Difference between revisions of "Part:BBa K2547004"

Revision as of 19:40, 21 October 2019

Carbonic anhydrase 2 (L203K)

This part is the coding sequence (CDS) of the mutant human carbonic anhydrase 2 (CA2 (L203K)) with a His-tag attached. Because wild-type CA2 has the fastest reaction rate at 37 °C and loses its activity at 50 °C, so it may be not suitable for using wild type CA2 to capture CO₂ under industrial operating conditions. Therefore, we use molecular simulation to design new high-efficiency and stable carbonic anhydrases by improving their catalytic properties and biostability. We have found that when the amino acid encoded by the 203th codon is mutated from leucine to lysine, the resulting CA2 is more thermostable than wild type CA2.

Sequence and Features

Construction of mutant human carbonic anhydrase 2 (CA2 (L203K)) expression plasmid

Because wild-type CA2 has the fastest reaction rate at 37 °C and loses its activity at 50 °C, so it may be not suitable for using wild type CA2 to capture CO2 under industrial operating conditions. Therefore, we use molecular simulation to design new high-efficiency and stable carbonic anhydrases by improving their catalytic properties and stability. Basing on the simulation results above, we finally determined that the suitable mutation site of CA2 with high and stable activity was L203K (the 203th leucine mutated into lysine).

Therefore, we constructed an expression vector containing CA2 (L203K) coding sequence for following activity assay (Fig. 1). The obtained recombinant vector was verified by restriction enzyme digestion (Fig. 2) and sequencing.

    

Induced expression of CA2 (L203K) protein

The CA2 (L203K) expression plasmid was transformed into E. coli BL21 (DE3), and its expression was induced with IPTG, and identified by SDS-PAGE analysis. The results showed that CA2 (L203K) could be expressed in BL21 (DE3) strain and existed in soluble form in the cell lysate supernatant (Fig. 3).

    

Purification of CA2 (L203K) protein

In order to detect the enzyme activity of CA2 (L203K) protein, we further purify the crude protein extract by nickel column to obtain purified CA2 (L203K) protein. CA2 (L203K) was purified with high purity as indicated by a significant single protein band after SDS-PAGE and Western blot (Fig. 4).

    

Enzyme activity assay of CA2-WT and CA2 (L203K) protein

Next, we determined the enzymatic activities of wild-type and mutant CA2 by colorimetric and esterase methods. As indicated in Fig. 5, specific activity of mutant CA2 was about 2 times greater than that of wild-type enzyme. The kinetic constants (Km and Vmax) were calculated for esterase activity assay, and the result showed that CA2 (L203K) protein has a higher activity than CA2-WT (Fig. 6).

    

    

Thermal stability studies of CA2-WT and CA2 (L203K) protein

We then investigated the effect of temperature on CA2 activity by esterase activity assay. As shown in Fig. 7, as the temperature increases, especially at 55 °C and 65 °C, the enzymatic activity of CA2-WT was significantly decreased, while the mutant CA2 still retain relatively high activity, indicating that CA2 (L203K) was more stable at high temperature and retained its activity.

    

User Reviews

Contribution from iGEM2019 AHUT_China

nina_wang

In 2019, AHUT_China iGEM team has contructed a new biobick by connecting the C-terminal of this part coding sequences with the six-residue sulfatase submotif(LCTPSR), then achieved enzyme immobilization and tested its activity by esterase method, the reuse ability of the immobilized enzyme has been tested by the designed simulation model for CO2 capture. The result was documented in the experience page and the main page of BBa_K2949013.

1. Engineered E.coli TB1

1.1 Construction of CA2(L203K)-C-LCTPSR expression plasmid

The coding sequence of CA2(L203K)-C-LCTPSR was synthesized, and then cloned into pET-30a(+) expression vector.

Fig.1 Map of CA2(L203K)-C-LCTPSR recombinant vector

The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2) and sequencing(Fig.3).

