반복단위 단백질 고분자의 유전공학적 합성 및 응용

Genetic Synthesis and Applications of Repetitive Protein Polymers

  • 박미성 (서울대학교 화학생물공학부) ;
  • 최차용 (서울대학교 화학생물공학부) ;
  • 원종인 (홍익대학교 화학공학과)
  • Park, Mi-Sung (School of Chemical and Biological Engineering, Seoul National University) ;
  • Choi, Cha-Yong (School of Chemical and Biological Engineering, Seoul National University) ;
  • Won, Jong-In (Department of Chemical Engineering, Hongik University)
  • 발행 : 2007.08.30

초록

본 연구는 특정 아미노산들로 구성된 단위체가 반복되는 형태를 가지는 반복단위 단백질을 유전공학적으로 합성하는 방법들과 응용사례들을 소개하고 있다. 유전공학적 합성법은 단위체의 반복횟수를 정확하게 제어하면서 인식부위의 제한을 없애서 원하는 단백질만을 발현할 수 있도록 발전해왔으며, 최근 소개된 RDL과 CCM 방법에 의하여 가능해졌다. 반복단위 단백질의 응용사례로는 대표적으로 ELP, SLP, Prolamin 등의 단백질을 합성하여 생체재료나 약물전달시스템을 개발하는데 응용하거나, ELFSE의 drag-tag 개발에 응용되는 연구들이 진행되고 있다. 화학적으로 합성된 고분자에 비해 유전공학적으로 합성된 반복단위 고분자의 경우, 고유의 물리적 성질과 함께 환경에 미치는 유해함이 상대적으로 적다는 점 때문에 미래의 신소재로 기대되고 있다.

This study introduces the characteristics and some applications of repetitive polypeptides, especially to the biomaterial, tissue engineering scaffolds, drug delivery system, and DNA separation systems. Since some fibrous proteins, which consist of repeating peptide monomers, have been reported that their physical properties are changed dramatically by means of temperature alteration or pH shifting. For that reason, fibrous protein-mimetic polypeptides, which are produced by the recombinant technology, can be applied to the diverse biological fields. Repetitive polypeptides can also be used in the bioseparation area such as DNA sequencing, because they make DNA separation possible in free-solution electrophoresis by conjugating DNA fragments to them. Moreover, artificial synthesis of repetitive polypeptides helps to demonstrate the correlations between mechanical properties and structures of natural protein polymer, which have been proven that repetitive domains are affected by the sequence of the repeating domains and the number of repeating subunits. Repetitive polypeptides can be biologically synthesized using some special cloning methods, which are represented here. Recursive directional ligation (RDL) and controlled cloning method (CCM) have been proposed as excellent cloning methods in that we can control the number of repetition in the multimerization of polypeptides and the components of repetitive polypeptides by either method.

