Navigation Links
Continuous-Elution Electrophoresis for Purification of the Baculovirus-Expressed Coronavirus Structural Proteins, Rev A

K. Sestak, L. Kim, K.-O. Chang and L. J. Saif, Food Animal Health Research Program, Ohio Agricultural Research and Development Program, The Ohio State University, Wooster, OH 44691

Summary
Three major structural proteins of the porcine coronavirus, transmissible gastroenteritis virus (TGEV), membrane (M), nucleocapsid (N), and spike (S), were expressed in a baculovirus expression system. A method for rapid purification of the virus-specific proteins from cellular components was developed by using continuous-elution polyacrylamide gel electrophoresis (CE-PAGE). This approach might be a valuable preparative tool prior to immunization studies, monoclonal antibody production, subunit vaccine preparation, quantification of protein expression and for other experimental scenarios where purified viral proteins are required.


Introduction

TGEV causes diarrhea and vomiting in pigs of all ages and produces 90100% mortality in 12 week old sero-negative pigs (Bohl et al., 1972). The economic impact of TGEV on the U.S. pork industry was estimated as $200 million per year (Saif and Wesley, 1992). A possible vaccine includes the three purified recombinant TGEV structural proteins embedded in immuno-stimulating complexes (ISCOMs), microcapsules or other carriers.


Materials and Methods

The Sf9 insect cells containing the TGEV proteins were harvested from 150 cm2 stationary cell culture flasks (1.8 x 107 cells) and resuspended in 1 ml of phosphate buffer saline (PBS), pH 7.3, mixed with 4 parts of sample buffer (0.06 M Tris, pH 6.8; 10% glycerol; 2% (w/v) SDS; 5% mercaptoethanol and 0.05% (w/v) bromophenol blue), heated at 95 C for 4 min and stored at 20 C until used for preparative electrophoresis.

A Mini Prep Cell (Bio-Rad Laboratories, Hercules, CA) system was used to perform continuous-elution polyacrylamide gel electrophoresis (CE-PAGE) with conditions modified for each TGEV protein to be purified. For the TGEV M-protein, the sample (0.5 ml, corresponding to 18 x 105 cells) was layered onto a 4% (1.5 cm) polyacrylamide stacking gel with a 10% (8 cm) polyacrylamide separating gel. Proteins were electrophoresed under denaturing conditions at constant voltage (200 V) for 3.5 h with a flow rate of 0.05 ml/min. After 3.5 h, fractions were collected in 5 min intervals (0.25 ml/fraction) with a total of 30 fractions. For the TGEV N-protein electrophoresis was performed as described above, but for 4.5 h prior to collection of 30 fractions. For the TGEV S-protein; the sample (0.5 ml, corresponding to 18 x 105 cells) was layered onto a 4% (1.5 cm) polyacrylamide stacking gel with a 6% (8 cm) polyacrylamide separating gel in the Mini Prep Cell gel tube assembly. Electrophoresis was performed as described above for 8.5 h prior to collection of the 30 fractions.

Selected protein fractions were examined by electrophoresis in analytical polyacrylamide slab gels and stained with Coomassie blue (Wu and Welsh, 1996). The fractions of desired molecular weight were pooled and concentrated using Centricon devices (Amicon, Beverly, MA) with a 10 kDa cutoff for the M-protein, and a 30 kDa cutoff for the N- and S-proteins. Virus specificity of the M-, N-, and S-proteins and conformational stability of the N-protein and epitopes A and D of the S-protein were confirmed by immunoblotting using porcine hyperimmune antiserum to TGEV (Stott, 1989) or a TGEV neutralizing monoclonal antibody specific for epitope A of the S-protein (25C9) and non-neutralizing monoclonal antibodies specific for epitope D of the S-protein (44C11) and N-protein (25H7) (Lanza et al., 1995). The protein concentration was estimated with spectrophotometer readings at 280 nm.


