Difference between revisions of "Part:BBa K5136015"

Revision as of 15:37, 30 September 2024

CYP199A4 T253M-His tag

Biology

CYP199A4 is a NADH-dependent cytochrome P450 monooxygenase from Rhodopseudomonas palustris cytochrome P450, a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations (1). CYP199A4 can also function as peroxygenase. The engineered CYP199A4 peroxygenases showed good functional group tolerance and preferential O-demethylation at the meta- or para-position, indicating potential O-demethylation of H- and G-type lignin monomers (1).

Usage and design

CYP199A4 can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2). It has been pointed out that position T253 of CYP199A4 can affect the coordination environment of the active center (1). Therefore, we carried out saturation mutagenesis on this site, hoping to screen out enzymes with higher activity.
Partial amino acid sequence of CYP199A4 T253M: …AGLDTMVNGI…

Construction

We use pET-28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 Gene circuit of CYP199A4 T253M-His tag.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1412 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136015_pET-28a(+).

The pET-28a (+) vectors containing CYP199A4 variants were transformed into E. coli BL21(DE3), and the cells were cultivated in LB medium containing 50 μg/mL kanamycin. The cultures were grown at 37 °C with vigorous shaking (~200 rpm). When the OD₆₀₀ of the cultures reached 0.8~1.0, the temperature was cooled to 20 °C, and the expression was induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic acid hydrochloride (0.5 mM). Following 16-20 h of expression, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 45.8 kDa).

Figure 3 SDS-PAGE analysis of CYP199A4 T253M-His tag protein.

Deinking Experiments

Reference

1. P. Zhao, Y. Jiang, Q. Wang, J. Chen, F. Yao, Z. Cong, Crucial gating residues govern the enhancement of peroxygenase activity in an engineered cytochrome P450 O-demethylase. Chemical Science 15, 8062-8070 (2024).
2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 150
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 831
Illegal XhoI site found at 1231
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 150
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 150
Illegal AgeI site found at 691
1000
COMPATIBLE WITH RFC[1000]

@@ Line 21: / Line 21: @@
 <center><b>Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136015_pET-28a(+).</b></center>
-The pET-28a (+) vectors containing CYP199A4 variants were transformed into E. coli BL21(DE3), and the cells were cultivated in LB medium containing 50 μg/ml kanamycin. The cultures were grown at 37 °C with vigorous shaking (~200 rpm). When the OD 600 of the cultures reached 0.8~1.0, the temperature was cooled to 20 °C, and the expression was induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic acid hydrochloride (0.5 mM). Following 16-20 h of expression, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 45.8 kDa).
+The pET-28a (+) vectors containing CYP199A4 variants were transformed into <i>E. coli</i> BL21(DE3), and the cells were cultivated in LB medium containing 50 μg/mL kanamycin. The cultures were grown at 37 °C with vigorous shaking (~200 rpm). When the OD<sub>600</sub> of the cultures reached 0.8~1.0, the temperature was cooled to 20 °C, and the expression was induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic acid hydrochloride (0.5 mM). Following 16-20 h of expression, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 45.8 kDa).
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