Construction Of Vectors For Protein Expression In Pseudomonas Sp. And Cell Surface Display Of Metallothionein

A stable Pseudomonas expression vector was constructed with the promoter of oprL gene cloned from Pseudomonas putida AB92019.Expression efficiency of the vector is determinated by employing green fluorescent protein(GFP) gene as the reporter,and the stability of the vector in the host strain is determinated as well. Further more,a cell surface display vector for Pseudomonas on the basis of expression vector was constructed by using ice nucleation protein N-domain(InaK-N) as anchoring motif.Green fluorescent protein and metallothionein were successfully displayed on the surface of Pseudomonas putida,respectively,by the way of C-terminal fusion to the N-domain of ice nucleation protein,and the abilities of bioadsorption of several heavy metal ions of recombinant strains were determined.A 380-bps of PCR-amplified promoter sequence of peptidoglycan-associated lipoprotein encoding gene(PoprL) from P.putida wild-type strain AB92019,and a MCS fragment from Escherichia coli plasmid vector pTrcHis-B,were inserted into EcoRâ… /Hindâ…¢sites of plasmid vector pUCP18 to generate a Pseudomonas shuttle expression vector pYMB03.Expression determination by employing green fluorescence protein(GFP) as the reporter,showed that GFP can be constitutionally expressed either in recombinant P.putida YMB001 or transformed E.coli DH5αharboring the recombinant plasmid pYMBP,which initiated by the introduced promoter(PoprL) of pYMB03.Consequently,the hosts are illuminant through the optical fluorescent microscopic examination.SDS-PAGE analysis confirmed that expressed GFPs comprising approximately 12.5%and 5%of the total cellular proteins, respectively,in YMB001 and transformed E.coli DH5α.GFP yielding in YMB001 was modulated by the culture time,and it reached the maximum level when cells grown into the stationary phase,whereas relatively little impact was found by the culture temperature.Additionally,by determining the successive growth of the recombinant strain YMB001/YMB002 in the absence of the antibiotic,it showed that the stability of the shuttle expression vector pYMB03 was 100%when inoculating in succession for 7 times and culturing for 168 h. A Pseudomonas cell surface display vector pYN was constructed by inserting inaK-n gene into the direct downstream of PoprL of pYMB03.GFP successfully displayed on the cell surface of P.putida by inserting gfp gene into the vector pYN, consequently,transforming into the P.putida host strain.The Location of fusion protein InaK-N/GFP on the cell surface was proven by cell fractionation and Western-Blot.Aiming at applicating the cell surface display vector more widely,a metallothionein(SmtA) can bioaccumulate various toxic metal ions was fused with the InaK-N,and a series of recombinant plasmids(pYNS,pYN2S,pYN3S,pYN4S), 1 to 4 tandem repeats smtA gene was fused with inaK-n gene respectively,were constructed.We report abilities of bioaccumulation of Cd²⁺,Cu²⁺,Mn²⁺ of the recombinant P.putida strains(YN-S,YN-2S,YN-3S,YN-4S) harboring these plasmids,respectively,in the different cultural temperature.It shows that YN-2S(two tandem repeats SmtA cell surface displayed) can bioaccumulate best(every mg dry weight can bioaccumulate 62.10±6.70 nmol Cd²⁺,71.54±5.98 nmol Cu²⁺,89.86±1.05 nmol Mn²⁺) when it was cultured at 24℃,and it shows much more tolerance to environment with high concentration of these toxic metals.