Difference between revisions of "Part:BBa K3034000"
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===Expression of CrpP-Histag=== | ===Expression of CrpP-Histag=== | ||
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)''. We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression. | 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)''. We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression. | ||
| − | [[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE of CrpP-Histag. 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. | + | [[File: SDS-PAGE of CrpP-Histag.png|500px|thumb|center|'''Fig. 1''' SDS-PAGE of CrpP-Histag. 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=== | ===Enzyme activity of CrpP=== | ||
Revision as of 00:45, 12 October 2019
CrpP：Ciprofloxacin-Modifying Enzyme
CrpP is a novel ciprofloxacin-modifying enzyme which can phosphorylate ciprofloxacin(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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contents
Characterization
Molecular weight
This gene codes for a protein of 65 amino acids with a molecular mass about 15 kDa.
Expression of CrpP-Histag
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). We use IPTG to induce its overexpression and SDS-PAGE is used to detect target protein's expression.
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, 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.