Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Production of miraculin protein in suspension cell lines of transgenic rice using Agrobacterium

Agrobacterium을 이용한 형질전환 벼 현탁 세포주에서 miraculin 단백질의 생산

  • Kim, Hee Kyoung (Division of Horticultural Biotechnology, Hankyong National University) ;
  • Go, Ji Yun (Division of Horticultural Biotechnology, Hankyong National University) ;
  • Park, So-Young (Department of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University) ;
  • Kang, Kwon Kyoo (Division of Horticultural Biotechnology, Hankyong National University) ;
  • Jung, Yu Jin (Division of Horticultural Biotechnology, Hankyong National University)
  • 김희경 (한경대학교 생명공학부 원예생명공학전공) ;
  • 고지윤 (한경대학교 생명공학부 원예생명공학전공) ;
  • 박소영 (충북대학교 응용생명공학부 원예과학전공) ;
  • 강권규 (한경대학교 생명공학부 원예생명공학전공) ;
  • 정유진 (한경대학교 생명공학부 원예생명공학전공)
  • Received : 2020.09.21
  • Accepted : 2020.09.23
  • Published : 2020.09.30
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Abstract

To produce the miraculin protein in suspension cultures, rice (Oryza sativa L.) was transformed with Agrobacterium tumefacience EHA105 containing the miraculin AB512278 gene. The cell suspension cultures were established using cell lines selected from transgenic rice callus. The integration of the miraculin gene into the rice chromosome was confirmed using genomic PCR analysis. In addition, RT-PCR analysis indicated that the miraculin gene is expressed in the selected suspension cell lines. Thus, the recombinant miraculin was expressed in the transgenic suspension cell line, HK-2. Therefore, we have successfully developed a HK-2 line that produces miraculin. These results demonstrate that transformed cell suspension cultures can be used to produce a taste-modifying protein such as miraculin.

벼(Oryza sativa L.)에서 세포 현탁 배양을 통한 miraculin 단백질의 생산을 위해 miraculin 유전자(AB512278)가 도입된 Agrobacterium tumefacience EHA105를 매개로 벼 캘러스에 형질전환하였다. 현탁배양세포주는 형질전환 캘러스를 이용하여 몇번의 선발과정 및 계대배양을 통해 선발하였고, 게놈 PCR 분석을 통해 miraculin 유전자가 벼 염색체에 안정적으로 도입된 것을 확인하였다. 또한, RT-PCR 분석을 통해 형질전환 세포주에서 도입된 miraculin 유전자가 과발현 되었다. 재조합 miraculin은 형질전환 현탁배양 HK-2 세포주에서 가장 높게 발현되어 total soluble protein (TSP) 대비 2.0%를 보였다. 이러한 결과는 형질전환 현탁세포배양이 miraculin과 같은 미각 수식 단백질의 대량생산 시스템을 구축하는데 이용 가능 할 것으로 사료된다.

