Difference between revisions of "Part:BBa K4202009"

Revision as of 10:51, 12 October 2022

Amorphous Calcium CarbonateBinding Protein(ACCBP)(without signal peptite)

Amorphous calcium carbonate binding protein(ACCBP) is a protein that can help to form and transform amorphous calcium carbonate(ACC).

In calcium carbonate solution，the ions can automatically aggregate and form prenucleation clusters, which further form ACC through certain pathways. ACC will transform into minerals of different crystal forms. In this process, organisms mainly regulate the biological mineralization process by regulating ACC formation and transformation. ACCBP is an important protein that can help to form ACC and transform ACC into calcite at specific conditions.

To ensure that this part can express active protein, the corresponding plasmid should be transformed into the E.coli, and the protein is induced at specific conditions.

Sequence and Features

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

Result: Acorrding to the result of SDS-PAGE, we can found that the more protein were both in the supernatant and precipitation. And the more protein are in the precipitation, indicating that under this condition majority of the protein are insoluble.

Fig 1 SDS-PAGE and Coomassie brilliantblue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .

3.Protein expression and purification

Result : The purified ACCBP and Δ16 ACCBP protein were obtained by multiple combination of plasmids and strains. The protein will be used in further experiments. </p>

Fig 2 SDS-PAGE and Coomassie brilliantblue staining results of protein purification. A: The purification result of ACCBP; B: The purification result of Δ16 ACCBP. The results show that both proteins have a considerable abundance in eluent 2 and 3 although there are some non-objective bands shown due to proteolysis.

4.Verification of ACCBP structure

Result: According to the result of SDS-PAGE, we found that that the band of 140kDa was the most significant one of all samples. As the apparent molecular weight of ACCBP monomer is about 25kDa，we predicted that ACCBP probably assembles as pentamers in the solution. And the cross-linking results were better with less non-objective bands when higher glutaraldehyde concentration was used. </p>

Fig 3 SDS-PAGE and Coomassie brilliantblue staining results of ACCBP chemical crosslinking experiment.

5.Extracellular crystallization assay

Result: For better images, the calcium carbonate crystals were observed in different magnifications. It was obvious that when the concentration of Mg²⁺ was low, ACCBP induced the formation of typical square calcite crystals. When the concentration of Mg²⁺ reached 40mM, ACCBP induced the formation of more aragonite particles. Our results also indicated that the deletion of 16 N-terminal amino acids of ACCBP had no significant effect on the protein function. </p>

Fig 4 The influence of Mg²⁺ to the crystallization results of ACCBP