Difference between revisions of "Part:BBa K2547004"
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<p>We first synthesized the sequence of the mutant CA2, 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).<br></p> | <p>We first synthesized the sequence of the mutant CA2, 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).<br></p> | ||
<div align="center"> https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg | <div align="center"> https://static.igem.org/mediawiki/parts/7/7f/T--AHUT_China--_par1t.jpg | ||
Revision as of 12:56, 13 October 2018
Carbonic anhydrase 2 (L203K)
This part is the coding sequence (CDS) of the mutant carbonic anhydrase (CA2), because wild type carbonic anhydrase (CA2) has the fastest reaction rate at 37 ° C and loses its activity at 50 ° C, so it is chosen to be applied. CO2 capture is not suitable for industrial production. We use molecular simulation to design new high-efficiency/stabilized carbonic anhydrases by improving their catalytic properties and improving their biostability. First, the analysis of carbonic anhydrase (CA2) was carried out by means of computer-aided analysis software, and the design principles were initially established. Second, enzyme-substrate molecules docked; third, enzyme-solvent kinetics simulation. We have found that when the amino acid encoded by the 203th codon is mutated from leucine to lysine, the resulting carbonic anhydrase is more thermostable and encoded. Simultaneous addition of a histidine tag (His-Tag) after the sequence (CDS) facilitates purification of the carbonic anhydrase protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
We first synthesized the sequence of the mutant CA2, 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).
Induced expression of CA2(L203K)
The CA2(L203K) expression plasmid was transformed into E. coli BL21 (DE3), and the cultured liquid was subjected to IPTG-induced CA2 (L203K) expression, and the bacterial solution was sonicated, followed by SDS-PAGE(figure 3), the size of CA2(L203K) is known to be 30.6 kDa, which is compared with Marker. The position indicated by the arrow in the figure is the CA2(L203K) band. It can be seen from lanes 1 and 2 in the figure that the IPTG condition is significant to the expression of CA2 which was induced, and it can be seen from lanes 3-6 that the induced expression of CA2 was mainly expressed in soluble form in the supernatant of the bacterial liquid. The above results indicated that we successfully obtained E. coli expressing CA2(L203K).
Purification of CA2(L203K) protein
After confirming that CA2(L203K) can be induced by E. coli BL21(DE3), we will further purify the crude protein extract by nickel column purification to obtain purified CA2(L203K) protein. Figure 4 shows the results. We have obtained a highly purified mutant CA2 protein.
Determination of protease activity of CA2 and CA2 (L203K)
We determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As shown in Figure 5 and Figure 6, mutant CA2 (L203K) has higher enzymatic activity than wild-type CA2.
Analysis of Thermal Stability of CA2 and CA2 (L203K)
We examined the activity of carbonic anhydrase in wild-type and mutant CA2 at different times and temperatures by esterase method. The results are shown in Figure 7. As the temperature increases, especially at 55 ° C and 65 ° C, The enzymatic activity of wild type CA2 was significantly decreased, while the mutant CA2 still had higher activity, indicating that CA2 (L203K) has better thermal stability.