Results
By extrapolating the molecular size to prestained protein standards or to native coronavirus proteins from virus (Wesley et al., 1991) cell culture adapted TGEV (Miller strain), the recombinant TGEV M-protein (2831 kDa) was expected to be eluted within fraction range 913 (Figure 1A). Fractions 913 were pooled and concentrated. The presumed M-protein was electrophoresed and detected by Coomassie blue stain (Figure 1B, lane 5) and its virus specificity was confirmed by western blot (Figure 1C, lane 5). After concentration of fractions 913, the final protein concentration was 0.4 mg/ml or 5% of the initial solubilized cell lysate protein.

The recombinant TGEV N-protein (47 kDa) was expected to be within fraction range 2527 (Figure 2A). These fractions were pooled and concentrated. The concentrated sample was analyzed as described for the recombinant TGEV M-protein (Figure 2B, C). The resulting purified N-protein concentration was 0.5 mg/ml, or 8% of the initial baculovirus lysate.

The recombinant TGEV S-protein (150 kDa) was expected to be within fraction range 2030 (Figure 3A). Fractions 2030 were pooled and concentrated. The concentrated S-protein sample was further analyzed as described for the M- and N-proteins (Figure 3B, C). In addition to the major band of 150 kDa, a minor, virus-specific band of 100 kDa was detected on both polyacrylamide gels and nitrocellulose membranes (Figure 3B, C, lane 5). The resulting purified S-protein concentration was 0.15 mg/ml or 2% of the initial baculovirus lysate.


Discussion

The production of subunit viral vaccines requires the purification of the immunogens of interest from other cellular proteins. The CE-PAGE represents an approach whereby proteins are electrophoretically separated based on their molecular size and collected in fractions.

The yields of purified M-, N- and S-proteins corresponding to the 18 x 105 Sf9 insect cells (0.5, 0.4 and 0.15 mg/ml, respectively) are reflective of the levels of recombinant protein expected (28%) and in agreement with data published on the efficiency of the baculovirus expression system (Luckow et al., 1988). Elution of small volumes of purified, concentrated proteins (0.20.5 ml) reflects the capacity of the Mini Prep Cell system, but can be scaled up when larger amounts are required by using the same conditions and a Model 491 Prep Cell.

By western blotting of the purified proteins with porcine hyperimmune antiserum to TGEV (S, M, N) in western blot and the ELISA reaction with monoclonal antibodies (S, N), we demonstrated that immunogenic and neutralizing epitopes were preserved on the purified proteins. On both polyacrylamide gels and nitrocellulose membranes, a minor 100 kDa protein band in addition to the major 150 kDa protein was detected for the TGEV-S protein (Figure 3B, C, lane 5). This most likely reflected the presence of an immature, posttranslationally non-modified form of S-glycoprotein as previously reported (Shoup et al., 1997) or cleavage product. Similarly, a minor protein band in addition to the full size recombinant TGEV-M protein was detected by western blot (Figure 1C, lanes 3 and 5).

The results from this study suggest that CE-PAGE can be a useful semi-preparative tool to purify baculovirus-expressed TGEV viral proteins.


Acknowledgements

The authors thank Drs. H. Laude, D. J. Jackwood and D. I. Shoup for providing the recombinant baculovirus plasmid (N) or constructs (S, M).


References

1. Bohl, E. H., et al., Antibody responses in serum, colostrum and milk of swine after infection or vaccination with transmissible gastroenteritis virus, Infect. Immun., 6, 289301 (1972).

2. Lanza, I., et al., Lactogenic immunity and milk antibody isotypes to transmissible gastroenteritis virus in sows exposed to porcine respiratory coronavirus during pregnancy, Am. J. Vet. Res., 56, 739748 (1995).

3. Luckow, V. A. and Summers, M. D., Trends in the development of baculovirus expression vectors, Biotechnol., 6, 4755 (1988).

4. Saif, L. J. and Wesley, R. D., Transmissible gastroenteritis. In: Leman, A.D., et al., (Eds), Diseases of Swine. 7th edn., Iowa State University Press, Ames, pp. 362386 (1992).

