Constructing The Prokaryotic Expression Model Of Outer Membrane Protein OprM Of Pseudomonas Aeruginosa And Inducing Its Expression

Objective This study was to construct the prokaryotic expression model of OprM that is an outer membrane protein of active efflux system of pseudomonas aeruginosa,and induce expression of protein OprM.Methods The research uses genetic engineering technology.Through exploring the conditions of the experiment,the PCR amplification conditions of oprM gene of pseudomonas aeruginosa with EcoR-â… and Not-â… -restriction sites and each protective bases were determined.The recombinants of oprM gene and vector pET28a-c(+) would be converted into bacteria BL21(DE3) after invitro recombination.The correct prokaryotic expression model of OprM protein was selected using these methods such as resistance screening,colony PCR amplification,restriction enzyme digestion and DNA sequencing analysis.Then,determine the optimal induction conditions by exploring some important factors impacting the expression of OprM protein,and induce plentiful expression of OprM protein with His-Tag.SDS-PAGE and western blot technique were used to verify the expressive protein.Results1.Through exploring the PCR amplification conditions of oprM gene with EcoR-â… and Not-â… -restriction sites and each protective bases,we showed that the optimized reaction mixtures contained the forward and reverse primers at 400 nmol/L each;200μmol/L dNTP; 1.5 mmol/L MgSO₄;2×Enhancer solution;1×PCR buffer;pfu DNA polymerase 0.1 U/μl; templates 1μl;the total reaction volume was 50μl.The PCR was performed under the following conditions:95℃for 5 minutes,followed by 35 cycles of 95℃for 45 seconds,50℃for 1 minute and 72℃for 2 minutes;then,72℃for 7 minutes.After PCR finished,add 0.6μl (3U) Taq DNA polymerase to the reaction mixtures and keep 72℃for 20 minutes to acquire PCR production with A tail. 2.Test the sequence of oprM gene in plasmid pET28a-c-oprM and contrast it with oprM gene published in GeneBank by blast 2.The result showed there was only one synonymous codon mutation and the base mutation didn't affect the protein translation.3.By exploring the expression conditions of OprM protein,the optimized inducement conditions were found that when the OD₆₀₀ of BL21-pET28a-c-oprM growing in LB culture medium with Kanamycin(final concentration of 0.05mg/ml) got to a concentration of 0.5,add to IPTG inducer to final concentration of 1.5mmol/L and oscillate at 25℃overnight.4.The test results of SDS-PAGE and western blot showed that the expression protein was the insoluble OprM protein with His-Tag.Conclusions Outer membrane protein OprM's prokaryotic expression model named BL21-pET28a-c-oprM was successfully constructed.And the OprM protein with His.Tag was induced expression.