Part:BBa_K4202009:Experience

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1.Construction of Pet28a-His tag-ACCBP

We obtained the CDS and protein sequence of ACCBP and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-ACCBP. The codon of SP-ACCBP was optimized for Bacillus subtilis and synthesized by GenScript, and the plasmid PHY-300PLK-SP-ACCBP(PHY-ACCBP) was constructed.
Considering the large quantity of protein we needed for function verification, we constructed the expression plasmid PET-28a(+)-ACCBP for E.coli in order to obtain the purified protein of high concentration for later experiments. Besides, we also constructed the palsmid Pet28a(+)-Δ16 ACCBP（BBa_K4202010）to explore the influence of N-terminal amino acids to protein folding.

2.Protrein expression verification

To confirm that the optimized sequence could be produced by E. coli, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 ^o C overnight. The next day, the overnight-cultured solution was transferred into 5 ml kanamycin resistant LB medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl₂ was added to induce the protein expression at 8^oC

At the end of induction, the bacterial solution was centrifuged at 12000 rpm at 4°C and the bacteria were collected. After washing the precipitation once with PBS, the precipitation were resuspended in a 1.5ml EP tube with 1mL PBS (appropriate amount of PMSF can be added).The bacteria were disrupted by ultrasonic disruption until the suspension was clear. The products are used for SDS-PAGE.

3.Protein expression and purification

The overnight-cultured bacterial fluid was inoculated into 500 ml Kanamycin resistant LB medium at the ratio of 1:100. The solution was cultured at 37 ^° C , 220rpm until OD₆₀₀ reached 0.6-0.8 and protein was induced at 8 ^oC with 1mM IPTG, 1% glycerol and 1mM CaCl2 for 48h. Then we collect the bacterium and resuspend in a special protein extract buffer (containing 10% glycerol, 500 mM PH 7.5 Tris-HCl buffer, and 0.01mM PMSF ). The next steps are same as 1 above .
Notes: In order to get a better protein expression effect, we also use palsmids Pet28a(+)-ACCBP, Pet28a(+)-Δ16 ACCBP(BBa_K4202010), strains BL21(DE3) and shuffle T7 for this experiment .

4.Verification of ACCBP structure

In order to verify the structure of active ACCBP protein, we conducted chemical crosslinking experiment. Add 0.1% and 0.5% glutaraldehyde to 25μl ACCBP protein eluent, mix and let it stand at room temperature. Take samples at regular intervals. After terminating the reaction with loading buffer, run 12% SDS-PAGE to detect the cross-linking structure.

5.Extracellular crystallization assay

Add an appropriate amount of ACCBP solution, water and MgCl₂ solution into the newly prepared saturated Ca(HCO₃)₂ to prepare 20μl solution containing 0, 8.0, 16.0, 40.0 mM MgCl₂. Drip the 20μl reaction system onto the cover glass. After 24 hours of reaction, observe the calcium carbonate crystals on the slides were studied by SEM.

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