DOI QR코드

DOI QR Code

Saccharomyces cerevisae에서 한국산 겨우살이 유래 lectin A 및 B 유전자의 발현

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

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

초록

본 연구는 한국산 겨우살이 lectin유전자 (A 및 B chain) 을 효모 (Saccharmyces cerevisiae) 에 형질 전환시키는 시스템을 사용한 것으로, 효모내 효과적인 lectin유전자 발현을 위하여 유전자 상에 Kozak translation initiation sequence를 PCR을 이용 삽입, 변형시켜 재 클로닝 하였다. 변형된 lectin A 및 B 유전자를 포함하는 재조합 플라스미드는 S. cerevisiae INVSc (MATa, his3 $\Delta$l, leu2, trpl-289, ura3-52) 에 형질전환 되었다. 형질전환된 효모는 ABI 3700 system을 이용한 DNA 염기서열 분석을 통해 확인되었고 재조합 한국산겨우살이 lectin 발현을 위해 2% galactose를 사용하여 유도발현되었다. 재조합 lectin A 및 B 단백질은 SDS-PACE 및 western blotting 분석을 수행한 결과 약 29kDa 크기로 확인되었다. 재조합 lectin은 세포내 가용성 단백질 1mg중 1.24∼l.75 $\mu\textrm{g}$ 수준으로 발현되어짐을 ELISA 분석을 통해 확인하였다. 한편 lectin 유전자는 galartose 유도발현 후 36시간이 되 었을 때 발현량이 최대가 되었으며 lectin A 유전자의 경우 48 시간 이후에는 발현이 억제되었다.

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.

키워드

참고문헌

  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