Difference between revisions of "Part:BBa K3034000"

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===Expression of CrpP-Histag===
 
===Expression of CrpP-Histag===
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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.
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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.
 
[[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''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]]
 
[[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''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]]
  

Revision as of 15:21, 18 October 2019

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

Enzyme activity of CrpP

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.