5. Shoup, D. I., et al., Active and passive immune responses to transmissible gastroenteritis virus (TGEV) in swine inoculated with recombinant baculovirus-expressed TGEV spike glycoprotein vaccines, Am. J. Vet. Res., 58, 242250 (1997).

6. Stott, D. I., Immunoblotting and dot blotting, J. Immunol. Methods, 119, 153187 (1989).

7. Wesley, R. D., et al., Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe, J. Vet. Diagn. Invest., 3, 2932 (1991).

8. Wu, W. and Welsh, M. J., Rapid Coomassie blue staining and destaining of polyacrylamide gels, Biotechniques, 20, 386388 (1996).


Coomassie is a trademark of ICI.


back to top
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. A Versatile Power Supply for All Electrophoresis Applications
2. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
3. Detection of K-ras Point Mutations in the Pancreas by Constant Denaturing Gel Electrophoresis Using the DCode System
4. Detection of Mutations in the Fas Antigen of Lymphoma Tumors by RT-PCR and Denaturing Gradient Gel Electrophoresis
5. Detection of Variation in Highly Polymorphic Mhc Genes by Denaturing Gradient Gel Electrophoresis Using the DCode System
6. Detection of p53 Gene in Breast Cancer by Denaturing Gradient Gel Electrophoresis and the DCode System
7. Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System
8. Microbial Diversity in Ground and Surface Water Analyzed by Denaturing Gradient Gel Electrophoresis Using the DCode System
9. Oligonucleotide Purity Analysis by Capillary Electrophoresis
10. Preparative Nondenaturing Gel Electrophoresis Used in the Purification of an Esterase Involved in Insecticide Resistance, Rev C
11. Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B
Post Your Comments:
(Date:8/28/2014)... research platform uses a laser to measure ... stress and heating, an approach likely to ... and batteries., This new technique, called nanomechanical ... and the surface stress of microscale structures ... the merits of surface-stress influence on mechanical ...
(Date:8/28/2014)... The ability for organizations to attract ... allow individuals to perform at the highest of levels, ... looking to compete in this post–recession era. The ... as the sector increasingly focuses on the importance of ... have real results on the organization's development and the ...
(Date:8/28/2014)... August 28, 2014 Sterlitech is proud ... membranes to its stable of products . ... weight cut-offs and include membrane filters with surface charges. ... in customer demand for our membrane process testing equipment, ... of flat sheet membranes,” explains Sterlitech President Mark Spatz. ...
(Date:8/28/2014)... BELLINGHAM, Washington, USA (PRWEB) August 27, 2014 ... Colorado next month for SPIE Laser Damage 2014 ... on optical materials for high-power lasers will run 14-17 ... international society for optics and photonics . , The ... key for understanding laser damage to optical materials will ...
Breaking Biology Technology:New analytical technology reveals 'nanomechanical' surface traits 2New analytical technology reveals 'nanomechanical' surface traits 3Klein Hersh International Leads Philadelphia Area Life Science Industry in Company Culture 2Klein Hersh International Leads Philadelphia Area Life Science Industry in Company Culture 3Sterlitech Corporation Enhances Its Selection of Flat Sheet Membrane Filters 2Laser Damage to Draw Researchers, Engineers, Scientists to 46th Annual Conference 2Laser Damage to Draw Researchers, Engineers, Scientists to 46th Annual Conference 3
... Therapeutics, Inc. (Nasdaq: TSRX ) announced today that ... present "Innovative Antibacterial Drugs by Design; The Trius Therapeutics Story" ... October 26, 2010 from 5:30-9:30 p.m. PT. ... . About Trius Therapeutics ...
... Pa., Oct. 22 Encorium Group, Inc. (Nasdaq: ... research organization (CRO) conducting studies in over 30 ... and biotechnology companies, today announced that, on October ... Listing Qualifications Panel (the "Panel") has determined to ...
... in German . Jena (21 October ... improving a fabrication process for Atomic Force Microscopy (AFM) probe ... surfaces so that even tiniest nano structures become visible. Knowledge ... of new materials and carrier systems for active substances. The ...
