Part:BBa_K5136000

CYP199A4 WT-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 WT: …AGLDTTVNGI…

Construction

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

Figure 1 Gene circuit of CYP199A4 T253T-His tag.

Routine Characterization

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). The strain is cultured at 37 °C, 200 rpm until OD₆₀₀ reaches 0.8~1.0, then IPTG (final concentration at 0.2 mM) and δ-aminolevulinic acid hydrochloride (final concentration at 0.5 mM) are added. After 16-20 h of expression at 25 °C, 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 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136000_pET-28a(+).

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, 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 (45.8 kDa).

Figure 3 SDS-PAGE analysis of CYP199A4 T253T-His tag protein.

Reference

1. M. Jovanovic, E. Lawton, J. Schumacher, M. Buck, Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system. Fems Microbiology Letters 356, 201-211 (2014).
2. B. Wang, R. I. Kitney, N. Joly, M. Buck, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nature Communications 2, 508 (2011).

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]