title: Lactococcus lactis M4, a potential host for the expression of heterologous proteins.
abstract: BACKGROUND
  Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.
  
  
  RESULTS
  Several bacterial strains were isolated from cow's milk and eight of those were identified as Lactococcus lactis by 16S rRNA sequence analysis. Antibiotic susceptibility tests that were carried out showed that 50% of the isolates had almost identical antibiotic resistance patterns compared to the control strains MG1363 and ATCC 11454. Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4. Competent L. lactis M4 and MG1363 were prepared and electrotransformed with several lactococcal plasmids such as pMG36e, pAR1411, pAJ01 and pMG36e-GFP. Plasmid isolation and RE analyses showed the presence of these plasmids in both M4 and the control strain after several generations, indicating the ability of M4 to maintain heterologous plasmids. SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4.
  
  
  CONCLUSIONS
  Based on the 16S rRNA gene molecular analysis, eight Gram-positive cocci milk isolates were identified as L. lactis subsp. lactis. One of the strains, L. lactis M4 was able to maintain transformed low molecular weight plasmid vectors and expressed the GFP gene. This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.
authors.forename: Nanyan
authors.initials: N
authors.lastname: Noreen
authors.forename: Wei Yeng
authors.initials: WY
authors.lastname: Hooi
authors.forename: Ali
authors.initials: A
authors.lastname: Baradaran
authors.forename: Mohamad
authors.initials: M
authors.lastname: Rosfarizan
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Sieo
authors.forename: Md Illias
authors.initials: MI
authors.lastname: Rosli
authors.forename: Khatijah
authors.initials: K
authors.lastname: Yusoff
authors.forename: Abdul Rahim
authors.initials: AR
authors.lastname: Raha
mesh_terms: D000818:Animals; D002417:Cattle; D052978:Disk Diffusion Antimicrobial Tests; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D013294:Lactococcus lactis / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D008892:Milk / Q000382:microbiology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins