Hostname: page-component-78c5997874-t5tsf Total loading time: 0 Render date: 2024-11-08T06:24:21.476Z Has data issue: false hasContentIssue false
  • English
  • Français

Expression and Self Assembly of Cowpea Chlorotic Mottle Virus Capsid Proteins in Pichia pastoris and Encapsulation of Fluorescent Myoglobin

Published online by Cambridge University Press:  28 January 2011

Yuanzheng Wu ,
Hetong Yang  and
Hyun-Jae Shin
Yuanzheng Wu
Affiliation:
Department of Chemical and Biochemical Engineering, Chosun University, #375 Seosuk-dong, Dong-gu, Gwangju, 501-759, Republic of Korea Biotechnology Center of Shandong Academy of Sciences, No. 19 Keyuan Road, Jinan, 250014, China, P. R.
Hetong Yang
Affiliation:
Biotechnology Center of Shandong Academy of Sciences, No. 19 Keyuan Road, Jinan, 250014, China, P. R.
Hyun-Jae Shin
Affiliation:
Department of Chemical and Biochemical Engineering, Chosun University, #375 Seosuk-dong, Dong-gu, Gwangju, 501-759, Republic of Korea
Get access

Abstract:

Cowpea chlorotic mottle virus (CCMV) has been a model system for virus studies for over 40 years and now is considered to be a perfect candidate as nanoplatform for applications in materials science and medicine. The ability of CCMV to self assemble in vitro into virus-like particles (VLPs) or capsids makes an ideal reaction vessel for nanomaterial synthesis and entrapment. Here we report expression of codon optimized CCMV coat protein in Pichia pastoris and production of self assembled CCMV VLPs by large-scale fermentation. CCMV coat protein gene (573 bp) was synthesized according to codon preference of P. pastoris and cloned into pPICZA vector. The recombinant plasmid pPICZA-CP was transformed into P. pastoris GS115 by electroporation. The resulting yeast colonies were screened by PCR and analyzed for protein expression by SDS-PAGE. After large-scale fermentation CCMV coat protein yields reached 4.8 g L−1. The CCMV VLPs were purified by modified PEG precipitation followed by cesium chloride density gradient ultracentrifugation, and then analyzed by size exclusion fast performance liquid chromatography (FPLC), UV spectrometry and transmission electron microscopy. Myoglobin was used as a model protein to be encapsulated in CCMV VLPs. The fluorescence spectroscopy showed that inclusion of myoglobin had occurred. The results indicated the production of CCMV capsids by P. pastoris fermentation now available for utilization in pharmacology or nanotechnology fields.

Keywords

biological synthesis (assembly)nanostructuretransmission electron microscopy (TEM)
Type
Articles
Information
MRS Online Proceedings Library (OPL) , Volume 1317: Symposium RR – Interdisciplinary Approaches to Safe Nanotechnologies , 2011 , mrsf10-1317-rr03-05
Copyright
Copyright © Materials Research Society 2011

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

References:

