Molecular Cloning And Functional Study Of A Novel Human Gene Encoding Antimicrobial Peptide

A human derived glycine—rich gene (designated as hGlynchin) was obtained after PCR amplification of human fetal liver cDNA library based on our first cloned mouse gene, mGlyrichin, (GenBank^TMnumber:AY028425). The encoding sequences of hGlynchin and mGlyrichin are both 79 amino acids long and rich in glycine and methionine, hGlyrichin showed 100% homology with mGlyrichin in protein sequence and 93.4% homology in nucleotide sequence, NCBI electronic PCR demonstrated that hGlyridhin gene was localized in human chromosome 20 q11.22 with 2 exons and 1 intron; Northern Bolt result showed hGlyrichin has a single transcription in 4 human tumors cell lines tested, which is about 600bp in length. Results of RT-PCR in four differnet embryotic tissues tested was to demonstrated the natural characterization further, hGlyrichin gene was inserted into the expression vectors pEGFP-N1 and pEGFP-C1, and then transfected into HepG₂ cells, the subcellular location of hGlyrichin-GFP fusion protein was different if the gene was inserted into the up-stream or down-stream of GFP, why it was different when the gene was inserted into different plasmids was left unknown. These results have demonstrated the natural characterizations of hGlyrichin gene but no any functional clue could be found by searching for the publications and motifs. Structurally analysis seems suggest that all characterizations of relatively small molecular weight 8.18Kd, rich-in glycine (21.5%), high isoelectropoint (9. 58) and positive charge (5 net charges) are coincidence with that of known antimicrobial peptides.A peptide was synthesizd according to the result of bioinformatic analysis and found to have a good activity, and functionally confirmed by inhibition of the growth of E.coli BL-21 (DE3) after transformed with pET22b-hGlyrichin and induced expression with IPTG.The use of methylotrophic yeast, Pichia pastoris, as a cellular host for the expression of heterologous protein has become increasing popular in recent times and offer significant advantages over E.coli expression system for the production of many heterologous eukaryotic proteins,So we try to establish techniques to express hGlyrichin in the pichia pastoris expression system, hGlyrichin was amplified and cloned into two pichia pastoris expression vetors,pPIC9 and pPIC9K plasmids, for secreted expression, then transformed into expression strain (GS115) with lithium chloride transformation method and confirmed integration of hGlyrichin in recombinants by PCR ,The recombinant pichia pastoris strains, selected by G418 could contain multiple copies of the expression cassette, culture supernatant was saved to test whether it had antibacterial activity after the recombinants was cultured and induced with methanol.Using the entire ORF sequence of hGlyrichin to do the alignment analysis, the blastP resulting sequences with the alignment score more than 40 were used for the multiple sequence alignment analysis by DNAMAN software. So we found that 29 protein sequences were highly homologue to this gene. The secondary structure predictions of all the homologues were analyzed by using DNASTAR5.0 software. Other characterizations of all homologues, such as signal peptides, net charges, and isoelectropoints were analyzed by using other public softwares. Four peptides representing 9 members of the family were synthesized according to the result of multiple sequences alignment, and they have antibacterial activities more or less. A novel Caenorhabditis elegans derived gene (named as cele52) , which is highly homologous to hGlyrichin, was cloned and expressed with fusion protein in prokaryotic expression system to test its function.In conclusion, A novel human glycine-rich protein with antibacterial activity and a potential antimicrobial peptide family crossing wide species of organisms was determined.