• BMB Reports
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• v.56 no.7
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• pp.392-397
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• 2023

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacterium-mediated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.327-333
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• 1988

For optimal production of recombinant human interleukin-2 (IL-2) in E. coli the effect of fermentation conditions on cell growth, IL-2 production, and stability of recombinant cells were investigated. Among the complex nutrients tested in this work, yeast extract, peptone and corn steep liquor were found to be effective for recombinant cell growth. The recombinant cells were maintained stably under repression condition (3$0^{\circ}C$), but the stability of recombinant cells were drastically reduced upon induction of IL-2 expression (42$^{\circ}C$) even under the selection pressure. Addition of antibiotics to the culture medium resulted in the cell growth inhibition without significant improvement in recombinant stability. When the expression of IL-2 gene was induced at different growth phases, highest IL-2 production was achieved by the induction of IL-2 at the middle-exponential growth phase. It was found that the production of IL-2 significantly inhibited the cell growth and the ex-pression of other genes in the plasmid.

• Journal of Life Science
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• v.30 no.3
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• pp.304-312
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• 2020

Agarase can be used in the field of basic science, as well as for production of agar-derived high-functional oligosaccharides and bioenergy production using algae. In 2012, we summarized the classification, origin, production, and applications of agar. In this paper, we briefly review the literature on the recombinant expression of agarases from 2012 to the present. Agarase genes originated from 19 genera, including Agarivorans, Flammeovirga, Pseudoalteromonas, Gayadomonas, Catenovulum, Microbulbifer, Cellulophaga, Saccharophagus, Simiduia, and Vibrio. Of the 47 recombinant agarases, there were only two α-agarases, while the rest were β-agarases. All α-agarases produced agarotetraose, while β-agarases yielded many neoagarooligosaccharides ranging from neoagarobiose to neoagarododecaose. The optimum temperature ranged between 25 and 60℃, and the optimum pH ranged from 3.0 to 8.5. There were 14 agarases with an optimum temperature of 50℃ or higher, where agar is in sol state after melting. Artificial mutations, including manipulation of carbohydrate-binding modules (CBM), increased thermostability and simultaneously raised the optimum temperature and activity. Many hosts and secretion signals or riboswitches have been used for recombinant expression. In addition to gene recombination based on the amino acid sequence after agarase purification, recombinant expression of the putative agarase genes after genome sequencing and metagenome-derived agarases have been studied. This study is expected to be actively used in the application fields of agarase and agarase itself.

• Analytical Science and Technology
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• v.12 no.6
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• pp.565-570
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• 1999

The alcohol dehydrogenase (ADH) gene from Bacillus stearothermopilus was amplified by the polymerase chain reaction. The amplified DNA was inserted into the expression vector pGEX-KG, and expressed it as a fusion protein with glutathione S-transferase (GST) in E. coli. The recombinant ADH was produced by induction with 1 mM isopropyl-${\beta}$-D-thiogalactopyranoside at $37^{\circ}C$ and purified by glutathione affinity chromatography. The recombinant ADH exhibited high substrate specificity for ethanol. The activity of the recombinant ADH proceeded optimally at pH 9.0 and $70^{\circ}C$. The recombinant ADH was highly stable against high temperature. This thermostable alcohol dehydrogenase can be used for the enzymatic determination of alcohol and for the industrial production of alcohol.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.541-547
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• 1999

Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.

• Korean Journal of Veterinary Research
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• v.61 no.2
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• pp.19.1-19.7
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• 2021

Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, an accidental laboratory-mediated infection is concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:2¹⁵. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HI-FPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.146-149
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• 2000

Biologically active porcine Interleukin-2(poIL-2) was produced from in vitro and in viva baculovirus expression system, namely the Autographa californica nuclear polyhedrosis virus (ACNPV)-cell culture system and the Hybrid nucler polthedrosis virus (HyNPV)-sillkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.620-623
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• 2003

The delta sleep-inducing peptides (DSIP, Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) is an important regulatory hormone, controlling hypothalamus and pituitary functions. In the current study, an expression system was designed for the rapid and economic expression oi recombinant DSIP for biophysical studies. Artificially synthesized oligonucleotides encoding DSIP were cloned into a pGEX-KG vector and expressed in E. coli (BL21). The recombinant GST-DSIP was then readily purified using a GST affinity column. To obtain intact DSIP from the GST-DSIP, thrombin cleavage and a CNBr reaction were successively carried out. The DSIP in the CNBr reaction mixture was subjected to RP-HPLC purification to yield 1.2 mg DSIP from a 1 liter culture of E. coli. Identification of the DSIP was peformed using MALDI-MS and an amino acid composition analysis.

• Microbiology and Biotechnology Letters
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• v.22 no.2
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• pp.152-157
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• 1994

Inulinase of Kluyveromyces marxianus origin was produced by recombinant yeast Saccharomyces cerevisiae under the control of GAL1 promoter, to examine the expression and localization of inulinase in a repressed(galactose-free) or derepressed(galactose-containinga) medium. The inulinase gene(INU1A) was constitutively expressed at 6.7 units/ml in a repressed medium. When the cell started to utilize galactose in a derepressed medium, the INU1A gene began to be expressed, and the final expression level reached about 45 units/ml. According to be the nondenaturingPAGE analysis, inulinase produced by S. cerevisiae was found to be less glycosylated than the bakers yeast invertase. In addition, its glycosylation pattern was less heterogeneous than the K. marxianus inulinase. The supplementation of inulin or raffinose into the derepressed medium increased the cell growth rate, while the expression of INU1A was repressed. Regardless of the carbon sources examined, most of inulinase activity (more than 98%) was found in the extracellular medium, indicating excellent secretion efficiency.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.266-273
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• 1991

Hirudin, a 65-amino acid protein isolated from the salivary gland of the bloodsucking leech, Hirudo medicinalis, is a potent thrombin-specific inhibitor and blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation. We have studied the gene expression and secretion of hirudin in yeast. Saccharomyces cerevisiae. A gene coding for hirudin was synthesized based on the amino acid sequence and cloned into a yeast expression vector $YEG{\alpha}-1$ containing the ${\alpha}-mating$ factor pre-pro leader sequence and galactose-inducible promoter, GALl0. Recombinant S. cerevisiae was found to secrete biologically active hirudin into the extracellular medium. The secreted recombinant hirudin was recovered from the culture medium and purified with ultrafiltration and reverse phase high performance liquid chromatography. Approximately 1 mg of hirudin per liter was produced under suboptimal culture conditions and brought to about 90% purity in two steps of purification.