키워드

참고문헌

  1. Gragory, H. A., D. Frank, J. Caroline, C. Tara, H. L. Rebecca, C. Jingsong, L. Helen, R. John, and K. L. David (2003), Silk-based biomaterials, Biomaterials 24, 401-416 https://doi.org/10.1016/S0142-9612(02)00353-8
  2. Cappello, J. (1990), The biological production of protein polymers and their use, Trends in Biotechnology 8, 309-911 https://doi.org/10.1016/0167-7799(90)90207-E
  3. McGrath, K. P., M. J. Fournier, T. L. Mason, and D. A. Tirrell (1992), Genetically directed syntheses of new polymeric materials. Expression of artificial genes encoding proteins with repeating -$(AlaGly)_3$ProGluGly - elements, J. Am. Chem. Soc. 114, 727-733 https://doi.org/10.1021/ja00028a048
  4. Prince, J. T., K. P. McGrath, C. M. DiGirolamo, and D. L. Kaplan (1995), Construction, cloning, and expression of synthetic genes encoding spider dragline silk, Biochemistry 94, 10879-10885
  5. McMillan, R. A., T. A. T. Lee, and V. P. Conticello (1999), Rapid assembly of synthetic genes encoding protein polymers, Macromolecules 32, 3643-3648 https://doi.org/10.1021/ma981660f
  6. Meyer, D. E. and A. Chilkoti (2002), Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system, Biomacromolecules 3, 357-367 https://doi.org/10.1021/bm015630n
  7. Won, J. I. and A. E. Barron (2002), A new cloning method for the preparation of long repetitive polypeptides without a sequence requirement, Macromolecules 35, 8281-8287 https://doi.org/10.1021/ma020892i
  8. Wang, Xianyan, H. J. Kim, J. Wong, C. Vepari, A. Matsumoto, and D. L. Kaplan (2006), Fibrous proteins and tissue engineering, Materials today 12, 44-53
  9. Won, J. I., R. J. Meagher, and A. E. Barron (2005), Protein polymer drag-tags for DNA separations by end-labeled free-solution electrophoresis, Electrophoresis 26, 2138-2148 https://doi.org/10.1002/elps.200410042
  10. Meagher, R. J., J. A. Coyne, C. N. Hestekin, T. N. Chiesl, R. D. Haynes, J. I. Won, and A. E. Barron (2007), Multiplexed p53 mutation detection by free-solution conjugate microchannel electrophoresis with polyamide drag-tags, Anal. Chem. 79, 1848-1854 https://doi.org/10.1021/ac061903z
  11. Won, J. L., R. J. Meagher, and A. E. Barron (2004), Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis, Biomacromolecules 5, 618-627 https://doi.org/10.1021/bm034442p
  12. Topilina, N. I., S. Higashiya, N. Rana, V. V. Ermolenkov, C. Kossow, A. Carlsen, S. C. Ngo, C. C. Wells, E. T. Eisenbraun, K. A Dunn, I. K. Lednev, R. E. Geer, A. E. Kaloyeros, and J. T. Welch (2006), Bilayer fibril formation by genetically engineered polypeptides: preparation and characterization, Biomacromolecules 7, 1104-1111 https://doi.org/10.1021/bm0509016
  13. Hamada, D., I. Yanagihara, and K. Tsumoto (2004), Engineering amyloidogenicity towards the development of nanofibrillar materials, Trends in biotechnology 22, 93-97 https://doi.org/10.1016/j.tibtech.2003.12.003
  14. Patacchini, C., S. Masci, R. D'Ovidio, and D. Lafiandra (2003), Heterologous expression and purification of native and mutated low molecular mass glutenin subunits from durum wheat, Journal of chromatography B-analytical technologies in the biomedical and life science 786, 215-220 https://doi.org/10.1016/S1570-0232(02)00734-1
  15. Meyer, D. E. and A. Chilkoti (1999), Purification of recombinant proteins by fusion with thermally-responsive polypeptides, Nature Biotechnology 17, 1112-1115 https://doi.org/10.1038/15100
  16. Trabbic-Carlson, K., L.. Liu, B. J. Kim, and A. Chilkoti (2004), Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptides fusion with an oligohistidine fusion, Protein Science, 13, 3274-3284 https://doi.org/10.1110/ps.04931604
  17. Meyer, D. E., K. Trabbic-Carlson, and A. Chilkoti (2001), Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin, Biotechnology Progress 17, 720-728 https://doi.org/10.1021/bp010049o
  18. Shimazu, M., A. Mulchandani, and W. Chen (2003), Thermally triggered purification and immobilization of elastin-OPH fusions, Biotehnol. Bioeng. 81, 74-79 https://doi.org/10.1002/bit.10446
  19. Kim, J. Y., S. O'Malley, A. Mulchandani, and W. Chen (2005), Genetically engineered elastin-protein A fusion as a universal platform for homogeneous phase-separation immunoassay, Anal. Chem. 77, 2318-2322 https://doi.org/10.1021/ac0484326
  20. Huang, J., C. Wong, A. George, and D. L. Kaplan (2007), The effect of genetically engineered spider silk-dentin matrix: protein 1 chimeric protein on hydroxyapatite nucleation, Biomaterials 28, 2358-2367 https://doi.org/10.1016/j.biomaterials.2006.11.021
  21. Szela, S., P. Avtqes, R. Valluzzi, S. Winkler, D. Wilson, D. Kirschner, and D. L. Kaplan (2000), Reduction-oxidation control of beta-sheet assembly in genetically engineered silk, Biomacromolecules 1, 534-542 https://doi.org/10.1021/bm0055697
  22. Oroudjev, E, J. Soares, S. Arcdiacono, J. B. Thompson, S. A. Fossey, and H. G. Hansma (2002) Proc. Natl. Acad Sci. USA 99, 6460-6465
  23. Valluzzi, R., S. Szela, P. Avtges, D. Kirschner, and D. Kaplan (1999), Methionine redox controlled crystallization of biosynthetic silk spidroin, J. Phys. Chem. B 103, 11382-11392 https://doi.org/10.1021/jp991363s
  24. Lazaris, A., S. Arcidiacono, Y. Huang, J. F. Zhou, F. Duguay, N. Chretien, E. A. Welsh, J. W. Soares, and C. N. Karatzas (2002), Spider silk fibers spun from soluble recombinant silk produced in mammalian cells, Science 295, 472-476 https://doi.org/10.1126/science.1065780
  25. Nagarsekar, A., J. Crissman, M. Crissman, F. Ferrari, J. Cappello, and H. Ghandehari (2003), Genetic engineering of stimuli-sensitive silkelastin-like protein block copolymers, Biomacromolecules 4, 602-607 https://doi.org/10.1021/bm0201082
  26. Mayer, P., G. W. Slater, and G. Drouin (1994), Theory of DNA sequencing using free-solution electrophoresis of protein-DNA complexes, Anal. Chem. 66, 1777-1780 https://doi.org/10.1021/ac00082a029
  27. Sourice, S., ANisole, J. Gueguen, Y. Papineau, and K. Elmorjani (2003), High microbial production and characterization of strictly periodic polymers modelled on the repetitive domain of wheat gliadins, Biochemical and Biophysical Research Communications 312, 989-996 https://doi.org/10.1016/j.bbrc.2003.11.028
  28. Mangavel, C., J. Barbot, Y. Papineau, and J. Gueguen (2001), Evolution of wheat gliadins conformation during film formation: a Fourier transform infrared study, J. Agric. Food Chem. 49, 867-872 https://doi.org/10.1021/jf0009899
  29. Sanchez, A. C., Y. Papineau, C. Mangavel, C. Larre, and J. Gueguen (1998), Effect of different plasticizers on the mechanical and surface properties of wheat gliadins films, J. Agric. Food Chem. 46, 4539-4544 https://doi.org/10.1021/jf980375s
  30. Ezpeleta, I., J. M. Irache, J. S. Stainmesse, C. Chabenat, J. Gueguen, Y. Papineau, and A. M. Orecchioni (1996), Gliadin nanoparticles for the controlled release of all-trans-retinoic acid, Int. J. Pharm. 131, 191-200 https://doi.org/10.1016/0378-5173(95)04338-1
  31. Herrero-Vanrell, R., A.C. Rincon, M. Alonso, V. Reboto, J. T. Molina-Martinez, and J. C. Rodriguez-Cabello (2005), Self-assembled particles of an elastin-like polymer as vehicles for controlled drug release, Journal of Controlled Release 102, 113-122 https://doi.org/10.1016/j.jconrel.2004.10.001