• The Journal of Natural Sciences
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• v.17 no.1
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• pp.103-111
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• 2006

Regulation of invertase biosynthesis was studied with the E. coil harboring recombinant plasmid, pYC17. Biosynthesis of invertase in the recombinant E. coil was effectively induced in the presence of 30mM of sucrose for 3h. Glucose also repressed the invertase induction in the recombinant E. coil at 10 mM, lower than that of parent strain (30 mM).

• KSBB Journal
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• v.11 no.2
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• pp.132-139
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• 1996

The growth of recombinant E. coil and by-product production were investigated. D-cycloserine was added to increase the secretion of ${\alpha}$-amylase from recombinant E.coil. Even though cell growth was increased for optimal d-cycloserine concentration $0.1g/\ell$, extracellular ${\alpha}$-amylase activity remained almost the same as the case without d-cycloserine. It is important to achieve the high cell density for commercial production of methaboliles including ${\alpha}$-amylase. To achieve this goal, culture conditions should be selected carefully and optimized considering cell growth and by-products production. In cultivating retombinant E. coli, lactic acid and acetic acid turned out to be important by-product which affected cell yield and growth rate.

• Microbiology and Biotechnology Letters
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• v.18 no.4
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• pp.394-400
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• 1990

Maximization of productivity of recombinant cell fermentations requires consideration of the inverse relationship between the host cell growth rate and product formation rate. The problem of maximizing a weighted performance index was solved by using optimal control theory for recombinant E. coli fermentation. Concentration of a growth inhibitor was used as a control variable to manipulate the specific growth rate, and consequently the cloned-gene expression rate. Using a simple unstructured model to describe the main characteristics of this system, theoretical analysis showed that the optimal control profile results in an initial high growth rate phase followed by a low growth rate and high product formation rate phase. Numerical calculations were done to determine optimal switching times from the growth to the production stage for two representative cases corresponding to different dependency of the product formation rate on the growth rate. For the case when product formation rate is sensitive to the specific growth rate, the optimized operation yields about 60% increase in the final product concentration compared with a simple batch fermentation.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.318-322
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• 2006

The intracellular invertase gene of alkalophilic and thermophilic Bacillus sp. TA-11 which was isolated from compost was cloned and expressed in E. coil HB101 using pUC19 as a vector. The invertase of the recombinant E. coli (pYC 17) was maximally produced when it was incubated at 37$^{\circ}C$ for 9 h in a SY medium containing 0.25% sucrose, 0.5% yeast extract, 0.1% each of $K_2HPO_4$ and $KH_2PO_4$, with an initial pH of 8.0. The final enzyme activity under the above condition was 47.7 U per ml of cell-free extract.

• Journal of Microbiology
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• v.40 no.1
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• pp.77-81
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• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematspoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator, In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His. Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-so1ub1e form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.107-113
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• 1995

Basement membrane laminin is a multidomain glycoprotein that interacts with itself, heparin and cells. The distal long arm plays major cell and heparin interactive roles. The long arm consists of three subunits (A, B1, B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). The globule is in turn subdivided into five subdomains (Gl-5). In order to analyze the functions of this region, recombinant G domains (rG, rAiG, rG5, rGΔ2980-3028) were expressed in Sf9 insect cells using a baculovirus expression vector. A hybrid molecule (B-rAiG), consisting of recombinant A chain(rAiG) and the authentic B chains (E8-B)was assembled in vitro. The intercalation of rAiG into E8-B chains suppressed a heparin binding activity identified in subdomain Gl-2. By the peptide napping and ligand blotting, the relative affinity of each subeomain to heparin was assigned as Gl> G2= G4> G5> G3, such that G1 bound strongly and G3 not at all. The active heparin binding site of G domain in intact laminin appears to be located in G4 and proximal G5. Cell binding was examined using fibrosarcoma Cells. Cells adhered to E8, B-rAiG, rAiG and rG, did not bind on denatured substrates, poorly bound to the mixture of E8-B and rG. Anti-${\alpha}$6 and anti-${\beta}$1 integrin subunit separately blocked cell adhesion on E8 and B-rAiG, but not on rAiG. Heparin inhibited cell adhesion on rAiG, partially on B-rAiG, and not on E8. In conclusion, 1) There are active and cryptic cell and heparin binding activities in G domain. 2) Triple-helix assembly inactivates cell and heparin binding activities and restores u6131 dependent cell binding activities.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.297-302
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• 1985

The 7.1 Mdal Xbaf fragment of Bacillus pasteurii ATCC 11859 containing gene for urease was inserted into the Xbal site of bifunctional plasmid pGR71, and its urease gene was cloned and expressed in E. coil RRI. But the cloned gene was not expressed in Bacillus subtilis BR151 in consequence of deletion of inserted DNA fragment. The recombinant plasmid thus formed was named pGU66. The restriction map of the plasmid pGU66 was determined, and the size of the plasmid was estimated to be 12.6 Mdal by double digestion of restriction enzymes of the plasmid. The urease of the cloned strain was accumulated in periplasmic space and very similiar to that of donor strains in their enzymatic properties.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.228-234
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• 1991

- A synthetic gene coding for human interleukin-2 (IL-2) was constructed from the oligonucleotides synthesized by an automatic DNA synthesizer. The nucleotide sequence of the synthetic gene was chosen considering the preferred codons of E. coEi by not changing the amino acid sequence of IL-2 polypeptide. The synthetic gene was expressed in E. coli by placing the gene under the control of the $\lambda$ PL promoter. IL-2 was produced in the E. coli cytoplasm in the form of inclusion bodies. The recombinant IL-2 showed growth promoting activity on the IL-2 dependent cell line.

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.434-442
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• 2008

We observed that an inclusion body (IB) of recombinant ${\beta}$-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the ${\beta}$-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific ${\beta}$-galactosidase production, although ${\beta}$-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of ${\beta}$-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of ${\beta}$-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.211-220
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• 1986

Nitrogen fixation (nif) genes of Rhizobium leguminosarum were transferred to nif Klebsiella pneumoniae and E. coli by conjugation after partial heat induction of $RP_4$ :: Mu cts in Rhizobium $R^+$ transconjugant, and the hybrid plasmids in the transconjugant strains were isolated and characterized. In order to transfer the nif genes from Rhizobium, the hybrid plasmid $RP_4$ :: Mu cts was transferred by conjugation from E. coil to the symbiotic nitrogen fixer, R. leguminosarum. After stabillity test, the $RP_4$ :: Mu cts in Rhixobium $R^+$ transconjugant was subjected to partial heat induction by culturing it statically at $38^{\circ}C$ for 16 hours, and then conjugated with the nif defective mutant strains of K. pneumoniae or nif mutant strains of E. coli having whole nif gene plasmid. Recombinant strains of K. pneumoniae, which could grow in a N-free medium and exhibit the nitrogenase activity were selected. However, in the case of E. coli, they could grow well in a NA medium containing antibiotices, but hardly frow in a N-free medium. The hybrid plasmids in these transconjugal strains were isolated by gel electrophoresis and compared their molecular sizes.