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.

BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0268-7) contains supplementary material, which is available to authorized users.

BackgroundMetabolic profile was initially designed as a presymptomatic diagnostic aid based on statistical analyses of blood metabolites to provide an early warning of certain types of metabolic disorder. However, there is little metabolic profile data available about Korean Hanwoo cows. Therefore, this study aimed to determine the reference intervals of metabolic profile for Korean Hanwoo cows.MethodsHealthy animals (2,205) were selected and divided into early (day 1 to 95), middle (day 96 to 190) and late (day 191 to 285) period according to their gestating period. Metabolic profile including total protein (TP), albumin (Alb), urea (UREA), glucose (Glu), total cholesterol (T-Cho), long-chain fatty acid (LCFA), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), creatinine (Crea), calcium (Ca), inorganic phosphorous (iP) and magnesium (Mg) were analyzed using a TBA-40FR automatic biochemical analyzer. The data of Korean Hanwoo cows were then compared to those of the Japanese Wagyu cows.ResultsMost of the data of the Korean Hanwoo cows were relatively higher than those of Japanese Wagyu cows, with the exception of Glu and GGT. This may indicate that the nutritional level of feed for the Korean Hanwoo cows was higher than that of the Japanese Wagyu cows because of the different feeding system. In particular, relatively higher levels of UREA and LCFA were observed in the Korean Hanwoo cows, and this may also contribute to the low reproduction efficiency.ConclusionsThese findings may provide some theoretical basis for understanding the reproductive and feeding situation of Korean Hanwoo cows.

The present study was conducted to examine the effect of a stair-stepped feed intake pattern on growth, feed efficiency, and meat quality of Hanwoo steers. Twenty-seven 11-month-old Hanwoo steers were randomly divided into three groups. The control group was fed according to the Korean steer feeding program, and the other two groups were fed according to an alternated feeding schedule of 3-2-4-2 months. During the first three months of the experiment, treatment group 1 (T1) and treatment group 2 (T2) were fed 20% and 30% less than the control group, respectively. For the following two months, the T1 group was fed 20% more than the control group while the T2 group was fed 20% less than the control group. In the third step, T1 and T2 groups were fed 20% and 10% less, respectively, than the control group for four months. In the last two months, T1 and T2 groups were fed 20% more than the control group. After the stair-step feeding trial, steers were fed concentrated feed ad libitum for five months. The altered feed intake pattern did not affect daily body weight gain. However, daily feed intake tended to decrease and growth efficiency tended to increase in the two treatment groups compared to the control group. Altered feed intake also affected blood metabolite levels. The serum glucose and BUN level of the T1 group increased in the first re-fed period compared to the T2 and control groups. The serum cholesterol level of the T2 group decreased in the first restricted-re-fed growth period compared to the T1 and control groups. The serum NEFA levels of the two treatment groups increased from the first restricted period compared to the controls. The serum insulin level of the T2 group increased in the last period compared to the T1 and control groups. Regarding meat yield index, the control group was significantly higher than the T2 group (p<0.05). Regarding meat yield grade, the carcass back fat thickness of the T2 group was significantly higher than the control group (p<0.05). In marbling score, the T1 group was the highest (4.9), followed by the control group (4.1) and the T2 group (4.0). These results indicate that using a stair-stepped growth pattern (T1) can contribute to improvements in growth efficiency and muscle marbling.