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The Multifaceted Benefits of Protein Co-expression in Escherichia coli
Published on: February 5, 2015
[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its
Chen-Li Yue1, Jie-Ran Shi, Chang-Hong Shi
1Respiratory Division of Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Objective:
To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity.
Methods:
The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis.
Results:
The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity.
Conclusions:
Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.
Insights
The Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants were successfully expressed and purified. The Rpf domain stimulated Mycobacterium smegmatis resuscitation, while mutations E54A and E54K inhibited this activity.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Expression
Background:
- The resuscitation promoting factor (Rpf) is crucial for reviving dormant bacteria.
- Understanding the structure-function relationship of Rpf is key to developing novel antimicrobial strategies.
Purpose of the Study:
- To express the Micrococcus luteus Rpf domain and its mutants (E54K, E54A) in prokaryotic cells.
- To investigate the bioactivity of the expressed Rpf domain and its mutants on Mycobacterium smegmatis resuscitation.
Main Methods:
- Polymerase chain reaction (PCR) amplification and cloning of Rpf domain and mutant genes.
- Subcloning into expression vector PGEX-4T-1 and transformation into E. coli DH5alpha.
- Purification using GST affinity chromatography, followed by SDS-PAGE and Western blot analysis.
- Bioactivity assessment by stimulating Mycobacterium smegmatis resuscitation.
Main Results:
- Successful expression and purification of the Micrococcus luteus Rpf domain and its E54K and E54A mutants.
- Confirmed protein identity and molecular mass via SDS-PAGE and Western blot.
- The purified Rpf domain demonstrated bioactivity in stimulating Mycobacterium smegmatis resuscitation.
- Mutations E54A and E54K significantly inhibited the Rpf resuscitation activity.
Conclusions:
- The study successfully obtained the Rpf domain and two mutant proteins from Micrococcus luteus.
- The findings clarify the effects of Rpf and its mutants on the resuscitation of dormant Mycobacterium smegmatis.
- This research provides insights into the functional role of specific residues within the Rpf domain.
