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Expression of bovine lactoferrin N-lobe by the green alga, Chlorella vulgaris

  • Received : 2013.09.10
  • Accepted : 2013.11.28
  • Published : 2013.12.15

Abstract

The purpose of this study was to express bovine lactoferrin N-lobe in Chlorella vulgaris, a green microalga, using the pCAMBIA1304 vector. Chlorella-codon-optimized bovine lactoferrin N-lobe (Lfb-N gene) was cloned in the expression vector pCAMBIA1304, creating the plasmid pCAMLfb-N. pCAMLfb-N was then introduced into C. vulgaris by electro-transformation. Transformants were separated from BG-11 plates containing 20 ${\mu}g\;mL^{-1}$ hygromycin. Polymerase chain reaction was used to screen transformants harboring Lfb-N gene. Finally, total soluble protein was extracted from the transformants, and the expression of Lfb-N protein was detected using western blotting. Using this method, we successfully expressed bovine lactoferrin in C. vulgaris. Therefore, our results suggested that recombinant lactoferrin N-lobe, which has many uses in the biomedical and pharmaceutical industries, can be produced economically.

Keywords

References

  1. Aisen, P. & Listowsky, I. 1980. Iron transport and storage proteins. Annu. Rev. Biochem. 49:357-393. https://doi.org/10.1146/annurev.bi.49.070180.002041
  2. Akhond, M. A. Y. & Machray, G. C. 2009. Biotech crops:technologies, achievements and prospects. Euphytica 166:47-59. https://doi.org/10.1007/s10681-008-9823-1
  3. Azencott, H. R., Peter, G. F. & Prausnitz, M. R. 2007. Influence of the cell wall on intracellular delivery to algal cells by electroporation and sonication. Ultrasound Med. Biol. 33:1805-1817. https://doi.org/10.1016/j.ultrasmedbio.2007.05.008
  4. Babinger, P., Kobl, I., Mages, W. & Schmitt, R. 2001. A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri. Nucleic Acids Res. 29:1261-1271. https://doi.org/10.1093/nar/29.6.1261
  5. Boynton, J. E. & Gillham, N. W. 1993. Chloroplast transformation in Chlamydomonas. Methods Enzymol. 217:510-536. https://doi.org/10.1016/0076-6879(93)17087-L
  6. Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. 1988. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534-1538. https://doi.org/10.1126/science.2897716
  7. Bradford, M. M. 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
  8. Brock, J. H. 1985. Metal proteins with non-redox roles (Part II). Topics in molecular and structural biology. In Harrison, P. (Ed.) Metalloproteins. Macmillan Press, Lodon, 183-262.
  9. Brown, L. E., Sprecher, S. L., & Keller, L. R. 1991. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol. Cell Biol. 11:2328-2332. https://doi.org/10.1128/MCB.11.4.2328
  10. Cannons, A. C. & Shiflett, S. D. 2001. Transcriptional regulation of the nitrate reductase gene in Chlorella vulgaris: indentification of regulatory elements controlling expression. Curr. Genet. 40:128-135. https://doi.org/10.1007/s002940100232
  11. Cerutti, H., Johnson, A. M., Gillham, N. W. & Boynton, J. E. 1997. Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9:925-945. https://doi.org/10.1105/tpc.9.6.925
  12. Chen, H. L., Li, S. S., Huang, R. & Tsai, H. -J. 2008. Conditional production of a functional fish growth hormone in the transgenic line of Nannochloropsis oculata (Eustigmatophyceae). J. Phycol. 44:768-776. https://doi.org/10.1111/j.1529-8817.2008.00508.x
  13. Choi, S. P. & Sim, S. J. 2009. Production of $CO_2$ by biological process using microalgae. KIC News 12:1-11.
  14. Chong, D. K. X. & Langridge, W. H. R. 2000. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res. 9:71-78. https://doi.org/10.1023/A:1008977630179
  15. Chow, K. -C. & Tung, W. L. 1999. Electrotransformation of Chlorella vulgaris. Plant Cell Rep. 18:778-780. https://doi.org/10.1007/s002990050660
  16. Dawson, H. N., Burlingame, R. & Cannons, A. C. 1997. Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene. Curr. Microbiol. 35:356-362. https://doi.org/10.1007/s002849900268
