• Applied Biological Chemistry
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• v.39 no.5
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• pp.343-348
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• 1996

Experiments were carried out to find possibility of economic production of SCP in mixed culture by Cellulomonas sp. KL-6 and E. coli LI-10. The best cell growth was obtained at the ratio of 1 : 1(v/v) in mixed culture. When these strains were mixed culture, cell growth was increased to about 63%, compared with those of single culture of strain KL-6. It was found that the majority of the population during growth in mixed culture consisted of strain KL-6. $CaCO_3$ added to the medium as the ratio of 0.1% was enhanced medium pH. Cell growth increased in that circumstances. These strains produced much amounts of cellobiose, but glucose was not detected in filter paper medium. When these organisms were cultured under the optimal medium for 4 days, cell mass was produced $1.0\;g/{\ell}$. The results showed the increase of cell mass up to 53% than those produced in CMC medium.

• BMB Reports
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• v.39 no.3
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• pp.263-269
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• 2006

In the genome of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, open reading frame 39 (Ha39) is the only gene predicted to encode an RNA recognition protein. Computer analysis revealed that Ha39 homologues were found in 15 NPVs, but not in GVs. Its transcripts were detected from 3 through 72 hours post infection (h p.i.) using RT-PCR and Northern blot analysis. The protein was detected in infected-cell lysates from 6 h p.i. Western blot assay of ODV and BV preparations revealed that Ha39 encodes a structural protein associated with BVs. Additionally, immunofluorescence microscopy demonstrated that the protein was present within cytoplasm in virus-infected cells, but not in the nuclear region.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1977-1988
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• 2015

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

• BMB Reports
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• v.32 no.6
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• pp.529-534
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• 1999

Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.284-288
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• 2005

Baker's yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to $50\%$(w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of $20\%$(ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to $85\% (v/v)$. Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than $20\%(w/v)$ and 10 m/s, respectively.

• Food Science of Animal Resources
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• v.35 no.2
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• pp.189-196
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• 2015

The aim of this study was to manufacture functional high protein fermented beverage, using whey protein concentrate (WPC) and Lactobacillus plantarum DK211 isolated from kimchi, and to evaluate the physicochemical, functional, and sensory properties of the resulting product. The fermented whey beverage (FWB) was formulated with whey protein concentrate 80 (WPC 80), skim milk powder, and sucrose; and fermented with Lactobacillus plantarum DK211 as single, or mixed with Lactococcus lactis R704, a commercial starter culture. The pH, titratable acidity, and viable cell counts during fermentation and storage were evaluated. It was found that the mixed culture showed faster acid development than the single culture. The resulting FWB had high protein (9%) and low fat content (0.2%). Increased viscosity, and antioxidant and antimicrobial activity were observed after fermentation. A viable cell count of 10⁹ CFU/mL in FWB was achieved within 10 h fermentation, and it remained throughout storage at 15℃ for 28 d. Sensory analysis was also conducted, and compared to that of a commercial protein drink. The sensory scores of FWB were similar to those of the commercial protein drink in most attributes, except sourness. The sourness was highly related with the high lactic acid content produced during fermentation. The results showed that WPC and vegetable origin lactic acid bacteria isolated from kimchi might be used for the development of a high protein fermented beverage, with improved functionality and organoleptic properties.

• BMB Reports
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• v.38 no.3
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• pp.354-359
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• 2005

Open reading frame 29 (ha29) is a gene specific for Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearSNPV). Sequence analyses showed that the transcription factor Tfb2 motif, bromodomain and Half-A-TPR (HAT) repeat were present at aa 66-82, 4-76, 55-90 of the Ha29 protein respectively. The product of Ha29 was detected in HearSNPV-infected HzAM1 cells at 3 h post-infection. Western blot analysis using a polyclonal antibody produced by immunizing a rabbit with purified GST-Ha29 fusion protein indicates that Ha29 is an early gene. The size of Ha29 product in infected HzAM1 cells was about 25 kDa, which was larger than the presumed size of 20.4 kDa. Tunicamycin treatment of HearSNPV-infected HzAM1 cells suggested that the Ha29 protein is N-glycosylated. Fluorescent confocal laser scanning microscope examination, and Western blot analysis of purified budded virus (BVs), occlusion-derived virus (ODVs), cell nuclear and cytoplasmic fraction, showed that the Ha29 protein was localized in the nucleus. Our results suggested that ha29 of HearSNPV encodes a non-structurally functional protein that may be associated with virus gene transcription in Helicoverpa hosts.

• Korean Journal of Environmental Agriculture
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• v.28 no.4
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• pp.435-441
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• 2009

In the present study, we analyzed the defensin protein deduced from Korean radish (Raphanus sativus L.) seeds.To express the genes in E. coli, we constructed a recombinant expression vector with a defensin gene, named rKRs-AFP gene isolated from Korean radish seeds. Over expressed rKRs-AFP proteins was separated by SDS-PAGE to determine the purity, and protein concentration was determined by the Bradford method. Antifungal activity was assessed by disk assay method against the tested fungi. As a result, when 500 mL of cell culture were disrupted by sonicator, 32.5 mg total proteins were obtained. The purified protein showed a single band on SDS-PAGE with estimated molecular weight about 6 KDa, consistent with the molecular mass calculated from the deduced amino acid sequence. The purified rKRs-AFP protein showed remarkable antifungal activities against several fungi including Aspergillus niger, Botrytis cinerea causing the gray mold disease, and Candida albicans. In field tests using the purified rKRs-AFP protein, the protein showed the reducing activity of disease spot and the mitigating effect of spreading of disease like agrichemicals. The immuno-assay of rKRs-AFP protein showed that the purified protein entirely accumulated at B. cinerea cytoplasm through the hyphal septa shown by fluorescence imaging. There was no fluorescence inside the cell, when the hypha was incubated without the protein. These all results indicate that the recombinant rKRs-AFP proteins can be utilized as a potential antifungal drug to control harmful plant fungal pathogens.

• BMB Reports
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• v.46 no.2
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• pp.65-72
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• 2013

In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demon-strating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells.

• BMB Reports
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• v.44 no.9
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• pp.584-589
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• 2011

The mammalian ste20-like kinase (MST) pathway is important in the regulation of apoptosis and cell cycle and emerges as a novel tumor suppressor pathway. MST-induced phosphorylation of Salvador homolog 1 (SAV1), which is a scaffold protein, has not been evaluated in detail. We performed a mass spectrometric analysis of the SAV1 protein that was co-expressed with MST2. Phosphorylation was detected at Thr-26, Ser-27, Ser-36 and Ser-269. Although single or double mutations had little effects, the mutation of all four residues in SAV1 to Ala (SAV1-4A) had inhibitory effects on the MST pathway. MST2-mediated induction of SAV1-4A protein levels, SAV1-4A interaction with MST2 and the self-dimerization of SAV1-4A were weaker compared to those of wild-type SAV1. SAV1-4A inhibited MST2- and K-RasG12V-induced cell death of MCF7 cells. These results suggest that MST-mediated phosphorylation of four residues within SAV1 may be important in the induction of cell death by the MST pathway.