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Applications of BBa_K3656304

This year, we used Pyrococcus horikoshii OT3 carbonic anhydrase (OT3-CA-WT) as the research object because this carbonic anhydrase (CA) can catalyze the hydration of CO2 to HCO3- with high thermal stability. Based on its coding sequence, we constructed a new part OT3-CA-WT (BBa_K3656304) and obtained its protein with higher thermostability.

Engineered E. coli TB1(DE3) with high OT3-CA-WT expression

Construction of OT3-CA-WT expression plasmids

The coding sequences of OT3-CA-WT (BBa_K3656304) were both synthesized , then cloned into the expression vector pET-28a(+) (Fig.1). The length of digested OT3-CA-WT was 546 bp, confirmed by the band above 500 bp in electrophoresis as indicated in Fig. 2, which indicated that the pET-28a(+) OT3-CA-WT recombinant plasmid was successfully constructed.

Fig.1 Map of OT3-CA-WT recombinant plasmid

Fig.2 Enzyme digestion of OT3-CA-WT recombinant plasmid. Lane M: DL marker; Lane 1: OT3-CA-WT recombinant plasmid; Lane 2: the enzyme digested OT3-CA-WT recombinant plasmid, the length was 546 bp (the arrow indicated).

Induced expression of OT3-CA-WT protein

The OT3-CA-WT expression plasmid was transformed into E. coli TB1, and positive clones were screened for kanamycin resistance. The recombinant bacteria were expanded and cultured, and isopropyl-1-thio-β-Dgalactopyrasonide (IPTG) was used to induce the expression of OT3-CA-WT in E. coli TB1. Then the bacteria were lysed to extract the total protein, and SDS-PAGE and Western Blot were applied to verify the expression of OT3-CA-WT. As shown in Fig. 3 and Fig. 4 it showed that after induction by IPTG, a band about 19.5 KDa with a molecular weight similar to CA was appeared, as indicated by the arrow. This indicates that we have successfully constructed an engineered strain of E. coli TB1 expressing OT3-CA-WT.

Fig.3 Induced expression of OT3-CA-WT in TB1 strain verified by SDS-PAGE

Fig.4 Induced expression of OT3-CA-WT in TB1 strain verified by Western blot

OT3-CA-WT purification

After confirming that OT3-CA-WT can be expressed in E. coli TB1, due to the nickel chloride in the nickel column can bind to the protein with His (histidine) tag, we used nickel column to further purify the OT3-CA-WT, then we used the Bradford method to test the concentration of the purified OT3-CA-WT protein. The analysis showed that the purified protein concentration of OT3-CA-WT was 7.7 mg·mL-1 (Fig. 5).

Fig.5 Evaluation of OT3-CA-WT protein concentration via Bradford method

Enzyme activity assay of OT3-CA-WT

We measured the enzymatic activity of OT3-CA-WT protein at 37°C, 45°C and 70°C by esterase activity assay. As shown in Fig. 6, the result showed that the enzyme activity at 45°C is almost or slightly higher than that at 37°C. Although the enzyme activity of OT3-CA-WT at a higher temperature of 70°C slightly decreases, it still has a higher activity. This shows that we have obtained carbonic anhydrase with specific high-temperature resistance and good thermal stability, which is also in line with our previous assumptions.

Fig.6 Esterase activity analysis of OT3-CA-WT protein at different temperatures

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