Difference between revisions of "Part:BBa K2949013"

Latest revision as of 02:06, 22 October 2019

Carbonic Anhydrase 2(L203K)-C-LCTASR

We constructed this new biobrick by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized support (Unisil 30-100 NH2) through the Schiff base reaction.

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]

In order to further improve the industrial application of CA2 for CO₂ capture, basing on the existing part we designed last year, we have constructed a new biobrick[CA2(L203K)-C-LCTPSR] by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)](BBa_K2547004) coding sequences with the six-residue sulfatase submotif(LCTPSR) in a way similar to that of CA2(L203K)-N-LCTPSR, to achieve enzyme immobilization and maintain high thermal stability and CA2 reuse by modifying its gene sequence.

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).

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 TB1 were detected by SDS-PAGE. The results showed that CA2(L203K)-C-LCTPSR could be successfully expressed in our chassis E.coli TB1.(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 vectpr 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 and CA2(L203K) protein 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 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

4. Conclusion

Our results demonstrated that the function of CA2(L203K)-C-LCTPSR 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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2949013 short</partinfo>
-we constructed this new biobrick by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized carriers (Unisil 30-100 NH2) through the Schiff base reaction.
+We constructed this new biobrick by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) with the six-residue sulfatase submotif (LCTPSR). Formylglycine generating enzyme can selectively recognize and oxidize the cysteine residues in the sulfatase subunit of the protein terminal to form formylglycine (FGly) residues containing aldehyde groups, which can be immobilized via forming covalent bond with amino functionalized support (Unisil 30-100 NH2) through the Schiff base reaction.
 <!-- Add more about the biology of this part here
@@ Line 17: / Line 17: @@
 <partinfo>BBa_K2949013 parameters</partinfo>
 <!-- -->
+In order to further improve the industrial application of CA2 for CO<sub>2</sub> capture, basing on the existing part we designed last year, we have constructed a new biobrick[CA2(L203K)-C-LCTPSR] by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)](BBa_K2547004) coding sequences with the six-residue sulfatase submotif(LCTPSR) in a way similar to that of CA2(L203K)-N-LCTPSR, to achieve enzyme immobilization and maintain high thermal stability and CA2 reuse by modifying its gene sequence.
-In order to the further improve the industrial application of CA2 for CO2 capture, basing on the existing part (BBa_K2547004) we designed last year, we have constructed a new biobrick[CA2(L203K)-C-LCTPSR](BBa_K2949013) by connecting the C-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)](BBa_K2547004) coding sequences with the six-residue sulfatase submotif(LCTPSR) in a way similar to that of CA2(L203K)-N-LCTPSR, to achieve enzyme immobilization and maintain high thermal stability and CA2 reuse by modifying its gene sequence.
+===1. Engineered E.coli TB1===
+====1.1 Construction of CA2(L203K)-C-LCTPSR expression plasmid====
-The coding sequence of CA2(L203K)-C-LCTPSR(BBa_K2949013) was synthesized, and then cloned into pET-30a(+) expression
+The coding sequence of CA2(L203K)-C-LCTPSR was synthesized, and then cloned into pET-30a(+) expression vector (Fig.1).
-vector.
-The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.10) and sequencing(Fig.11).
-Subsequently, 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 E.coli.(Fig. 12)
-To use for the following CA2(L203K)-C-LCTPSR immobilization, the improve part of CA2(L203K)-C-LCTPSR protein was purified through nickel column and detected by SDS-PAGE, as shown in Fig.13.
