Abstract:To achieve soluble expression of the Lactococcus lactis anchoring protein cA (C-terminal region of N-acetylmuraminidase [AcmA]) in Escherichia coli, and elucidate its biological activity, the AcmA gene was amplified by polymerase chain reaction (PCR) using L. lactis MG1363 as the template. The C-terminal of AcmA was fused with green fluorescent protein (GFP), followed by ligation of the chimeric cA-GFP into the E. coli expression vector pET28a, yielding the recombinant plasmid pET28a-cA-GFP. Subsequently, the recombinant plasmid was transformed into E. coli strain BL21 (DE3), and expression of the recombinant cA-GFP protein was induced at low temperature. After ultrasonic disruption of the recombinant strains, the supernatant was directly incubated with acid-pretreated L. lactis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence microscopy analyses were conducted. The results showed that the cA-GFP fusion protein with a molecular weight (MW) of about 53 ku (as expected) was expressed in the engineered bacteria. cA-GFP was successfully docked onto the cell surface of L. lactis via the inverse docking directed by the anchoring protein cA. One gram of dry bacteria could load 121 mg of cA-GFP protein. The fusion protein (cA-GFP) was very stable and the fluorescence intensity could be maintained at 82.2% when the proteins were stored at 4 ℃ for six days. The cA-GFP soluble protein with biological function was successfully obtained, which provides a solid basis for developing exogenous proteins with biological functions.
