Cross-reactivity of Human Polyclonal Anti-GLUT1 Antisera with the Endogenous Insect Cell Glucose Transporters and the Baculovirus-expressed GLUT1

Lee, Chong-Kee 대한의생명과학회 2001 Biomedical Science Letters Vol.7 No.4

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression for the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthemore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization fo the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression in insect cells relies upon their specificity for the human protein and lack of cross-reactionwith endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected for a protein of M_r comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

Cross-reactivity of Human Polyclonal Anti-GLUT1 Antisera with the Endogenous Insect Cell Glucose Transporters and the Baculovirus-expressed GLUT1

Lee, Chong-Kee The Korean Society for Biomedical Laboratory Scien 2001 Journal of biomedical laboratory sciences Vol.7 No.4

Most mammalian cells take up glucose by passive transport proteins in the plasma membranes. The best known of these proteins is the human erythrocyte glucose transporter, GLUT1. High levels of heterologous expression far the transporter are necessary for the investigation of its three-dimensional structure by crystallization. To achieve this, the baculovirus expression system has become popular choice. However, Spodoptera frugiperda Clone 9 (Sf9) cells, which are commonly employed as the host permissive cell line to support baculovirus replication and protein synthesis, grow well on TC-100 medium that contains 0.1% D-glucose as the major carbon source, suggesting the presence of endogenous glucose transporters. Furthermore, very little is known of the endogenous transporters properties of Sf9 cells. Therefore, human GLUT1 antibodies would play an important role for characterization of the GLUT1 expressed in insect cell. However, the successful use of such antibodies for characterization of GLUT1 expression m insect cells relies upon their specificity for the human protein and lack of cross-reaction with endogenous transporters. It is therefore important to determine the potential cross-reactivity of the antibodies with the endogenous insect cell glucose transporters. In the present study, the potential cross-reactivity of the human GLUT1 antibodies with the endogenous insect cell glucose transporters was examined by Western blotting. Neither the antibodies against intact GLUT1 nor those against the C-terminus labelled any band migrating in the region expected fur a protein of M$_r$ comparable to GLUT1, whereas these antibodies specifically recognized the human GLUT1. Specificity of the human GLUT1 antibodies tested was also shown by cross-reaction with the GLUT1 expressed in insect cells. In addition, the insect cell glucose transporter was found to have very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

Production of Recombinant Protein, Human Stem Cell Factor, Using Insect Cell Line

Sang-Mi Park,Kisang Kwon,Tae Won Goo,Eun Young Yun,Seok Woo Kang,Sung Wan Kim,Kweon Yu,O-Yu Kwon 대한의생명과학회 2009 Biomedical Science Letters Vol.15 No.1

Insect cell cultures have become important tools in the production of biological substances for use in a variety of research, human and veterinary medicine, and pest control applications. These applications often require the introduction of foreign DNA into the cells and have generally used methods originally developed for use with human and other mammalian cell cultures. While these methods can be successfully employed, they are often less efficient with insect cells and frequently involve complex procedures or require specialized equipment. Even when they do work, they may require substantial modification because of differences in the culture medium or growth patterns of insect cells. In this study, We have optimized transfection conditions of Sf9 cell line using insect expression vector pIZT/V5-His which expresses green fluorescent protein effectively. Human stem cell factor (hSCF) is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a key role in mast cell development, gametogenesis, and melanogenesis. It can exist in membrane-bound form and in proteolytically released soluble form. As determined by an enzyme-linked immunosorbent assay performed, hSCF level in supernatant averaged 995 ng/ml. The human hSCF was partially purified by immunoaffinity chromatography and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The results show that the hSCF has N-linked carbohydrate and corresponds to the soluble form, at or about 223 amino acids in length. The findings suggest functional importance for soluble hSCF in cells.

Cloning, Gene Expression and Enzyme Activity of Serine Proteases from Laccotrephes japonensis, Ranatra unicolor, Muljarus japon

Kwan-Ho Park,Young-Cheol Choi,Jong-Gil Kim,Ji-Young Choi,Won-Tae Kim,Si-Kab Nho,Jae-Sam Hwang 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease. There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects. RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant protein showed activity in the protease enzyme assay using gelatin as a substrate.

