Cloning and prokaryotic expression of the N--terminal fragment of mouse long type peptidoglycan recognition protein (mPGRP--L)

A cDNA fragment of about 500 bp encoding the N-terminal region of mouse long type peptidoglycan recognition protein (mPGRP-L) was amplified by RT-PCR from the total RNA in liver tissues of BALB/c mice, and the recombinant plasmid, pmGN, was constructed through linkage of the DNA fragment with pUCm-T vector. Result of DNA sequencing of a selected clone indicated that the length of the cloned gene fragment was 530 bp and its sequences were identical to the data published in GenBank. The target gene fragment was amplified from recombinant plasmid pmGN by PCR technique, and inserted into expression vector pET-28a to construct the recombinant expression vector pET-GN, and then introduced to E. coli BL21 strain in order to induce the expression of the target proteins. The recombinant expression products were purified by Ni~(+)-NTA agarose chromatography. As demonstrated by SDS-PAGE and Western blot analysis, the expressed products existed mainly in the inclusion bodies with a relative molecular mass of 29 000 Mr. These products could be recognized by the mono-epitope multiclonal antibody against N-terminal region of mPGRP-L, as demonstrated by ELISA assay. The results in the present study could provide the foundation for the further studies of PGRP molecule.