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Updated: Aug 9, 2026
Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX
Published on: June 27, 2017
[Cloning and prokaryotic expression of the gene encoding PGRP domain of mouse long peptidoglycan recognition protein]
Zhi He1, Da-ming Zuo, Zheng-liang Chen
1Department of Immunology, Southern Medical University, Guangzhou 510515, China. hezhi@fimmu.com
Objective:
To clone the gene coding for the peptidoglycan recognition protein (PGRP) domain (PGRPd) of mouse long PGRP (mPGRP-L) and express the protein in E. coli.
Methods:
The cDNA fragment encoding PGRPd of mPGRP-L was obtained by RT-PCR from the total RNA of Balb/C mouse liver cells and cloned into pUCm-T vector. The recombinant plasmid were identified by PCR, restriction endonucleases and sequence analysis. The PGRPd gene fragment was amplified by PCR from the recombinant plasmid, inserted into pQE-30 vector and transformed into E. coli strain M15, and the expressed PGRPd protein was purified.
Results:
A cDNA fragment of about 500 bp was amplified by RT-PCR and the recombinant plasmid, pmPGRPd, was constructed by linking the fragment to pUCm-T vector. The results of restriction mapping of the recombinant vector were consistent with those of computer analyses. Sequence analysis showed that the cloned gene fragment (518 bp) had identical sequence with the gene encoding PGRPd of mPGRP-L gene in GenBank. The recombinant expression vector pQE-PGRPd was constructed and expressed in E. coli M15. SDS-PAGE showed that the expressed product existed mainly in the lysate supernatant as a soluble protein with relative molecular mass of 29 kD.
Conclusion:
The PGRPd cDNA of mPGRP-L has been successfully cloned and expressed in E. coli, which provides the basis for further study of PGRP molecule.
Insights
Mouse long peptidoglycan recognition protein (PGRP-L) domain (PGRPd) cDNA was successfully cloned and expressed in E. coli. This achievement provides a foundation for future research into PGRP molecules.
Area of Science:
- Molecular Biology
- Immunology
- Protein Expression
Background:
- Peptidoglycan recognition proteins (PGRPs) are crucial components of the innate immune system.
- Mouse long PGRP (mPGRP-L) plays a role in recognizing bacterial peptidoglycans.
- Understanding the structure and function of PGRP domains is essential for deciphering immune responses.
Purpose of the Study:
- To clone the gene encoding the peptidoglycan recognition protein domain (PGRPd) of mouse long PGRP (mPGRP-L).
- To express the PGRPd protein in Escherichia coli (E. coli) for further study.
- To establish a foundation for investigating the mPGRP-L molecule's function.
Main Methods:
- Complementary DNA (cDNA) encoding PGRPd was amplified from mouse liver RNA using RT-PCR.
- The cDNA fragment was cloned into pUCm-T and subsequently into the pQE-30 expression vector.
- The recombinant vector was transformed into E. coli M15, and the expressed protein was purified.
Main Results:
- A 518 bp cDNA fragment encoding PGRPd was successfully amplified and sequenced, confirming its identity with mPGRP-L.
- The recombinant expression vector pQE-PGRPd was constructed and expressed in E. coli.
- SDS-PAGE analysis revealed a soluble expressed protein of approximately 29 kD.
Conclusions:
- The PGRPd cDNA of mPGRP-L was successfully cloned and expressed in E. coli.
- This successful expression facilitates further investigation into the PGRP molecule.
- The study provides a valuable resource for research on peptidoglycan recognition proteins.
