Cloning and Characterization of a Novel tuf Promoter from Lactococcus lactis subsp. lactis IL1403

Abstract

Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene expression system. Especially, a stable promoter is an important genetic element to induce gene expression in such a system. We report on a novel tuf promoter (Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected through analysis of previously published microarray data. Ptuf activity was examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA, and Pusp45) using different bacteria as expression hosts. Each promoter was, respectively, fused to the promoterless and modified bmpB gene as a reporter, and we estimated promoter activity through BmpB expression. All promoters were active in IL1403, and Ptuf activity was strongest among them. The activity of each promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus reuteri ATCC23272, and Escherichia coli Top10F'). Ptuf had the highest activity in IL1403 when growth reached late log phase. The activity of each promoter correlated with the expression of each cognate gene in the microarray data (R (2) = 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms and Ptuf can be used to develop a reliable gene expression system in L. lactis.

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization

“…S2), was adapted as a mother plasmid, which was previously constructed by our research group [17]. We generated the mouse IL-6 gene fragments with or without nucleotide sequences encoding Usp45-secretion signal peptide [18] and the M cell-targeting peptide, CKS9, through the oligonucleotide-assembly PCR using a series of synthetic overlapped oligonucleotides (IDT Inc., Belgium), which were computationally designed as approximately 60-base-pair long, according to a previously described method [19].…”

Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri

“…Signal peptides are short, hydrophobic peptide sequences at the N-termini of proteins that allow for the translocation of proteins across the membrane via the secretory pathway. The tuf gene promoter and RBS sequences were chosen because the tuf gene is known to be expressed at a high level in most bacteria [ 48 , 49 ]. In S. citri , the presence of −10 and −35 sequences, which are characteristic of bacterial promoters recognized by RNA polymerase associated with its general σ factor, as well as the detection of large amounts of Tuf protein in proteomic analyses, are consistent with the assumption that the tuf gene in S. citri is constitutively expressed at a high level.…”

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant

“…The recombinant L. lactis IL 1403 expressing secretory form of 181 amino acid sRANKL [ 15 ] (sRANKL-LAB) was prepared using pILPtuf vector system previously constructed by our group [ 26 ]. To permit the production and secretion of protein, sRANKL was conjugated with Usp45 signal peptide [ 3 ].…”

“…We measured a 24 % secretion efficiency (60 ng/ml) when analyzed by western blot and ELISA. In addition, strong and constitutive expression of sRANKL was driven by a Ptuf promoter, which was previously isolated by our group [ 26 ] and determined to work in vitro and in the gastrointestinal tract [ 3 ]. The high-level protein expression can overburden the metabolic capacities of the host and may inhibit normal physiology.…”

“…To construct recombinant sRANKL-LAB, the pILPtuf vector system previously constructed by our group was used as a backbone [ 26 ]. The 181 amino acid sequence of soluble RANKL was obtained from the literature [ 15 ] and the codon usage was optimized for expression by L. lactis .…”