Part:BBa_K2547003

Carbonic anhydrase (csoS3)-His

The carbonic anhydrase from Halothiobacillus neapolitanus converts the incoming bicarbonate into carbon dioxide in the carboxyl group, a step that is essential for CO 2 fixation. We have codon-optimized the coding sequence (CDS) to make the carbonic anhydrase gene more suitable for expression in E. coli. Simultaneously linking a histidine tag (His-Tag) to the coding sequence (CDS) is beneficial to carbonic anhydride. Purification of enzyme protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The coding sequence of Carbonic anhydrase csoS3 from original part was codon-optimized, and also a His tag was added to the end, to ensure that Carbonic anhydrase csoS3 could be expressed in E. coli BL21 (DE3) and retained potent carbonic anhydrase activity (Fig. 1).

    

Fig. 1 Map of Carbonic anhydrase csoS3-His expression vector

First, the original coding sequence of csoS3 and the coding sequence with codon optimization were synthesized, and cloned into the pET-30a (+) expression vectors, respectively. The correctness of the two recombinant plasmids was verified by PCR (Fig. 2).

Fig. 2 Agarose Gel Electrophoresis of Carbonic anhydrase csoS3 expression vectors and its identification by PCR. Lane M: DL marker; Lane 1: expression vector of csoS3 new part; Lane 2: PCR band of expression vector of csoS3 new part, the length was 1620 bp; Lane 3: expression vector of csoS3 original part; Lane 4: PCR band of expression vector of csoS3 original part, the length was 1620 bp.

Subsequently, the expression of two csoS3 plasmids in E. coli was detected via SDS-PAGE and Coomassie blue staining. As shown in Fig. 3, the result presented that the expression of csoS3 original part in E. coli was relatively low, and the expression of codon-optimized csoS3 new part in E. coli was higher than original part.

    

Fig. 3 SDS-PAGE and Coomassie blue staining of Carbonic anhydrase csoS3 plasmids expressed in E. coli BL21(DE3) strains. The arrow indicated was the bands of csoS3. Lane 1: Negative control (cell lysate without IPTG induction) of new part; Lane 2: Cell lysate with induction for 6 h at 37 ℃ of new part; Lane 3: Negative control (cell lysate without IPTG induction) of original part; Lane 4: Cell lysate with induction for 6 h at 37 ℃ of original part.

To further demonstrate the activity of our new part, new part of csoS3 carbonic anhydrase was purified through Ni-chelating affinity chromatography and detected by SDS-PAGE and Coomassie blue staining, as shown in Fig. 4. Then, the activity of csoS3 was measured via esterase method, and the enzyme activity was about 22.84 U/mL.

    

Fig. 4 SDS-PAGE analysis of purified Carbonic anhydrase csoS3 protein.

In conclusion, our results demonstrated that the function of csoS3 new part has been improved with higher expression and activity than original part.