Over-expression of Chlamydia psittaci MOMP in Escherichia coli and its purification

대장균에서 Chlamydia psittaci MOMP 유전자의 과발현과 순수분리

  • Accepted : 2006.01.22
  • Published : 2006.03.29

Abstract

Generally known psittacosis or ornithosis is a disease of birds caused by the bacterium Chlamydia psittaci. Humans are accidential hosts and are most commonly infected from avian sources. It raises hepatitis or neurosis. As major outer membrane protein (MOMP) of Chlamydia psittaci has been known to play a role in the avoidance of host immune defenses, research on developing a Chlamydia vaccine has focused on the MOMP. In this study, the gene encoding the major outer membrane protein (MOMP) of the Chlamydia psittaci strain 6BC was cloned and expressed in Escherichia coli strain M-15. The recombinant DNA was cloned by fusion prokaryotic expression vector pQE30-GFPII. Expression of the recombinant protein was performed in E. coli and was induced by IPTG. The size of expressed recombinant protein is 74.220 kDa (MOMP, 43.260 kDa; GFP expression region, 30 kDa; $6{\times}His$ tag, 960Da). This protein was purified by using his-tagging-inclusion body. Recombinant protein was reconfirmed through ELISA test and western blot with antibody against pQE30-GFPII. It will be useful antibody development.

References

  1. 윤병수. Chlamydia 형질전환을 위한 GFP-plasmid vector의 개발. In: 윤병수 (ed.). 분자생물학연구방법론 2. pp. 70-78, 경기대학교 연구지원팀, 1999
  2. Moulder JW. Why is Chlamydia sensitive to penicillin in the absence of peptidoglycan? Infect Agents Dis 1993, 2, 87-99
  3. Verkooyen RP, Willemse D, Hiep-van Casteren SC, Joulandan SA, Snijder RJ, van den Bosch JM, van Helden HP, Peeters MF, Verbrugh HA. Evaluation of PCR, culture, and serology for diagnosis of chlamydia pneumoniae respiratory infections. J Clin Microbiol 1998, 36, 2301-2307
  4. Hatch TP. Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae. J Bacteriol 1996, 178, 1-5 https://doi.org/10.1128/jb.178.1.1-5.1996
  5. Newhall WJ V. Biosynthesis and disulfide crosslinking of outer membrane components during the growth cycle of chlamydia trachomatis. Infect Immu 1987 55, 162-168
  6. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 1992, 111, 229-233 https://doi.org/10.1016/0378-1119(92)90691-H
  7. Vanrompay D, Cox E, Volckaert G, Goddeeris B. Turkeys protected from infection with chlamydia psittaci by plasmid DNA vaccination against the major outer membrane protein. Clin Exp Immunol 1999, 118, 49-55 https://doi.org/10.1046/j.1365-2249.1999.01024.x
  8. Vanrompay D, Mast J, Ducatelle R, Haesebrouck F, Goddeeris B. Chlamydia psittaci in terkeys: pathogenesis of infections in avian serovars A, B and D. Vet Microbiol 1995, 47, 245-256 https://doi.org/10.1016/0378-1135(95)00125-5
  9. Newhall WJ, Jones RB. Disulfied-linked oligomers of the major outer membrane protein of chlamydia. J Bacteriol 1983, 154, 998-1001
  10. Zhang YX, Stewart S, Joseph T, Taylor HR, Caldwell HD. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of chlamydia trachomatis. J Immunol 1987, 138, 575-581
  11. Freidank HM, Vogele H, Eckert K. Evaluation of a new commercial microimmunofluorescence test for detection of antibodies to Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci. Eur J Clin Microbiol Infect Dis 1997, 16, 685-688 https://doi.org/10.1007/BF01708561
  12. Westbay TD, Dascher CC, Hsia RC, Bavoil PM, Zauderer M. Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis. Infect Immu 1994, 62, 5614-5623
  13. Hatch TP, Allan I, Pearce JH. Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of chlamydia spp. J Bacteriol 1984, 157, 13-20
  14. Lambden PR, Everson JS, Ward ME, Clarke IN. Sulfur-rich proteins of Chlamydia trachomatis: developmentally regulated transcription of polycistronic mRNA from tandem promoters. Gene 1990, 87, 105-112 https://doi.org/10.1016/0378-1119(90)90500-Q
  15. Wang S, Grayston JT. Chlamydia trachomatis immunotype J. J Immunol 1975, 115, 1711-1716
  16. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed, Cold Spring Harbor Laboratory Press, New York, 1989
  17. Hatch TP, Miceli M, Sublett JE. Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis. J Bacteriol 1986, 165, 379-385 https://doi.org/10.1128/jb.165.2.379-385.1986
  18. 한상훈, 정규회, Stokes GV, 윤병수. Chlamydia psittaci strain fransis의 plasmid pCpA1과 C. psittaci strain 6BC의 plasmid의 염기서열 상동성 분석. 경기대학교 논문집 1999, 43, 59-69
  19. Everett KD, Hatch TP. Architecture of the cell envelope of Chlamydia psittaci 6BC. J Bacteriol 1995, 177, 877-882 https://doi.org/10.1128/jb.177.4.877-882.1995
  20. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science 1994. 263, 802-805 https://doi.org/10.1126/science.8303295
  21. Colligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W. Current protocols in immunology. John Wiley & Sons, New York, 1992