  17. Dreesen, I. A. J., Charpin-El Hamri, G. & Fussenegger, M. 2010. Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J. Biotechnol. 145:273-280. https://doi.org/10.1016/j.jbiotec.2009.12.006
  18. Ebrahimi, N., Memari, H. R., Ebrahimi, M. A. & Ardakani, M. R. 2012. Cloning, transformation and expression of human gamma interferon gene in tomato (Lycopersicon esculentum Mill.). Biotechnol. Biotechnol. Equip. 26:2925-2929. https://doi.org/10.5504/bbeq.2012.0004
  19. El-Sheekh, M. M. 2000. Stable chloroplast transformation in Chlamydomonas reinhardtii using microprojectile bombardment. Folia Microbiol. 45:496-504. https://doi.org/10.1007/BF02818717
  20. Feng, S., Xue, L., Liu, H. & Lu, P. 2009. Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Mol. Biol. Rep. 36:1433-1439. https://doi.org/10.1007/s11033-008-9333-1
  21. Franklin, S., Ngo, B., Efuet, E. & Mayfield, S. P. 2002. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J. 30:733-744. https://doi.org/10.1046/j.1365-313X.2002.01319.x
  22. Fuhrmann, M., Oertel, W. & Hegemann, P. 1999. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 19:353-361. https://doi.org/10.1046/j.1365-313X.1999.00526.x
  23. Geng, D. -G., Han, Y., Wang, Y. -Q., Wang, P., Zhang, L. -M., Li, W. -B. & Sun, Y. -R. 2004. Construction of a system for the stable expression of foreign genes in Dunaliella salina. Acta. Bot. Sin. 46:342-346.
  24. Heitzer, M., Eckert, A., Fuhrmann, M. & Griesbeck, C. 2007. Influence of codon bias on the expression of foreign genes in microalgae. Adv. Exp. Med. Biol. 616:46-53. https://doi.org/10.1007/978-0-387-75532-8_5
  25. Kindle, K. L. 1990. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U. S. A. 87:1228-1232. https://doi.org/10.1073/pnas.87.3.1228
  26. Kindle, K. L., Richards, K. L. & Stern, D. B. 1991. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U. S. A. 88:1721-1725. https://doi.org/10.1073/pnas.88.5.1721
  27. Kovar, J. L., Zhang, J., Funke, R. P. & Weeks, D. P. 2002. Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. Plant J. 29:109-117. https://doi.org/10.1046/j.1365-313x.2002.01193.x
  28. Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J. & Rajam, M. V. 2004. Genetic transformation of the green alga-Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 166:731-738. https://doi.org/10.1016/j.plantsci.2003.11.012
  29. Ladygin, V. G. 2003. The transformation of the unicellular alga Chlamydomonas reinhardtii by electroporation. Microbiology 72:585-591. https://doi.org/10.1023/A:1026051402366
  30. Ladygin, V. G. 2004. Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation. Process Biochem. 39:1685-1691. https://doi.org/10.1016/j.procbio.2003.07.001
  31. Li, Y., Geng, Y., Song, H., Zheng, G., Huan, L. & Qiu, B. 2004. Expression of a human lactoferrin N-lobe in Nicotiana benthmiana with potato virus X-based agroinfection. Biotechnol. Lett. 26:953-957. https://doi.org/10.1023/B:BILE.0000030038.27358.20
  32. Lorimer, D., Raymond, A., Walchli, J., Mixon, M., Barrow, A., Wallace, E., Grice, R., Burgin, A. & Stewart, L. 2009. Gene composer: database software for protein construct design, codon engineering, and gene synthesis. BMC Biotechnol. 9:36. https://doi.org/10.1186/1472-6750-9-36
  33. Mayfield, S. P. & Kindle, K. L. 1990. Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker. Proc. Natl. Acad. Sci. U. S. A. 87:2087-2091. https://doi.org/10.1073/pnas.87.6.2087
  34. Metz-Boutigue, M. -H., Jolles, J., Mazurier, J., Schoentgen, F., Legrand, D., Spik, G., Montreuil, J. & Jolles, P. 1984. Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. Eur. J. Biochem. 145:659-676. https://doi.org/10.1111/j.1432-1033.1984.tb08607.x
  35. Nakamura, Y., Gojobori, T. & Ikemura, T. 2000. Codon usage tabulated from international DNA sequence databases: status of the year 2000. Nucleic Acids Res. 28:292. https://doi.org/10.2216/06-83.1