Keywords

References

  1. Al Bachchu MA, Jin SB, Park JW, Boo KH, Sun HJ, Kim YW, Lee HY, Riu KZ, Kim JH (2011) Functional expression of Miraculin, a taste-modifying protein, in transgenic Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.). J Korean Soc Appl Biol Chem 54:24-29
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Brouwer JN, Van Der Wel H, Francke A, Henning GJ (1968) Miraculin, the sweetness-inducing protein from miracle fruit. Nature 220:373-374 https://doi.org/10.1038/220373a0
  4. Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219-226 https://doi.org/10.1016/S1360-1385(01)01922-7
  5. Hood EE, Jilka JM (1999) Plant-based production of xenogenic proteins. Curr Opin Biotechnol 10:382-386 https://doi.org/10.1016/S0958-1669(99)80069-X
  6. Hood EE, Woodard SL, Horn ME (2002) Monoclonal antibody manufacturing in transgenic plants-myths and realities. Curr Opin Biotechnol 13:630-635 https://doi.org/10.1016/S0958-1669(02)00351-8
  7. Horn ME, Woodard SL, Howard JA (2004) Plant molecular farming: systems and products. Plant Cell Rep 22:711-720 https://doi.org/10.1007/s00299-004-0767-1
  8. Ito K, Asakura T, Morita Y, Nakajima KI, Koizumi A, Shimizu-Ibuka A, Masuda K, Ishiguro M, Terada T, Maruyama J, Kitamoto K, Misaka T, Abe K (2007) Microbial production of sensory-active miraculin. Biochem Bioph Res Co 360:407-411 https://doi.org/10.1016/j.bbrc.2007.06.064
  9. Jung YJ., Bae S, Lee GJ, Seo PJ, Cho YG, Kang KK (2017) A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system. J Plant Biotechnol 44:89-96 https://doi.org/10.5010/JPB.2017.44.1.089
  10. Jung YJ, Kang KK (2018) Stable expression and characterization of brazzein, thaumatin and miraculin genes related to sweet protein in transgenic lettuce. J Plant Biotechnol 45:257-265 https://doi.org/10.5010/JPB.2018.45.3.257
  11. Jung YJ, Nou IS, Kang KK (2014) Overexpression of Oshsp16.9 gene encoding small heat shock protein enhances tolerance to abiotic stresses in rice. Plant Breed Biotech 2:370-379 https://doi.org/10.9787/PBB.2014.2.4.370
  12. Kajiura H, Hiwasa-Tanase K, Ezura H, Fujiyama K (2018) Comparison of the N-glycosylation on recombinant miraculin expressed in tomato plants with native miraculin. Plant Biotechnol 18:1023-1027
  13. Kaman-Toth E, Pogany M, Danko T, Szatmari A, Bozso Z (2018) A simplified and efficient Agrobacterium tumefaciens electroporation method. 3 Biotech 8:148-153 https://doi.org/10.1007/s13205-018-1171-9
  14. Kant R (2005). Sweet proteins-potential replacement for artificial low calorie sweeteners. Nutr J 4:5-10 https://doi.org/10.1186/1475-2891-4-5
  15. Kim KY, Kwon SY, Lee HS, Hur Y, Bang JW, Kwak SS (2003) A novel oxidative stress-inducible peroxidase promoter from sweetpotato: molecular cloning and characterization in transgenic tobacco plants and cultured cells. Plant Mol Biol 51:831-838 https://doi.org/10.1023/A:1023045218815
  16. Kurihara Y (1992) Characteristics of antisweet substances, sweet proteins, and sweetness-inducing proteins. Crit Rev Food Sci Nutr 32:231-252 https://doi.org/10.1080/10408399209527598
  17. Kurihara Y (1997) Structures and activities of sweetness-inducing substances (miraculin, curculin, strogin) and the heat-stable sweet protein, mabinlin. FFI J Jpn 174:67-74
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 https://doi.org/10.1038/227680a0
  19. Lee YR, Akter S, Lee IH, Jung YJ, Park SY, Cho YG, Kang KK, Jung YJ (2018) Stable expression of brazzein protein, a new type of alternative sweetener in transgenic rice. J Plant Biotechnol 45:63-70 https://doi.org/10.5010/JPB.2018.45.1.063
  20. Ming D and Hellekant G (1994) Brazzein, a new high‐potency thermostable sweet protein from Pentadiplandra brazzeana B. FEBS lett 355:106-108 https://doi.org/10.1016/0014-5793(94)01184-2
  21. Park YJ, Han JE, Lee H, Jung YJ, Murthy HN, Park SY (2020) Large-scale production of recombinant miraculin protein in transgenic carrot callus supension cultures using air-lift bioreactors. Amb expr 10:140-146 https://doi.org/10.1186/s13568-020-01079-3
  22. Ruiz-Canada C, Kelleher DJ, Gilmore R (2009) Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms. Cell 136:272-283 https://doi.org/10.1016/j.cell.2008.11.047
  23. Shirasuka Y, Nakajima KI, Asakura T, Yamashita H, Yamamoto A, Hata S, Nagata S, Abo M, Sorimachi H, Abe K (2004) Neoculin as a new taste-modifying protein occurring in the fruit of Curculigo latifolia. Biosci Biotechnol Biochem 68:1403-1407 https://doi.org/10.1271/bbb.68.1403
  24. Shrimal S, Cherepanova NA, Gilmore R (2015) Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum. Sem Cell Dev Biol 41:71-78 https://doi.org/10.1016/j.semcdb.2014.11.005
  25. Sun HJ, Cui ML, Ma B, Ezura H (2006) Functional expression of the taste‐modifying protein, miraculin, in transgenic lettuce. FEBS lett 580:620-626 https://doi.org/10.1016/j.febslet.2005.12.080
  26. Sun HJ, Kataoka H, Yano M, Ezura H (2007) Genetically stable expression of functional miraculin, a new type of alternative sweetener, in transgenic tomato plants. J Plant Biotechnol 5:768-777 https://doi.org/10.1111/j.1467-7652.2007.00283.x
  27. Theerasilp S, Hitotsuya H, Nakajo S, Nakaya K, Nakamura Y, Kurihara Y (1989) Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin. J Biol Chem 264:6655-6659 https://doi.org/10.1016/S0021-9258(18)83477-9
  28. Theerasilp S, Kurihara Y (1988) Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem 263:11536-11539 https://doi.org/10.1016/S0021-9258(18)37991-2
  29. Yoshida K, Shinmyo A (2000) Transgene expression systems in plant, a natural bioreactor. J Biosci Bioeng 90:353-362 https://doi.org/10.1016/S1389-1723(01)80001-3