A simple method for constructing polymerized genes using only restriction
enzymes and commercially available cloning systems was established. In this
system, gel isolations or purifications of target genes after restriction
enzyme digestions or PCR amplifications, which often cause errors and mutations
in the target gene sequence, are not necessary. To verify the usefulness of
this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression
gene in Escherichia coli were
sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E.
coli showed an increasing trend in accordance with the copy numbers of the
gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining,
no expressed protein could be seen in E.
coli cells harboring plasmids that contained one or two copies of the gene.
However, expressed protein bands in E.
coli cells were clearly detected with 4 copies of the gene. In quantitative
analyses involving green fluorescence intensities per culture volume, the expression
level in E. coli with 16 copies of
the gene was 36.3-fold higher than that in E.
coli with one copy at 22 hours after induction.
Cite this paper
Shibui, T. , Sakaguchi, D. and Hara, H. (2014) A New Tandem Gene Construction Method Involving a Cloning System Using Poxvirus DNA polymerase
, and Its Application to Gene Expression. Advances in Bioscience and Biotechnology
, 838-845. doi: 10.4236/abb.2014.510098
 Shibui, T. and Nagahari, K. (1992) Secretion of a Functional Fab Fragment in Escherichia coli and the Influence of Culture Conditions. Applied Microbiology and Biotechnology, 37, 352-357. http://dx.doi.org/10.1007/BF00210991
 Dolgikh, V.V., et al. (2014) Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli. Acta Naturae, 6, 106-109.
 Rosano, G.L. and Ceccarelli, E.A. (2014) Recombinant Protein Expression in Microbial Systems. Frontiers in Microbiology, 5, 1-2.
 Goodrick, J.C., et al. (2001) High-Level Expression and Stabilization of Recombinant Human Chitinase Produced in a Continuous Constitutive Pichia pastoris Expression System. Biotechnology and Bioengineering, 74, 492-497.http://dx.doi.org/10.1002/bit.1140
 Farhadi, B., Shekari Khaniani, M. and Mansoori Derakhshan, S. (2014) Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor. Advanced Pharmaceutical Bulletin, 3, 303-308.
 Fazel, R., et al. (2014) Cloning and Expression of Aspergillus flavus Urate Oxidase in Pichia pastoris. SpringerPlus, 3, 395.
 Nevalainen, K.M., Te’o, V.S. and Bergquist, P.L. (2005) Heterologous Protein Expression in Filamentous Fungi. Trends in Biotechnology, 23, 468-474. http://dx.doi.org/10.1016/j.tibtech.2005.06.002
 Caron, A.W., Archambault, J. and Massie, B. (1990) High-Level Recombinant Protein Production in Bioreactors Using the Baculovirus-Insect Cell Expression System. Biotechnology and Bioengineering, 36, 1133-1140.http://dx.doi.org/10.1002/bit.260361108
 Kito, M., et al. (2002) Construction of Engineered CHO Strains for High-Level Production of Recombinant Proteins. Applied Microbiology and Biotechnology, 60, 442-448. http://dx.doi.org/10.1007/s00253-002-1134-1
 Aflakiyan, S., et al. (2013) Expression of the Recombinant Plasminogen Activator (Reteplase) by a Non-Lytic Insect Cell Expression System. Research in Pharmaceutical Sciences, 8, 9-15.
 Yu, H., et al. (2013) Large-Scale Production of Functional Human Lysozyme in Transgenic Cloned Goats. Journal of Biotechnology, 168, 676-683.
 Fussenegger, M. and Hauser, H. (2007) Protein Expression by Engineering of Yeast, Plant and Animal Cells. Current Opinion in Biotechnology, 18, 385-386. http://dx.doi.org/10.1016/j.copbio.2007.10.002
 Strasser, R., Altmann, F. and Steinkellner, H. (2014) Controlled Glycosylation of Plant-Produced Recombinant Proteins. Current Opinion in Biotechnology, 30C, 95-100.
 Tuse, D., Tu, T. and McDonald, K.A. (2014) Manufacturing Economics of Plant-Made Biologics: Case Studies in Therapeutic and Industrial Enzymes. BioMed Research International, 2014, Article ID: 256135.
 Somers, J., Poyry, T. and Willis, A.E. (2013) A Perspective on Mammalian Upstream Open Reading Frame Function. The International Journal of Biochemistry & Cell Biology, 45, 1690-1700.http://dx.doi.org/10.1016/j.biocel.2013.04.020
 Shibui, T. and Nagahari, K. (1994) Antibody Produced by Using Escherichia coli Expression Systems. Food and Bioprocess Technology, 19, 253-268.
 Patterson, S.S., et al. (2005) Codon Optimization of Bacterial Luciferase (Lux) for Expression in Mammalian Cells. Journal of Industrial Microbiology Biotechnology, 32, 115-123. http://dx.doi.org/10.1007/s10295-005-0211-8
 Shibui, T., et al. (1991) High-Level Secretion of Human Apolipoprotein E Produced in Escherichia coli: Use of a Secretion Plasmid Containing Tandemly Polymerized ompF-Hybrid Gene. Journal of Biotechnology, 17, 109-120.http://dx.doi.org/10.1016/0168-1656(91)90002-D
 Koga, H., Misawa, S. and Shibui, T. (2009) A Wheat Embryo Cell-Free Protein Synthesis System Not Requiring an Exogenous Supply of GTP. Biotechnology Progress, 25, 1322-1327. http://dx.doi.org/10.1002/btpr.230
 Zhu, B., et al. (2007) In-Fusion Assembly: Seamless Engineering of Multidomain Fusion Proteins, Modular Vectors, and Mutations. BioTechniques, 43, 354-359. http://dx.doi.org/10.2144/000112536