• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.789-796
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• 2000

In order to analyze the effects of tktA, $aroF^{FBR}$, and aroL expression in a tryptophan-producing Escherichia coli, a series of plasmids carrying the genes were constructed. Introduction of tktA, $aroF^{FBR}$, and aroL into the E. coli strain resulted in approximately 10-20 fold increase in the activities of transketolase, the feedback inhibition-resistant 3-deoxy-D-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of $aroF^{FBR}$ in the aroB mutant strain of E. coli resulted in the accumulation of 10 mM of 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and $aroF^{FBR}$ in the strain further increased the amount of excreted DAHP to 20 mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5 mM of DAHP. However, the expression of tktA and $aroF^{FBR}$ in a tryptophan-producing E. coli strain did not lead to the increased production of tryptophan, but instead, a significant amount of shikimate, which is an intermediate in the tryptophan biosynthetic pathway, was excreted to the growth medium. Despite the fact that additional expression of shikimate kinase in the strain could possibly remove 90% of excreted shikimate to 0.1 mM, the amount of tryptophan produced was still unchanged. Removing shikimate using a cloned aroL gene caused the excretion of glutamate, which suggests disturbed central carbon metabolism. However, when cultivated in a complex medium, the strain expressing tktA, $aroF^{FBR}$, and aroL produced more tryptophan than the parental strain. These data indicate that additional rate-limiting steps are present in the tryptophan biosynthetic pathway, and the carbon flow to the terminal pathway is strictly regulated. Expressing tktA in E. coli cells appeared to impose a great metabolic burden to the cells as evidenced by retarded cell growth in the defined medium. Recombinant E. coli strains harboring plasmids which carry the tktA gene showed a tendency to segregate their plasmids almost completely within 24h.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.115-118
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• 2000

Recombinant plasmids harboring a heterologous gene coding for the enhanced green fluorescent protein (EGFP) were transfected and expressed in Drosophila melanogaster S2 cells. A stable transformation of polyclonal cell populations expressing EGFP were isolated after 4 weeks of selection with hygromycin B. The recombinant EFGP expressed in transformed S2 cells consisted of a molecular weight of 27 kDa. EGFP expression was also confirmed by fluorometric measurement. The maximum EGFP concentration was about 9.3 mg/I. The present findings demonstrate not only the successful stable expression of EGFP in Drosophuila was about 9.3 mgI. The present findings demonstrate not only the successful stable expression of EGFP in Drosophila S2 cells, but also the use of EGFP as a reporter to analyze gene expression, with its potential of a Drosophila cell expression system for recombinant protein production being an alternative to a baculovirus-insect cell expression system.

• KSBB Journal
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• v.8 no.2
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• pp.133-142
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• 1993

The effects of genetic and environmental factors on productivity of a cloned protein were studied in recombinant Saccharomyces cerevisiae. Plasmid stability and copy level were very high for a $REP^+$ system(at ca. 10 generations, stability: 65-90%, plasmid copy number per cell: 40-200), whereas these were very low for a yep- system(at ca. 10 generations, stability: 30%, plasmid copy number per cell 20). In plasmids containing the $2{\mu}m$ circle genome, a $[cir^o]$ strain was a preferred host cell since the plasmid stability and the copy number in a $[cir^o]$ strain were higher than in a $[cir^+]$strain. Cloned gene expression was dependent on plasmid copy number and stability. The inducer (galactose) level played a very important role in cloned lacZ gene expression, showing that a galactose concentration of 0.8% was sufficient for induction of gene expression. Induction rate was very fast in the case of plasmids exhibiting high stability and copy number by a factor of 4 to 25. The time to reach the peak value of gene expression was longer when galactose was added at the start of fermentation (ca. 26 hours) than at the mid-exponential phase (ca. 6 hours). Glucose repression was reduced by a factor of 2 to 5 as the relative inducer level increased.

• Preventive Nutrition and Food Science
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• v.2 no.1
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• pp.61-65
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• 1997

Wild type nodulin 26(nod 26) cDNA(S262) and phodphorylation aite mutant(S262D) were constructed by a yeast expression system using pYES2 plasmids(pTES2-D262 and pTES2-S262D) were sc-reened by restriction mapping with BamHI of KpnI. S262 nod 26 contained a sreine residue at position 262 and S262D nod 26 contained the substitution mutation of serine to aspartic acid residue at position 262 were verified by automated floursent DNA sequencing.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.288-296
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• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• Annals of Laboratory Medicine
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• v.38 no.6
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• pp.555-562
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• 2018

Background: The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock. Methods: Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced. Results: Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid. Conclusions: mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.

• KSBB Journal
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• v.16 no.1
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• pp.15-23
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• 2001

The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.

• Journal of Microbiology
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• v.35 no.2
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• pp.149-151
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• 1997

A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The numan cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced .betha.-galactosidase, dimonstrating its dual host usage.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.871-874
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• 2010

Many studies require expression analysis of the same gene/promoter across a range of bacterial genera. However, there is currently a lack of availability of reporters based on the broad-host-range IncQ replicon, which is compatible with a popular improved IncP transfer system that is self-transfer defective. We report IncQ lacZ reporter plasmids with features including (1) compatibility with IncP, IncW, and pBHR/pBBR replicons, (2) a variety of antibiotic markers (Sp-r, Sm-r, Km-r, Cm-r), (3) convenient mobilization via a novel self-transfer-defective IncP conjugation system, and (4) GenBank DNA sequences. Utility is demonstrated using three different promoters in different Gram-negative genera.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.186-189
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a potent immunogen as well as major virulence factor. In order to express the recombinant urease in tobacco plants, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a plant expression vector. The recombinant vector was transformed to tobacco plants. The integration of the recombinant plasmids into tobacco chromosomal genome was verified by genomic PCR. Expression to mRNA was confirmed by Northern blot analysis, and expression to recombinant urease protein was observed by Western blot analysis. These results showed that the recombinant urease can be produced in tobacco plants and will be tested for immune response to use as an edible vaccine.