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.