Construction of Novel Phytochelatins by Overlap Oligonucleotides
Keywords:Design of oligonucleotide, Hexahistidine, Synthetic phytochelatin
Synthetic phytochelatins are protein analogs of phytochelatin with similar heavy metal binding affinities that can be easily produced from a synthetic DNA template. We design synthetic phytochelatin [(Glu-Cys)n Gly] linked to hexahistidine by viral linker peptide and then followed by gene synthesis and cloning of it. Then peptide coding gene (synthetic phytochelatin with linker and hexahistidine) was designed exactly and constructed with step by step methods by overlapping oligonucleotides using T4 DNA Ligase. Finally, synthesized gene amplified by PCR, cloned in pTZ57R/T and transformed to Escherichia coli (DH5?). The results of sequencing show that some types of synthetic phytochelatin (EC4, EC12, and EC20) with linker and hexahistidine were constructed and cloned in vector.
. Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., Bae, W., Mehra, R.K., Mortari, A., Csöregi, E. (2003). Novel synthetic Phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics, 18:547-553.
. Cobbett C.S. (2000). Phytochelatins and their roles in heavy metal detoxification. Plant Physiology, 123: 825-832.
. Bae, W., Mehra, R.K., Mulchandani, A. and Chen W. (2001). Genetic engineering of E. coli for enhanced uptake and bioaccumulation of mercury. Applied and Environmental Microbiology, 67: 5335-5338.
. Bae, W. and Mehra, R.K. (1997). Metal-binding characteristics of a phytochelatin analog (Glu-Cys)2Gly. Journal of Inorganic Biochemistry, 68: 201-210.
. W. Bae, W. Chen, A. Mulchandani, and R.K. Mehra (2000). Heavy metal removal using bacteria displaying synthetic phytochelatins. Environmental Chemistry, 40: 793-794.
. Cobbett, C., Goldsbrough, P. (2002). Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol., 53: 159-82.
. Xue, F., Gu, Z., Feng, J.A. (2004). LINKER: a web server to generate peptide sequences with extended conformation. Nucleic acids Research, 32: W562-5.
. Zhang, J., Yun, J., Shang, Z., Zhang, X., Pan, B. (2009). Design and optimization of a linker for fusion protein construction. Progress in Natural Science, 19: 1197-1200.
. George, R.A. and Heringa, J. (2002). An analysis of protein domain linkers: their classification and role in protein folding. Protein engineering, 15: 871-9.
. Mahnam, K., Saffar, B., Moboni, M., Mohamadi, A. Design and construction of a novel metal binding peptide for sequestering of heavy metals by molecular dynamics simulation. Unpublished.
. Borodina, T.A., Lehrach, H., Soldatov, A.V. (2003). Ligation-Based synthesis of oligonucleotides with block structure. Anal. Biochem., 318: 309–313.
. Rouillard, J., Lee, W., Truan, G., Gao, X., Zhou, X., Gulari, E. (2004). Gene2oligo: oligonucleotide design for in vitro gene synthesis. Nucleic Acids Research, 32: W176-180.
. Hoover, D.M., Lubkowski, J. (2002). DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res., 30: e34.
. Au, L.C., Yang, F.Y., Yang, W.J., Lo, S.H., Kao, C.F. (1998). Gene synthesis by an LCR-based approach: high-level production of leptin-L54 using synthetic gene in Escherichia coli. Biochem. Biophys. Res. Commun., 248: 200-203.
. Bae, W., Chen, W., Mulchandani, A., Mehra, R.K. (2000). Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and Bioengineering, 70: 518-524.
. B. Saffar, B. Yakhchali, M. Arbabi (2007). Bioaccumulation of heavy metal ions by E. coli cells displaying hexahistidine peptide using a novel display system based on CS3 pili. Current Microbiolog., 55: 273-277.