Molecular cloning, gene structure, expression and enzyme activity of a serine-like protease from Laccotrephes japonensis

Kwan-Ho Park,Seong-Ryul Kim,Eun-Young Yun,Hwa-Jin Suh,Si-Kab Nho,Jae-Sam Hwang 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.10

The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

Expression of Recombinant Proteins in Plants by Using Baculovirus Vectors

오은이,김영관,박다영,Zhe Lu,추영국,한연수,박정미,고기성 한국원예학회 2011 Horticulture, Environment, and Biotechnology Vol.52 No.1

Baculovirus has been widely used for the production of numerous recombinant proteins in insect cells. Baculovirus vectors have several advantages, including proper post-translational modification, biosafety, and multiple large gene expression ability. Most insect cell-produced proteins have been expressed by using the baculovirus expression vector system (BEVS) under the control of strong polyhedrin (Polh) or p10 promoters. There has been no report on the expression of recombinant proteins by baculovirus in plant cells. In this study, we used the baculovirus vector to express recombinant green fluorescent protein (GFP) in plants. To investigate the expression of GFP protein by baculovirus in plants, we cloned the gfp gene under the control of Polh promoter or Cauliflower Mosaic Virus (CaMV) 35S promoter to yield the Polh-GFP and 35SGFP bacmids carrying the GFP expression cassettes, respectively. The presence of Polh-GFP and 35S-GFP expression cassettes in the bacmids was confirmed by polymerase chain reaction (PCR). Subsequently, both the GFP bacmids and GFP baculovirus vectors generated from the bacmid-transfected Sf9 insect cells were inoculated into Nicotiana benthamiana leaves. Confocal microscopy revealed that the gfp gene expression was high in plant leaves at 48 and 72 h after bacmid and baculovirus inoculation. Reverse transcription-PCR (RT-PCR) and fluorescence microscopy confirmed that the gfp genes under the control of Polh or CaMV35S promoters were highly expressed in plant leaves inoculated with 40 μL of baculovirus solution. These results suggested that the baculovirus vector can be used to express recombinant proteins in plants. The baculovirus vector-mediated gene delivery and expression system could be used in plant biotechnology for fast and efficient production of recombinant proteins and for molecular virology studies in plants.

Molecular Cloning of the cDNA of Heat Shock Protein 88 Gene from the Entomopathogenic Fungus, Paecilomyces tenuipes Jocheon-1

Liu, Ya-Qi,Park, Nam Sook,Kim, Yong Gyun,Kim, Keun Ki,Park, Hyun Chul,Son, Hong Joo,Hong, Chang Ho,Lee, Sang Mong Korean Society of Sericultural Science 2014 International Journal of Industrial Entomology Vol.28 No.2

The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.

Large-Scale Production of Rotavirus VLP as Vaccine Candidate Using Baculovirus Expression Vector System (BEVS)

( Park Jin Yong ),( Hun Kim ),( Hi Ku Hwang ),( Su Jeen Lee ),( Hyun Sung Kim ),( Byung Ki Hur ),( Yeon Woo Ryu ),( Chang Nam An ),( Jong Soo Kim ) 한국미생물생명공학회 2004 Journal of microbiology and biotechnology Vol.14 No.1

Molecular Cloning of the cDNA of Heat Shock Protein 88 Gene from the Entomopathogenic Fungus, Paecilomyces tenuipes Jocheon-1

( Ya Qi Liu ),( Nam Sook Park ),( Yong Gyun Kim ),( Keun Ki Kim ),( Hyun Chul Park ),( Hong Joo Son ),( Chang Ho Hong ),( Sang Mong Lee ) 한국잠사학회 2014 International Journal of Industrial Entomology Vol.28 No.2

The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomycestenuipes Jocheon -1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes )Jocheon-1 Uni-Zap cDNA library and performing 5` RACE polymerase chain reaction(PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of2, 139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of theP. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipesJocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminalregion. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton(kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditionsfor the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stressinduced a higher level of mRNA expression compared to normal growth conditions.