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Applications of BBa_K2547000

Construction of wild-type human carbonic anhydrase 2 (CA2-WT) expression plasmid We first synthesized the sequence of CA2-WT, and then cloned it into the expression vector pET-30a(+), and identified the correctness of the obtained recombinant vector by restriction enzyme digestion and sequencing (Fig. 1 and Fig. 2).

    

Fig. 1 Map of CA2-WT recombinant vector

Fig. 2 Agarose Gel Electrophoresis of CA2-WT recombinant plasmid and its identification by enzyme digestion. Lane M: DL marker; Lane 1: CA2-WT recombinant plasmid; Lane 2: enzyme digestion band of CA2-WT plasmid digested by MluⅠ, the length was 1028 bp (the arrow indicated).

Induced expression of CA2-WT

The CA2-WT expression plasmid was transformed into E. coli BL21 (DE3), and positive clones were screened by kanamycin resistance. Then, the recombinant E. coli BL21 (DE3) were propagated and CA2-WT expression was induced at the fourth hour of cell cultivation using an IPTG concentration of 500 μM. Cells were lysed by sonication on ice, and the obtained crude extract was centrifuged to separate supernatant and debris, and both fractions were subjected to SDS-PAGE and Western Blot (Fig. 3 and Fig. 4). The arrow indicated in Fig. 3 was the band of CA2 protein as the molecular weight of CA2 is about 30.6 kDa, and the correctness of CA2 protein was also confirmed by Western blot assay in Fig. 4. It can be seen from lanes 1 and 2 in both Figures that the CA2-WT expression was significantly induced with IPTG incubation. Results from lanes 3-6 indicated that the induced expression of CA2 mainly existed in soluble form in the cell lysate supernatant. In consequence, the results above demonstrate that an engineered E. coli BL21 (ED3) strain that expresses CA2-WT has been constructed.

    

Fig. 3 SDS-PAGE analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain.

    

Fig. 4 Western blot analysis for CA2-WT cloned in pET-30a(+) and expressed in BL21(DE3) strain.

Purification of CA2-WT protein

After confirming that CA2-WT could be expressed in E. coli BL21 (DE3), CA2-WT protein was further purified with nickel column, and the resulting protein had a molecular mass corresponding to CA2-WT protein (Fig. 5 and Fig. 6). Western blot analysis showed the protein to be recognized by antibodies specifically recognizing histidine-tag (Fig. 6).

    

Fig. 5 CA2 was purified with Ni column; fractions were analyzed by SDS-PAGE. Lane M: Protein marker; Lane 1: Supernatant after cell lysate centrifugation; Lane 2: Flow through; Lane 3: Wash with 50mM Tris, 150mM NaCl, 20 mM Imidazole, pH 8.0; Lane 4: Elute with 50mM Tris, 150mM NaCl, 50 mM Imidazole, pH 8.0; Lane 5: Elute with 50mM Tris, 150mM NaCl, 500 mM Imidazole, pH 8.0.

    

Fig. 6 SDS-PAGE and Western blot analysis of CA2-WT protein. Lane 1: Positive control (BSA); Lane 2: purified CA2-WT; Lane 3: Purified CA2-WT.

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