New parts

This page is to record the new part BBa_K2949012 that we have done

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We found that formylglycine-generating enzymes (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.

So, in this year, we constructed a new biobrick [CA2(L203K)-N-LCTPSR] (BBa_K2949012) by connecting the N-terminal of the mutant human carbonic anhydrase 2 [CA2(L203K)] (BBa_K2547004) coding sequences with the six-residue sulfatase submotif (LCTPSR), to immobilize CA2(L203K) for efficient CO₂ capture.

1. Engineered E.coli TB1

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

The coding sequence of CA2(L203K)-N-LCTPSR (BBa_K2949012) was synthesized and cloned into the expression vector pET-30a(+), as presented in Fig.1:

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

Fig.3 Sequencing results

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

We induced pilot expression of CA2(L203K)-N-LCTPSR in E.coli TB1 by using isopropyl-1-thio-β-Dgalactopyrasonide(IPTG).

Briefly, recombinant plasmid of the CA2(L203K)-N-LCTPSR was transformed into E.coli TB1, and positive clone was screened by kanamycin resistance. Then, the recombinant E.coli TB1 were propagated and CA2(L203K)-N-LCTPSR expression were induced with IPTG. Cells were lysed by sonication on ice, and the obtained crude extracts were centrifuged to separate supernatant and debris, and the fraction was subjected to SDS-PAGE.

The results showed that CA2(L203K)-N-LCTPSR could be successfully expressed in TB1 strain (Fig.4).

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

After confirming that CA2(L203K)-N-LCTPSR could be expressed in our chassis E.coli TB1, we successfully co-transformed pBAD-FGE and pET-30a(+)-CA2(L203K)-N-LCTPSR expression vector into E.coli TB1. Then the protein of CA2(L203K)-N-LCTPSR was further purified with nickel column for the following enzyme immobilization.

The results showed that CA2(L203K)-N-LCTPSR was purified with high purity as indicated by a significant single protein band by SDS-PAGE (Fig.5).

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

2. Identification of the function for CO₂ capture

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

In order to improve the efficiency of the repeated utilization of the CA2(L203K)-N-LCTPSR protein, we achieved enzyme immobilization.

Briefly, we cultured CA2(L203K)-N-LCTPSR in shaking table with the immobilization support (Unisil 30-100 NH2), then added 10% sodium cyanoborohydrate and continued to immobilize it in shaking table culture.

Our formula for calculating the enzymatic immobilized efficiency is as follows：

$$\eta=\frac{W 1-W 2}{W 1}\times100\%$$

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

According to the formula, we got the efficiency of immobilized CA2(L203K)-N-LCTPSR protein is 32.35%.

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

The enzyme activity of immobilized CA2(L203K)-N-LCTPSR protein was tested experimentally by esterase activity assay at 37℃ and 50℃.

As indicated in Fig.6, the immobilized CA2(L203K)-N-LCTPSR protein were stable and retained their activity at high temperature.

Fig.6 Esterase activity analysis of immobilized CA2(L203K)-N-LCTPSR protein at 37℃ and 50℃

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Team:AHUT China/New parts

1. Engineered E.coli TB1

2. Identification of the function for CO2 capture

2. Identification of the function for CO₂ capture