  36. Picerce, A., Colavizza, D., Benaissa, M., Maes, P., Tartar, A., Montreuil, J. & Spik, G. 1991. Molecular cloning and seppquence analysis of bovine lactotransferrin. Eur. J. Biochem. 196:177-184. https://doi.org/10.1007/s00239-002-2313-5
  37. Puigbo, P., Bravo, I. G. & Garcia-Vallve, S. 2008a. CAI-cal: a combined set of tools to assess codon usage adaptation. Biol. Direct 3:38. https://doi.org/10.1186/1745-6150-3-38
  38. Puigbo, P., Bravo, I. G. & Garcia-Vallve, S. 2008b. E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI). BMC Bioinformatics 9:65. https://doi.org/10.1186/1471-2105-9-65
  39. Puigbo, P., Guzman, E., Romeu, A. & Garcia-Vallve, S. 2007. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res. 35S:W126-W131.
  40. Rose, T. M., Plowman, G. D., Teplow, D. B., Dreyer, W. J., Hellstrom, K. E. & Brown, J. P. 1986. Primary structure of the human melanoma-associated antigen p97 (melanotransferrin) deduced from the mRNA sequence. Proc. Natl. Acad. Sci. U. S. A. 83:1261-1265. https://doi.org/10.1073/pnas.83.5.1261
  41. Salmon, V., Legrand, M., Slominny, M. -C., El Yazide, I., Spick, G., Gruber, V., Bournat, P., Olagnier, D., Mison, M., Theisen, B. & Merot, B. 1998. Production of human lactoferrin in transgenic tobacco plants. Protein Expr. Purif. 13:127-135. https://doi.org/10.1006/prep.1998.0886
  42. Schroda, M. 2006. RNA silencing in Chlamydomonas: mechanisms and tools. Curr. Genet. 49:69-84. https://doi.org/10.1007/s00294-005-0042-1
  43. Shimogawara, K., Fujiwara, S., Grossman, A. & Usuda, H. 1998. High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148:1821-1828.
  44. Sun, G., Zhang, X., Sui, Z. & Mao, Y. 2008. Inhibition of pds gene expression via the RNA interference approach in Dunaliella salina (Chlorophyta). Mar. Biotechnol. 10:219-226. https://doi.org/10.1007/s10126-007-9056-7
  45. Sun, Y., Gao, X., Li, Q., Zhang, Q. & Xu, Z. 2006. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene. Gene 377:140-149. https://doi.org/10.1016/j.gene.2006.03.018
  46. Sun, Y., Yang, Z., Gao, X., Li, Q., Zhang, Q. & Xu, Z. 2005. Ex-pression of foreign genes in Dunaliella by electroporation. Mol. Biotechnol. 30:185-192. https://doi.org/10.1385/MB:30:3:185
  47. Takase, K., Hagiwara, K., Onodera, H., Nishizawa, Y., Ugaki, M., Omura, T., Numata, S., Akutsu, K., Kumura, H. & Shimazaki, K. 2005. Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance. Biochem. Cell Biol. 83:239-249. https://doi.org/10.1139/o05-022
  48. Tang, D. K. H., Qiao, S. Y. & Wu, M. 1995. Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem. Mol. Biol. Int. 36:1025-1035.
  49. Tran, M., Zhou, B. & Pettersson, P. L., Gonzalez, M. J. & Mayfield, S. P. 2009. Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol. Bioeng. 104:663-673.
  50. Xia, X. 2007. An improved implementation of codon adaptation index. Evol. Bioinform. Online 3:53-58.
  51. Walker, T. L., Becker, D. K., Dale, J. L. & Collet, C. 2005. Towards the development of a nuclear transformation system for Dunaliella tertiolecta. J. Appl. Phycol. 17:363-368. https://doi.org/10.1007/s10811-005-4783-5
  52. Wang, C., Wang, Y., Su, Q. & Gao, X. 2007a. Transient expression of the GUS gene in a unicellular marine green alga, Chlorella sp. MACC/C95, via electroporation. Biotechnol. Bioprocess Eng. 12:180-183. https://doi.org/10.1007/BF03028646
  53. Wang, T., Xue, L., Hou, W., Yang, B., Chai, Y., Ji, X. & Wang, Y. 2007b. Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions. Appl. Microbiol. Biotechnol. 76:651-657. https://doi.org/10.1007/s00253-007-1040-7
  54. Weickert, M. J., Doherty, D. H., Best, E. A. & Olins, P. O. 1996. Optimization of heterologous protein production in Escherichia coli. Curr. Opin. Biotechnol. 7:494-499. https://doi.org/10.1016/S0958-1669(96)80051-6
  55. Yang, Z., Li, Y., Chen, F., Li, D., Zhang, Z., Liu, Y., Zheng, D., Wang, Y. & Shen, G. 2006. Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast. Chin. Sci. Bull. 51:1703-1709. https://doi.org/10.1007/s11434-006-2041-0

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