1.Douglas, T. and Young, M., Science, 312: 872–875 (2006).CrossRefGoogle Scholar
2.Koudelka, K. J. and Manchester, M., Curr Opin Chem Biol, 14: 1–8 (2010).CrossRefGoogle Scholar
3.Zlotnick, A. and Stray, S. J., Trends Biotechnol, 21: 536–542 (2003).CrossRefGoogle Scholar
4.Wang, Q., Kaltgrad, E., Lin, T., Johnson, J. E. and Finn, M. G., Chem Biol, 9: 805–811 (2002).CrossRefGoogle Scholar
5.Carette, N., Engelkamp, H., Akpa, E., Pierre, S. J., Cameron, N. R., Christianen, P. C., Maan, J. C., Thies, J. C., Weberskirch, R., Rowan, A. E., Nolte, R. J., Michon, T. and Van Hest, J. C., Nat Nanotechnol, 2: 226–229 (2007).CrossRefGoogle Scholar
6.Loo, L., Guenther, R. H., Basnayake, V. R., Lommel, S. A. and Franzen, S., J Am Chem Soc, 128: 4502–4503 (2006).CrossRefGoogle Scholar
7.Minten, I. J., Hendriks, L. J., Nolte, R. J. and Cornelissen, J. J., J Am Chem Soc, 131: 17771–17773 (2009).CrossRefGoogle Scholar
8.Bancroft, J., Hills, G. and Markham, R., Virology, 31: 354–379 (1967).CrossRefGoogle Scholar
9.Speir, J. A., Munshi, S., Wang, G., Baker, T. S. and Johnson, J. E., Structure, 3: 63–78 (1995).CrossRefGoogle Scholar
10.Lucas, R. W., Larson, S. B. and McPherson, A., J Mol Biol, 317: 95–108 (2002).CrossRefGoogle Scholar
11.Tama, F. and Brooks, C. L. 3rd, J Mol Biol, 318: 733–747 (2002).CrossRefGoogle Scholar
12.Johnson, J. E. and Speir, J. A., J Mol Biol, 269: 665–675 (1997).CrossRefGoogle Scholar
13.Douglas, T. and Young, M., Nature, 393: 152–155 (1998).CrossRefGoogle Scholar
14.Gillitzer, E., Willits, D., Young, M. and Douglas, T., Chem Commun, 21:2390–2391 (2002).CrossRefGoogle Scholar
15.Ren, Y., Wong, S. M., Lim, L. Y., J Gen Virol, 87: 2749–2754 (2006).CrossRefGoogle Scholar
16.Aniagyei, S. E., Dufort, C., Kao, C. C. and Dragnea, B., J Mater Chem, 18: 3763–3774 (2008).CrossRefGoogle Scholar
17.Minten, I. J., Ma, Y., Hempenius, M. A., Vancso, G. J., Nolte, R. J. and Cornelissen, J. J., Org Biomol Chem, 7: 4685–4688 (2009).CrossRefGoogle Scholar
18.Adolph, D. W., Butler, P. J., J Mol Biol, 109: 345–357 (1977).CrossRefGoogle Scholar
19.Zhao, X., Fox, J. M., Olson, N. H., Baker, T. S. and Young, M., Virology, 207: 486–494 (1995).CrossRefGoogle Scholar
20.Brumfield, S., Willits, D., Tang, L., Johnson, J. E., Douglas, T. and Young, M., J Gen Virol, 85: 1049–1053 (2004).CrossRefGoogle Scholar
21.Phelps, J. P., Dao, P., Jin, H. and Rasochova, L., J Biotechnol, 128: 290–296 (2007).CrossRefGoogle Scholar
22.Allison, R. F., Janda, M. and Ahlquist, P., J Virol, 62: 3581–3588 (1988).CrossRefGoogle Scholar
23.Sreekrishna, K., Brankamp, R. G., Kropp, K. E., Blankenship, D. T., Tsay, J. T., Smith, P. L., Wierschke, J. D., Subramaniam, A. and Birkenberger, L. A., Gene, 190: 55–62 (1997).CrossRefGoogle Scholar
24.Sambrook, J. and Russell, D. W., Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory Press, 2001): 16.33-16.37.Google Scholar
25.Ali, A. and Roossinck, M. J., J Virol Methods, 141: 84–86 (2007).CrossRefGoogle Scholar
26.Comellas-Aragone, M., Engelkamp, H., Claessen, V.I., Sommerdijk, N.A.J.M., Rowan, A.E., Christianen, P.C.M., Maan, J.C., Verduin, B.J.M., Cornelissen, J.J.L.M. and Nolte, R.J.M., Nat Nanotechnol, 10: 635–639 (2007).CrossRefGoogle Scholar
27.Macauley-Patrick, S., Fazenda, M. L., McNeil, B. and Harvey, L. M., Yeast, 22: 249–70 (2005).CrossRefGoogle Scholar
28.Yokoyama, S., Curr Opin Chem Biol, 7: 39–43 (2003).CrossRefGoogle Scholar