Joint mLCC with another protein hydrophobins to create a fusion protein
Cell surface display of mLCC in B-subtilis
Joint mLCC with another protein hydrophobins to create a fusion protein
1. Molecular Cloning
For Molecular cloning, we selected pET28a plasmid. We successfully amplified fourgene segments of MLCC (as control group), mLCC-linker-mHFBI, mLCC-linker-mHGBIand mLCC-linker-BslA. Then we digested and connected the four segments to pET28a vector through two restriction enzymes of BamHI and XhoI. At present, four recombinant plasmids have been successfully constructed.
M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp
M:marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp
2. Protein Expression
We transformed four recombinant plasmids into BL21 expressing strains. For four recombinant strains, we tried three IPTG induction concentrations of 0.5mM,0.7mM, 1.0mM and three induction times of 16h, 20h and 24h, respectively. We found that the induction concentration of 0.5mM IPTG and the induction time of20h were the best.
pET28a-mLCC transformed into BL21 expressing strains
pET28a-mLCC-linker-mHGFI transformed into BL21 expressing strains
pET28a-mLCC-linker-mHFBI transformed into BL21 expressing strains
pET28a-mLCC-linker-BslA transformed into BL21 expressing strains
(Because the pET28a plasmid has His-tag), therefore we used affinity chromatography to purify the supernatant obtained after clastogenesis using nickel. The result of our proteo gels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified MLCC and MLCC-BslA. At the same time, we can also see that the two proteins, MLCC-mHGFI, MLCC-mHFBI, are expressed, but the protein expression is very low.
We perform PET degradation reaction to detect enzyme activity. We set several protein concentrations to detect enzyme activity, under the condition of pH8, 70°C, and18h of reaction time. Then we measured the absorption value at uv240nm by nanodroplets, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!
Method 2: Cellsurface display of mLCC in B-subtilis.
1. Molecular Cloning
During cloning we used pHT43 plasmid Method 2: Cell surface display. We successfully performed PCR amplification of all the four fusion genes. As we continued our lab, recombinant plasmid with CotB was successfully constructed after the double digestion verification (pHT43-cotB-linker-mLCC). However, the other three CotC, CotG, and CotX are still continuing to be tried out.
M:marker; Lane 1: cotB-linker-mLCC; Lane 2: cotC-linker-mLCC; Lane 3: cotG-linker-mLCC1426bp Lane 4: cotX-linker-mLCC 1357bp
M: marker; Lane 1: cotB-linker-mLCC 1986bp ; Lane 2: pHT43-cotB-linker-mLCC (after construction)
