Part:BBa_K3034000

CrpP：Ciprofloxacin-Modifying Enzyme

CrpP is a novel ciprofloxacin(CIP)-modifying enzyme which can phosphorylate CIP. Then the phosphorylated CIP goes through multiple steps of degradation spontaneously and produces 1,4-dihydroquinoline finally[1]. 1,4-dihydroquinoline is used as a kind of new carriers for specific brain delivery [2], so we can infer that it's nontoxic. This part is responsible for degradation of ciprofloxacin in our project.

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]

Characterization

Molecular weight

This gene codes for a protein of 65 amino acids with a molecular mass about 11 kDa.When with a 6×Histag, the weight reaches about 15kDa.

Expression of CrpP-Histag

Because expression of CrpP-Histag is unsatisfying in E.coli DH5α, so we clone pelB-5D, CrpP with 6×Histag and TagRFP CDS into an expression vector, then the vector is transformed into E.coli BL21(DE3). 0.5mM IPTG is used for protein's expression and (over)expression is monitered by 17% SDS-PAGE.

Fig. 1 SDS-PAGE of CrpP-His. M: Protein ladder; Lane 2、3: Experimental group 1 (0.5mM IPTG is added); Lane 3、4: Control group 1 (without 0.5mM IPTG); Lane 5、6: Experimental group 2; Lane 7、8: Control group 2. The protein between 25kDa and 33kDa is TagRFP

References

[1]Chávez-Jacobo, Víctor M., et al. CrpP is a novel ciprofloxacin-modifying enzyme encoded by the Pseudomonas aeruginosa pUM505 plasmid. Antimicrobial agents and chemotherapy 62.6 (2018): e02629-17.

[2]Foucout, Lénaïg, et al. Synthesis, radiosynthesis and biological evaluation of 1, 4-dihydroquinoline derivatives as new carriers for specific brain delivery. Organic & biomolecular chemistry 7.18 (2009): 3666-3673.