Cached Biology Technology:Encorium Announces NASDAQ Panel Decision to Delist Common Stock; Announces Changes to its Board of Directors 2Encorium Announces NASDAQ Panel Decision to Delist Common Stock; Announces Changes to its Board of Directors 3Encorium Announces NASDAQ Panel Decision to Delist Common Stock; Announces Changes to its Board of Directors 4AFM tips from the microwave 2
(Date:8/29/2014)... cell,s nucleus, chromosomal DNA is tightly bound to ... call chromatin. Until about two decades ago, histones ... packing material around which the glamorous DNA strands ... greater appreciation for how DNA/histone interactions govern gene ... institutions studying the sequence of the genome from ...
(Date:8/29/2014)... of the Tibetan plateau -- the largest topographic anomaly ... both its profound effect on climate and its reflection ... GSA Bulletin , Katharine Huntington and colleagues employ a ... modern and fossil snail shells to investigate the uplift ... , Views range widely on the timing of surface ...
(Date:8/28/2014)... pleased to announce the selection of Dr. Fred Baxendale ... acknowledges those who have served ESA for at least ... the Society that has reached an extraordinary level. Candidates ... Board and then voted on by the ESA membership. ... at Entomology 2014, ESA,s Annual Meeting in Portland, Oregon ...
Breaking Biology News(10 mins):'K-to-M' histone mutations: How repressing the repressors may drive tissue-specific cancers 2'K-to-M' histone mutations: How repressing the repressors may drive tissue-specific cancers 3Snails tell of the rise and fall of the Tibetan Plateau 2Snails tell of the rise and fall of the Tibetan Plateau 3Snails tell of the rise and fall of the Tibetan Plateau 4Snails tell of the rise and fall of the Tibetan Plateau 5Snails tell of the rise and fall of the Tibetan Plateau 6Snails tell of the rise and fall of the Tibetan Plateau 7Fred Baxendale selected as Honorary Member of the Entomological Society of America 2
... , April 27 ARES Corporation ... operations division has been certified to the rigorous ... ISO 9001:2008 quality standard.  This certification signifies ARES, ... standards and strengthens our competitive position by standardizing ...
... , HOUSTON , April 27 Landry,s ... "Company") announced today that after lengthy negotiations with the Special ... the plaintiff in a lawsuit pending in Delaware , ... with the plaintiff,s attorneys to settle the stockholder derivative claim ...
... NEW YORK, April 27, 2010 The Juvenile Diabetes ... Living Cell Technologies (LCT), a New Zealand-based biotechnology company ... II clinical trial to study the safety and effectiveness ... treatment for type 1 diabetes with significant hypoglycemia unawareness. ...
Cached Biology News:ARES Aerospace and Defense Division Receives AS9100 Certification 2Landry's Restaurants, Inc. Announces Status of Acquisition by Tilman J. Fertitta and Tentative Partial Settlement of Delaware Lawsuit 2Landry's Restaurants, Inc. Announces Status of Acquisition by Tilman J. Fertitta and Tentative Partial Settlement of Delaware Lawsuit 3Landry's Restaurants, Inc. Announces Status of Acquisition by Tilman J. Fertitta and Tentative Partial Settlement of Delaware Lawsuit 4JDRF partners with LCT to test encapsulated cell replacement therapy for the treatment of diabetes 2JDRF partners with LCT to test encapsulated cell replacement therapy for the treatment of diabetes 3
... can perform many sophisticated DNA analysis and protein ... than just another sequence analysis product. Imagine having ... analyses to be performed each time you have ... sheets which specify exactly how your output will ...
... does for enzyme-/substrate-based blotting what intensifying screens do ... up to 10-fold (or one order of magnitude) ... Signal Enhancer membrane treatment is a simple, 15-minute ... current Western blotting protocol. The result is an ...
These stopping screens are for the biolistic PDS-1000 He and Hepta systems. Supplied as 500 screens....
...
Biology Products: