eGFP on the surface of spores prepared from B. subtilis transformants was measured by flow cytometry. When pUB19 vector was used, the activities of ∆CotB-eGFP and CotB-eGFP were 17.9 and 5.6 times higher than those of the pHY300PLK vector, respectively. In addition, the activity of pUB19-∆CotB-eGFP was 1.76 times higher than that of pUB19-CotB-eGFP. Overall, the activity of eGFP was more stable under extreme conditions (heat, pH, and protease challenges) when ∆CotB was used as an anchoring motif instead of CotB. Compared to the control groups, the activities of ΔCotB-eGFP and CotB-eGFP were maintained at 56% and 41% at 80 °C and 88% and 55% at pH 10, respectively. The activities of ΔCotB-eGFP and CotB-eGFP were maintained at 62% and 41%, respectively, when treated with 0.03 U of proteinase K. In addition, the activities were maintained at 77% and 36%, respectively, when treated with 5.5 U of trypsin.

applied biological chemistry

Comparison of the stability of eGFP displayed on the Bacillus subtilis spore surface using CotB and C-terminally truncated CotB proteins as an anchoring motif under extreme conditions