Prokaryotic Expression And Purification Of CTX-M-3 Extended-spectrum β-lactamase.

Objective:To carry out prokaryotic expression and purification of CTX-M-3 extended-spectrumβ-lactamase (ESBLs)from Klebsiella pneumoniae.Method:The sequence of CTX-M-3 Extended Spectrumβ-Lactamase (ESBL)encoding gene wsa analysised from the preserved recombinant DH5α/pGEM-T-CTX-M-3. Recombinant plasmid(pET-28a(+)-CTX-M-3)were identified by restriction enzymes digestion,then transformed into E.coli strain BL21(DE3).Expression of CTX-M-3 fusion protein induced by IPTG,and then purified with Ni-NTA. The purification protein were identified by SDS-PAGE and Western-blot.Results:(1) The recombinant plasmids pGEM-T-CTX-M-3 is digested by NdeI and EcoRI and yields fragments. The size of the fragments were proximate 876 bp and 3000 bp. The result of the sequence analysis of Nucleotide demonstrates that the size of the fragment of the target gene was 876 bp. Aminoacid sequence analysis showed the target gene was 100% homologous to CTX-M-3 in GenBank.(2) Prokaryotic recombinant plasmid pET-28a(+)-CTX-M-3 has been constructed successfully ,it can be cut to two fragments about 876bp and 5000bp by NdeI and EcoRI.The size of them was proximate matches expectation, which proved that the target gene had already successfully inserted in pET-28a(+) vector.(3) The solubility test indicated that the expression product existed in both the soluble form and the inclusion body form. After optimized the experiment of the protein expression condition, we obtained the optimum condition of 18℃culture temperature and 0.8mmol/L IPTG for 24h induced density by SDS-PAGE.(4) The preliminary study of purification of recombinant plasmids was used His-bind Purification system. The results of SDS-PAGE and Western-blot tests indicated that we had obtained the 32KD recombinant protein. Conclusions:The prokaryotic expression recombinant plasmid pET-28a(+)- CTX-M-3 was constructed successfully.The CTX-M-3 gene was expressed in E.coli BL21(DE3). The CTX-M-3 fusion protein was obtained by His-bind Purification system. Obtained high-grade body as the main form of expression of the recombinant protein.The purification of blaCTX-M-3 laid a good foundation for molecular biology and its antibody′s study of CTX-M-3 extended-spectrumβ-lactamase.