Gene Clone And Pichia Methanolica Expression System Expression Of Porcine Lactoferrin N-lobe

Lactoferrin (LF) is a multifunctional glycolprotein in physiological fluids of mammals. Many functions of lactoferrin are thought to be involved in the N-lobe of this protein. In this study, porcine lactoferrin N-lobe gene was cloned from mammary gland cells of lactating sow, and expressed by using E.coli and P. methanolica expression system. Antimicrobial activity of recombinant expression products was studied. The results were summarized as follows:Total RNA in mammary gland cells of 15-day lactating York Shine sow was extracted and PLF-N gene was cloned by the reverse transcription-coupled polymerase chain reaction (RT-PCR). This gene contains a 1038bp coding sequence (cds) of PLF-N. An "agg" codon between 1039-1041bp in PLF gene was replaced with a "taa" stop codon through primers design and PCR. Therefore protein translated from the new ORF contains 346 amino acid residues including a 17-amino acid signal peptide. Subsequently, this gene was cloned into pGEM-3Z vector and sequenced. The sequencing results were aligned with the sequence (Genebank, L77887), indicating that the homology between these two sequences was 99%.The gene was cloned into E.coli expression vector pET-28a(+) and transformed into E.coli BL21DE3. The expression was induced with IPTG. Recombinant protein was analyzed with SDS-PAGE and Western Blot. Both results showed that the rPLF-N was well expressed in E.coli and molecular weight (MW) of the resombinant product was extimated to be approximately 42kDa, which was coinciding with predicted MW of rPLF-N.In order to express the PLF-N in yeast. Two new primers were designed. Using the recombinant plasmid pGEM-PLF-N as template, PLF-N gene was amplified again and subsequently cloned into P. methanolica expression vector PMET-B. DNA sequencing results showed that ORF was correct and PLF-N gene fused correctly with V5 epitope and 6His tag in PMET-B vector. After the recombinant plasmid pMET-PLF-N was linearized and transformed into PMAD11 compentent cells by electroporation, Ade+(Muts)recombinants were selected and induced with methanol. SDS-PAGE and Western Blot results of recombinant expression products showed that there was a distinct band which MW was very close to rPLF-N. These results clearly indicated that PLF-N was expressed successefully in P. methanolica, and was secreted into the culture medium. It was not glycosylated as its MW was 42kDa approximately. Time-course analysis of expression showed that amount of rPLF-N in the culture medium reach peak at 72 hours post-inducement. Continuous inducement had no remarkable effect on increasing the expression level.Antimicrobial activity of rPLF-N in the culture medium was also analyzed. Results showed that rPLF-N could inhibit the growth of E.coli ATCC25922.