Fig.2 Agarose Gel Electrophoresis of CA2(L203K)-C-LCTPSR recombinant plasmid and its identification by enzyme digestion.
Lane M: DL15000 marker; Lane 1: CA2(L203K)-C-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-C-LCTPSR recombinant plasmid, the length was 834 bp (the arrow indicated).

Fig.3 Sequencing results

1.2 Expression and purification of CA2(L203K)-C-LCTPSR protein in E.coli TB1

The expression of CA2(L203K)-C-LCTPSR in E.coli were detected by SDS-PAGE. The results showed that CA2(L203K)-C-LCTPSR could be successfully expressed in our chassis E.coli.(Fig.4)

Fig.4 SDS-PAGE analysis for CA2(L203K)-C-LCTPSR cloned in pET-30a(+) and expressed in E.coli TB1
Lane 1: CA2(L203K)-C-LCTPSR protein expression without IPTG induction; Lane 2: CA2(L203K)-C-LCTPSR protein expression wit IPTG induction.

We successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-C-LCTPSR expression vector into E.coli TB1 for the following CA2(L203K)-C-LCTPSR immobilization. Then the improve part of CA2(L203K)-C-LCTPSR protein was further purified through nickel column and detected by SDS-PAGE, as shown in Fig.5.

Fig.5 SDS-PAGE of purified CA2(L203K)-C-LCTPSR protein

2. Identification of the function for CO₂ capture

2.1 The efficiency of CA2(L203K)-C-LCTPSR protein immobilization

FGE can selectively identify and oxidize cysteine residues in the sulfatase subunit(LCTPSR) at the end of the protein to form aldehyde-containing formylglycine, which can be used for enzyme immobilization. Then we immobilized CA2(L203K)-C-LCTPSR protein, and our formula for calculating the enzyme immobilized efficiency is as follows：

η: The efficiency of immobilized CA2(L203K)-C-LCTPSR protein;
W1: The concentration of total CA2(L203K)-C-LCTPSR protein;
W2: The concentration of free CA2(L203K)-C-LCTPSR protein.

According to the formula, we got the efficiency of immobilized CA2(L203K)-C-LCTPSR protein is 39.09%.

2.2 Enzyme activity asssay of CA2(L203K)-C-LCTPSR protein

To further demonstrate the activity of our improved part, the enzyme activity of CA2(L203K)-C-LCTPSR (BBa_K2949013) and CA2(L203K) protein (BBa_K2547004) of CO₂ capture were tested experimentally by esterase activity assay at 37℃ and 50℃.

As shown in Fig.6 and Fig.7, immobilized CA2(L203K)-C-LCTPSR protein was stable at high temperature and retained its activity, and free CA2(L203K)-C-LCTPSR protein has a higher activity than CA2(L203K) protein.

Fig.6 Esterase activity analysis of free CA2(L203K), free CA2(L203K)-C-LCTPSR and immobilized CA2(L203K)-C-LCTPSR protein at 37℃

Fig.7 Esterase activity analysis of free CA2(L203K), free CA2(L203K)-C-LCTPSR and immobilized CA2(L203K)-C-LCTPSR protein at 50℃

3. Application Model for detecting CO₂ capture

Because the immobilized CA2(L203K)-C-LCTPSR protein have higher activity and reuse ability than immobilized CA2(L203K)-N-LCTPSR protein, so the reuse ability of the immobilized CA2(L203K)-C-LCTPSR was tested by our designed simulation model (Fig.8). Compared with the original enzyme, the immobilized enzyme still retained 54 percent activity after five times of repeated absorption experiments of CO₂, as indicated in Fig.9. The result showed that the immobilized CA2(L203K)-C-LCTPSR could absorb CO₂ under the simulation model and showed potential reuse ability.

Fig.8 Picture of our designed model

Fig.9 The reuse ability of CO₂ capture of the immobilized CA2(L203K)-C-LCTPSR under our designed model

In conclusion, our results demonstrated that the function of improved part has been improved with higher activity than original part, especially achieved enzyme immobilization, and the immobilized CA2(L203K)-C-LCTPSR protein showed reuse ability, which might be suitable for industrial production.