+[[File:T--AHUT China--ImproPart 01.jpg|900px|center|thumb|Fig.1 Map of CA2(L203K)-C-LCTPSR recombinant vector]]
-FGE can selectively identify and oxidize cysteine residues in the sulfatase subunit(LCTPSR) at the end of the protein to form aldehyde-containing formylglycinase,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：
+The correctness of the obtained recombinant vector was identified by restriction enzyme digestion (Fig.2) and sequencing (Fig.3).
-η: The efficiency of immobilized CA2(L203K)-N-LCTPSR protein;
-W1: The concentration of total CA2(L203K)-N-LCTPSR protein;
+[[File:T--AHUT China--ImproPart 02.png|400px|center|thumb| Fig.2 Agarose Gel Electrophoresis of CA2(L203K)-C-LCTPSR recombinant plasmid and its identification by
-W2:The concentration of free CA2(L203K)-N-LCTPSR protein.
+            enzyme digestion.<br>
+            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).]]
+[[File:T--AHUT China--ImproPart 03.png|500px|center|thumb|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 TB1 were detected by SDS-PAGE. The results showed that CA2(L203K)-C-LCTPSR could be successfully expressed in our chassis E.coli TB1.(Fig.4)
+[[File:T--AHUT China--ImproPart 04.png|350px|center|thumb|Fig.4 SDS-PAGE analysis for CA2(L203K)-C-LCTPSR cloned in pET-30a(+) and expressed in
+           E.coli TB1<br>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 vectpr 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.
+[[File:T--AHUT China--ImproPart 05.jpg|350px|center|frame|Fig.5 SDS-PAGE of purified CA2(L203K)-C-LCTPSR protein]]
+===2. Identification of the function for CO<sub>2</sub> 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：
+[[File:T--AHUT China--ImproPart fx.png|300px|center|thumb|&eta;: The efficiency of immobilized CA2(L203K)-C-LCTPSR protein;<br>
+W1: The concentration of total CA2(L203K)-C-LCTPSR protein;<br>
+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%.
-To further demonstrate the activity of our improved part, we compared the enzymatic activity of CA2(L203K)-C-LCTPSR (BBa_K2949013) with CA2(L203K) protein (BBa_K2547004) by esterase activity assay.
+====2.2 Enzyme activity asssay of CA2(L203K)-C-LCTPSR protein====
-As shown in Fig.14 and Fig.15, immobilized CA2(L203K)-C-LCTPSR protein retain its activity at 37℃and 50 °C, and free CA2(L203K)-C-LCTPSR protein has a higher activity than CA2(L203K) protein at 50℃, indicating that CA2 (L203K)-C-LCTPSR protein was stable at high temperature and retained its activity.
-Then, the reuse ability of the immobilized enzyme was tested by our designed simulation model (Fig.16), and the result showed that the immobilized CA2(L203K)-C-LCTPSR could absorb CO2 under the simulation model and showed potential reuse ability as indicated in Fig.17.
+To further demonstrate the activity of our improved part, the enzyme activity of CA2(L203K)-C-LCTPSR and CA2(L203K) protein of CO<sub>2</sub> capture were tested experimentally by esterase activity assay at 37℃ and 50℃.
-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.
+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.
+[[File:T--AHUT China--ImproPart 06.png|400px|center|thumb|Fig.6 Esterase activity analysis of free CA2(L203K), free CA2(L203K)-C-LCTPSR and immobilized CA2(L203K)-C-LCTPSR protein at 37℃]]
+[[File:T--AHUT China--ImproPart 07.png|400px|center|thumb|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 CO<sub>2</sub> capture===
+Because the immobilized CA2(L203K)-C-LCTPSR protein have higher activity 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 CO<sub>2</sub>, as indicated in Fig.9. The result showed that the immobilized CA2(L203K)-C-LCTPSR could absorb CO<sub>2</sub> under the simulation model and showed potential reuse ability.
+[[File:T--AHUT China--ImproPart 08.jpg|400px|center|thumb|Fig.8 Picture of our designed model]]
+[[File:T--AHUT China--ImproPart 09.png|400px|center|thumb|Fig.9 The reuse ability of CO<sub>2</sub> capture of the immobilized CA2(L203K)-C-LCTPSR under our designed model]]
+===4. Conclusion===
+Our results demonstrated that the function of CA2(L203K)-C-LCTPSR 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.