• Journal of Life Science
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• v.23 no.2
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• pp.319-323
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• 2013

Protein aggregation can cause diseases and hinder the production of useful recombinant proteins. The present study showed that at least three types of aggregates can be formed from tryptophan synthase ${\alpha}$-subunit (${\alpha}TS$) by varying conditions: (1) an opaque white precipitous aggregate, (2) a transparent gel-like precipitous aggregate, and (3) an unprecipitous aggregate. Macroscopically different aggregate types might suggest different mechanisms underlying aggregation processes.

• KSBB Journal
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• v.17 no.6
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• pp.577-581
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• 2002

In an attempt to produce recombinant proteins using the pollen enriched in some plant species, a 1.7 kb DNA encoding urease subunit B (UreB) amplified by PCR from Helicobacter pylori urease gene cluster in pH808 plasmid was cloned to be expressed under CaMV35S promoter in lily (Lilium longiflorum) pollen tubes elongated in vitro. Lily pollen at early germinating stage was transformed with the ureB DNA using Agrobacterium via vacuum infiltration and, incubated for a full pollen tube growth 16 - 24 h in the dark in the presence of kanamycin. DNA integration and expression in the transgenic pollen were analyzed by the standard molecular techniques and the results suggest that the pollen in vitro may be employed as a protein factory in a disposable fashion.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.279-285
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• 1993

Alterations of the p53 gene arc among the most frequent genetic changes in human cancer and often result in increased levels of p53 protein within the malignant cells. Detection of accumulated p53 protein can be a useful prognostic tool in human cancer. In order to make polyclonal antibodies for immunohistochemical screening. human p53 gene was expressed in E. coli in the form of GST (glutathione S-transfi.:rase) fusion proteins. Two p53 gene fragments. which were N('()I small fragment encoding amino acid residues of 1-151-: and Ncol large fragment of 159-393. were subeloned into the unique BamHI site present within the pGEX-2T vector using BamHI linker and recombinant plasmids pGTNS and pGTNL were constructed. respectively. The p53 cDNA fragment (from pC53-$SN_3$,) encoding amino acid 38-145 (proline at residue 72) was amplified by polymerase chain reaction(PCR). The amplified DNA was digested with BamHI and Prull and inserted into the BamHI-Smal sites of pG EX-2T and recombinant plasmid pGTBP was constructed. After IPTG induction of these plasmids for 4 hours. fusion proteins were purified from E. coli extracts with glutathione Sepharose beads. The bound proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and the molecular weights were 54 kDa. 53 kDa and 40 kDa. respectively. Approximately one milligram of fusion proteins were purified from 1 -liter cultures.

• Journal of Life Science
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• v.18 no.7
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• pp.912-917
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• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

• Korean Chemical Engineering Research
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• v.47 no.5
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• pp.624-629
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• 2009

It is the most important step to screen soluble and insoluble proteins when we attempt to improve the solubility of recombinant proteins through directed evolution approach. Here we show that the solubility of a recombinant protein in vivo can be examined by expressing the recombinant protein with beta-lactamase as a fusion partner. First we constructed an expression system which can produc a fusion protein with the C-terminal of beta-lactamase. Two soluble proteins, i.e. adenine deaminase and aspartate aminotransferase, and insoluble GlcNAc-2-epimerase were cloned into the developed expression vector, respectively. We investigated the effect of the expression of the three recombinant fusion proteins on the growth of E. coli, and confirmed that the solubilities of the recombinant proteins correlated with cell growth rates.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.425-428
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• 2000

To enhance the productivity of recombinant protein, ${\beta} -glucuronidase(GUS)$, by transgenic Nicotiana suspensions, perfusion culture was carried out in a 5-L stirred tank bioreactor. Maximum cell density of 20.0 gDCW/L was obtained and GUS activity was noticeably affected by medium composition, such as salt concentration. Effect of headspace purging by $CO_2-enriched$ air was also investigated. Aeration of $CO_2(5%)-enriched$ air with 0.2 vvm significantly promoted the cell growth in lag pahse and increased volumetric GUS activity from 0.69 U/mL to 5.76 U/mL. In contrast, aeration of $CO_2(5%)-enriched$ air with 0.1 vvm did not affect the cell growth but enhanced the GUS acitivity from 3.24 to 5.25 U/mL at the 4th day.

• Korean Journal of Microbiology
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• v.51 no.2
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• pp.115-125
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• 2015

A monoclonal antibody AP64 IgM binds to human acute nonlymphocytic leukemia (ANLL) cell line HL60 and also cross-reacts with the homologous antigen in a rat ANLL cell. This antibody mediated by complement, has leukemia a suppression effect. In this study, we generated a recombinant single-chain variable domain fragment (ScFv) which were derived from $V_H$ and $V_L$ cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker $(G_4S)_3$. This recombinant ScFv was expressed as a single polypeptide chain from Escherichia coli BMH 71-18. The recombinant ScFv was purified by applying the periplasmic extract to $Ni^+$-NTA-agarose affinity column and detected with westernblot. The purified recombinant ScFv recognized a surface antigen (about 30 kDa) of HL60 cell line which is the same antigen detected by parental AP64 IgM. But the affinity of ScFv for a surface antigen of HL60 was lower than that of the parental AP64 IgM, which needs to be further improved. Overall, the recombinant ScFv specific to HL60 might be a useful bioreagent for either diagnostic or therapeutic purposes.

• Korean Chemical Engineering Research
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• v.44 no.1
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• pp.40-45
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• 2006

The baculovirus-insect Drosophila melanogaster S2 cell system combines advantages of conventional baculovirus system and non-lytic S2 cell system because baculoviruses can infect non-permissive cells such as mammalian and Drosophila S2 cells but cannot replicate themselves inside the cells. In the present work, we investigated effects of infection conditions on production of green fluorescent protein (GFP) as a target protein using this baculovirus-S2 cell system. Even though higher MOI and longer baculovirus contact time showed better GFP expression yield during the shorter period, overall protein yield could be lower during the longer period due to the relatively higher cell detachment and lysis (lower cell viability). In addition, maintaining high MOI will be not practical for large-scale cell culture. Therefore, instead of maintaining high MOI, we found that high initial cell number and concentrated (10X) baculovirus volume can confer comparable protein expression even under the moderate MOI condition. Also, we found that the post-infection time that is connected to state of cells after infection was an important factor for production yield.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.271-276
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• 2008

Ribosome stalling by nascent sticky peptide has been reported in several organisms across the kingdom. To test whether small subunit (SSU) rRNA is involved in this phenomenon, we developed a genetic system that utilized the specialized ribosome system to isolate SSU rRNA mutants that enable ribosomes to bypass the SecM-derived sticky peptide in protein synthesis. In this system, CAT-SecM mRNA, which encodes CAT protein containing the sticky peptide derived from SecM, is only translated by specialized ribosomes. These ribosomes were shown to transiently stall on CAT-SecM mRNA followed by the synthesis of the sticky peptide. Expression of specialized ribosomes resulted in the decreased steady-state level of CAT-SecM mRNA, which is consistent with a notion that ribosome stalling induces mRNA degradation. Isolation and characterization of SSU rRNA mutations using this genetic system that are sufficient to circumvent ribosome stalling induced by the SecM-derived sticky peptide will provide evidence of SSU rRNA function in mRNA cleavage.