Construction of food-grade inducible expression system in Lactococcus lactis and expression of fusion OprF/H from Pseudomonas aeruginosa

Abstract:

The food-grade inducible gene expression system in L. lactis was constructed for expression of cytoplasmic and anchored heterologous proteins. Gene α-aga encoding α-galactosidase was used as food-grade selectable marker instead of antibiotic resistance gene. Firstly，a food-grade cytoplasmic inducible expression vector pRNA48 containing α-aga，theta replicon from pRAF800，and P_nisA-MCS-T_pepN from pNZ8048 was constructed. Then the cell wall anchored expression vector pRNV48 containing α-aga，theta replicon，and P_nisA-S_PUsp45-nucA-CWA_M6-t1t2 was constructed based on the plasmids pRNA48 and pVE5524，which was suitable for the heterologous proteins anchored to the cell wall of L.lactis NZ9000. The fusion OprF/H derived from Pseudomonas aeruginosa was cloned into plasmids pRNA48 and pRNV48 to construct the pRNA48-OprF/H and ３RNV48-OprF/H for the expression of OprF/H. OprF/H was produced by the recombinant strains when induced with nisin. The highest yield of active OprF/H was 9.6% of intracellular soluble protein and 9.8% of cell wall anchored protein in L.lactis NZ9000，respectively. The immunogenicity and specificity of the expressed protein from recombinant were tested by animal immunization and Western blot.