Create a fusion protein by jointing mLCC with hydrophobins
1. Molecular Cloning
For Molecular cloning, we selected pET28a as vector. We successfully amplified four gene segments of mLCC (as control group), mLCC-linker-mHFBI, mLCC-linker-mHGFI and 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.
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(Fig1. (a). M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp
(b). M: marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane 8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp
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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 of 20h were the best.
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Fig.2. (a). pET28a-mLCC transformed into BL21 expressing strains.
(b). pET28a-mLCC-linker-mHGFI transformed into BL21 expressing strains.
(c). pET28a-mLCC-linker-mHFBI transformed into BL21 expressing strains.
(d). pET28a-mLCC-linker-BslA transformed into BL21 expressing strains.
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Fig.3. (a).Induction time:16h; M: marker; (b).Induction time:20h; M: marker;(c). Induction time: 24h; For all lanes 1-12 in (a), (b), (c):
Lane 1,2,3: pET28a-mLCC-mHFBI, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 4, 5, 6: pET28a-mLCC-mHGFI, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 7, 8, 9: pET28a-mLCC, induction concentration: 0.5mM, 0.7mM,1.0mM;
Lane 10, 11, 12: pET28a-mLCC-BsIA, induction concentration: 0.5mM, 0.7mM,1.0mM;
3. Protein Purification
Because the pET28a vector has His-tag, therefore we used affinity chromatography to purify the supernatant obtained after bacteria clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified mLCC and mLCC-linker-BslA. At the same time, we can also see that the two proteins, mLCC-mHGFI, mLCC-mHFBI, are expressed, but the protein expression level is very low.
Fig.4. (a). M: marker;
Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
4. PET Degradation Reaction
We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8, 70℃, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, 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!
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Fig.
(a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.
(b) The relativity activity of mLCC, mLCC-Linker-BslA.
Method 2: Cell surface display of mLCC in B-subtilis.
1. Molecular Cloning
During cloning we used pHT43 plasmid. 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.
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Fig. 5. (a). M: marker; Lane 1: cotB-linker-mLCC; Lane 2: cotC-linker-mLCC; Lane 3: cotG-linker-mLCC 1426bp Lane 4: cotX-linker-mLCC 1357bp
(b). M: marker; Lane 1: cotB-linker-mLCC 1986bp ; Lane 2: pHT43-cotB-linker-mLCC (after construction);
(c). cotB-linker-mLCC transformed into pHT43 plasmid
