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Expression of Recombinant Korean Mistletoe(KM) Lectin and B genes in Saccharomyces cerevisiae

Saccharomyces cerevisae에서 한국산 겨우살이 유래 lectin A 및 B 유전자의 발현

  • 최윤혁 (한동대학교 생명공학연구소) ;
  • 김종배 (한동대학교 생명공학연구소) ;
  • 양웅석 ((주)미술 바이오텍) ;
  • 황철원 (한동대학교 생명공학연구소)
  • Published : 2004.10.01

Abstract

A study for expression of Korean Mistletoe (KM) lectin gene (A,B) in Saccharomyces cerevisiae was done using transforming system of yeast. In order to overexpress the genes efficiently in yeast, two lectin genes (A,B) were re-cloned and modified including Kozak translation initiation sequence using PCR amplification. The constructed plasmids containing modified lectin A and B genes were transformed to S. cerevisea INVSc (MAT G, his3 $\Delta$1, leu2, trpl-289, ura3-52). The transformed cells were identified by DNA sequencing with ABI3700 system and induced with 2% of galactose for recombinant KM lectin (rKM lectin) protein. The rKM lectin A and B proteins were determinated about 29kDa size of protein by SOS-P AGE and western blotting analysis. The expressed recombinant lectin was determinated 1.24∼1.75 $\mu\textrm{g}$ per 1 mg of cytosolic soluble protein by sandwich ELISA method. Moreover the lectin genes were expressed as maximum level at 36 h after galactose induction and lectin A gene was were repressed after 48 h.

References

  1. Agapov I. I., A. G. Tonevitsky, A. T. Shamshiev, E. Pohl, P. Pohl, R. A. Palmer and M. P. Kirpichnikov, 1997. The role of structural domains in RIP II toxin model membrane binding. FEBS Letters, 402(1), 91-93 https://doi.org/10.1016/S0014-5793(96)01452-4
  2. Bussing A., K. Suzart and K. Schweizer, 1997. Differences in the apoptosis-inducing properties of Viscum album L. extracts. Anti-Cancer Drugs, 8(Suppl. 1), 9-14 https://doi.org/10.1097/00001813-199704001-00003
  3. Eck J., M. Langer, B. Mockel, K. Witthon, H. Zinke, and H. Lentzen, 1999. Characterization of recombinant and plant-derived mistletoe lectin and their B-chains. Eur J Biochem., 265(2), 788-797 https://doi.org/10.1046/j.1432-1327.1999.00784.x
  4. Elsasser-Beile U., M. Voss, R. Schuhle, and U. Wetterauer, 2000. Biological effects of natural and recombinant mistletoe lectin and an aqueous mistletoe extract on human monocytes and lymphocytes in vitro. J Clin Lab Anal., 14(6), 255-259 https://doi.org/10.1002/1098-2825(20001212)14:6<255::AID-JCLA1>3.0.CO;2-J
  5. Franz H., P. Ziska and A. Kindt, 1981. Isolation and properties of three lectins from mistletoe (Viscum album L.). Biochem. J. 195, 481-484
  6. Gietz R. D. and R. H. Shiestl, 1995. Transforming yeast with DNA. Meth. Mol. Cell. Biol., 5, 255-269
  7. Gong Z. Z., E. Yamagishi, M. Yamazaki, and K. Saito, 1999. A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molec ular characterization, heterologous expression in transgenic plants and transactivation in yeast cells. Plant Mol Biol., 41(1), 33-44 https://doi.org/10.1023/A:1006237529040
  8. Hajto T., K. Hostanska, K. Frei, G. Rordorf and H. J. Gabius, 1990. Increased secretion of tumor necrosis factor-$\alpha$, interleukin 1, and interleukin 6 by human mononuclear cells exposed to $\beta$-Galactoside-specific galactoside-specific lectin from clinically applied mistletoe extract. Cancer Res., 50, 3322-3326
  9. Kearns M. A., D. E. Monks, M. Fang, M. P. Rivas, P. D. Courtney, J. Chen, G. D. Prestwich, A. B. Theibert, R. E. Dewey, and V. A. Bankaitis. 1998. Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast. EMBO J., 17(14), 4004-4017 https://doi.org/10.1093/emboj/17.14.4004
  10. Khwaja T. A., J. C. Varven, S. Pentecost and H. Pande, 1980. Isolation of biologically active alkaloids from Korean mistletoe Viscum album coloratum. Experientia, 36, 599-600 https://doi.org/10.1007/BF01965825
  11. Kozak, M., 1997. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J., 16, 2482-2492 https://doi.org/10.1093/emboj/16.9.2482
  12. Kourmanova A. G., O. J. Soudarkina, S. Olsnes, and J. V. Kozlov, 2004. Cloning and characterization of the genes encoding toxic lectins in mistletoe (Viscum album L). Eur J Biochem., 271(12), 2350-2360 https://doi.org/10.1111/j.1432-1033.2004.04153.x
  13. Lee J. H., J. L. Weickmann, R. K. Koduri, P. Ghosh- Dastidar, K. Saito, L. C. Blair, T. Date, J. S. Lai, S. M. Hollenberg, and R. L. Kendall, 1988. Expression of synthetic thaumatin genes in yeast. Biochemistry, 27(14), 5101-5107 https://doi.org/10.1021/bi00414a023
  14. Niwa, H., A. G. Tonevitsky, I. I. Agapov, S. Saward, U. Pfuller, and R. A. Palmer, 2003. Crystal structure at 3 A of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose. Eur. J. Biochem., 270, 2739-2749 https://doi.org/10.1046/j.1432-1033.2003.03646.x
  15. Olsnes S., F. Stirpe, K. Sandvig and A. Pihl, 1982. Isolation and characterization of viscumin, a toxic lectin from Viscum album L. (mistletoe). J. Biol. Chem. 257, 13263-13270
  16. Park C. H., D. W. Lee, T. B. Kang, K. H. Lee, T. J. Yoon, J. B. Kim, M. S. Do, S. K. Song, 2001. cDNA cloning and sequence analysis of the lectin genes of the Korean mistletoe (Viscum album coloratum). Mol. Cells. 31;12(2), 215 -220
  17. Rasmussen-Wilson S. J., J. S. Palas, V. J. Wolf , C. S. Taft, and C. P. Selitrennikoff, 1997. Expression of a plant protein by Neurospora crassa. Appl. Environ. Microbiol., 63(9), 3488-3493
  18. Sambrook J., E. F. Fritsch and T. Maniatis, 1989. A Laboratory Manual (second edition), Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring
  19. Yoon T. J., Y. C. Yoo, T. B. Kang, H. Erk, S. H. Kim, K. S. Kim, I. Azuma and J. B. Kim, 2001. Cellular and humoral adjuvant activity of lectins isolated from Korean mistletoe (Viscum album colaratum). International Immunopharmacology, 1(5), 881-889 https://doi.org/10.1016/S1567-5769(01)00024-8