Concentration of Sample and Sdspage

1. Concentrate about one-quarter of each eluted fractions with 10,000-MW cutoff membrane (Centricon, Millipore).

2. Wash concentrate one time with excess TE buffer (10 mM Tris-HCl, 10 mM EDTA), and reduce volume to < 20 mL.

3. Add SDS-PAGE sample buffer and process as usual for SDS-PAGE analysis, followed by fixation and fluorography.

4. Core protein and/or proteoglycan should be apparent in positive lanes (see Fig. 3).

3.10. Mass Production

The following protocols briefly describe methods for production of larger amounts of protein (milligrams and higher), and essentially comprise simple scale-up of the above protocols. The system lends itself to high-level batch production of recombinant proteins, and purification under native conditions.

1. Day 1: Set up cells. Seed roller bottles (or cell factory) with cells at 5 x 107 cells per bottle (or per layer of the cell factory). Use 200 mL of 10% DMEM per roller (100 mL per layer).

2. Allow 1-2 d to reach 80-90% confluence.

3. Day 2: Co-infection. Thaw recombinant viruses and add an equal volume of trypsin. Incubate at 37°C for 30 min, vortexing every 5 min.

4. Mix an appropriate amount of each virus into 10 mL of 2.5% DMEM for each roller to be infected.

5. Remove media from cells and wash cell layer twice with PBS.

6. Overlay cells with virus-DMEM cocktail and incubate at 37°C for 2 h in roller apparatus.

7. Add 50 mL of 2.5% DMEM for each roller, and incubate a further 2-4 h.

8. Optional: Biosynthetically label one bottle to use as a trace during purification, by incubating in the presence of 1 mCi per roller of 35S-Trans label in a final volume of 30 mL of methionine- and cysteine-free DMEM. For nonlabeled bottles, use 30 mL of DMEM SF per roller.

9. Incubate cells overnight, up to 30 h postinfection.

10. Harvest conditioned media and process by metal-chelating chromatography

11. Harvest the supernatant from the roller bottles and add solid imidazole up to a final concentration of 5 mM.

11. Add a detergent to reduce nonspecific binding (concentration should be the same as for the column buffer).

12. Adjust pH to 8.0 using concentrated NaOH.

13. Purify hexa-histidine tagged proteoglycan by affinity purification on Ni2+-charged metal-chelating column (see Subheading 3.9.), using imidazole gradient elution as shown in Fig. 3.

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Fig. 3. Purification of recombinant proteoglycan by metal-chelating affinity chromatography. (a) 35S-labeled proteoglycan (decorin) in conditioned media was applied to a Ni2+-charged iminodiacetic acid column (2 mL) in 20 mM imidazole buffer. Bound material was eluted by a stepwise increase of imidazole to 60 mM (proteoglycan elutes), and a linear gradient of imidazole from 60 to 200 mM (core protein elutes); see text for discussion. (b) Analysis of selected fractions (indicated by the asterisk in panel a) by SDS-PAGE and visualized by fluorography. Note that the separation of proteoglycan and core protein glycoforms is likely to be specific to decorin (and biglycan) due to the proximity of the glycosaminoglycan chain to the hexa-histi-dine tag resulting in a lower affinity interaction.

3.11. Characterization of Recombinant Proteoglycan

Following purification of recombinant proteoglycan and core protein by the procedures described above, further steps are dictated by the ultimate disposition of the protein. Simple carbohydrate analysis should yield information about glycosami-noglycan type, number, and size; more detailed analysis will show substitution with N- and 0-linked oligosaccharides; and finally, study of the structure (e.g., CD spectroscopy, NMR analysis, crystallization) and biological function (e.g., interaction with other matrix components, role in ECM assembly, in-vivo activity) will take advantage of the high-level expression and purification of native proteoglycan that is likely to closely resemble the in-vivo product. Many of these methods are detailed elsewhere in this book.

4. Notes

1. Constructs developed for expression in the vaccinia/T7 phage hybrid system have a T7 promoter driving expression of the target protein. Following construction of an expres sion vector in any of the pT-cam series of vectors, we recommend doing in-vitro transcription directly from the plasmid vector with T7 RNA polymerase, and in-vitro translation with a reticulocyte lysate system. This is in addition to direct sequencing through cloning sites or, optimally, through the entire coding region. A significant amount of wasted time can be avoided by following these two simple steps.

2. Since there is likely to be background wild-type virus as well as spontaneous deletion of the TK gene activity, it is recommended to take 8 plaques through to the second round. Some of these may not survive a second round of selection, and others may not express protein. Ideally, 50-75% of the first round plaques should be the desired recombinant; however, this efficiency can be somewhat less (ultimately, you need only one positive!).

3. If pg 1.1.3 is a positive, the name indicates it is derived from plaque number pg 1 from the pg recombination, and that it has gone through three rounds of selection. If pg 1.1.3 is lost through a laboratory accident, then it is likely that pg 1.1.1, pg 1.1.2, and pg 1.1.4 are identical clones and the positive can be recovered.

4. It is possible to screen plaques at this stage by PCR analysis. Our experience suggests that it is difficult to design appropriate positive and negative controls for this procedure. Since the ultimate test of a recombinant virus will always be expression of the protein of interest, we recommend amplifying the eight recombinants, determining the titration, and screening for protein expression. However, PCR screening can be useful, particularly when generating and screening a large number of constructs (truncations, deletions, mutations, etc.). Therefore this protocol describes the isolation of viral DNA for subsequent PCR by standard methods using appropriately designed oligonucleotides.

5. These procedures are followed to generate enough virus for an accurate titration and to screen for protein expression. We recommend amplifying 4-8 recombinants from the third-round plaque assay.

6. An accurate titration is required before any attempts to screen for protein expression. Titers can vary by 1-2 orders of magnitude, and optimal protein expression is achieved only within a discrete range of infection (5-30 pfu/cell). Infection with too little or too much virus will not yield detectable levels of protein expression.

7. Following identification of a positive recombinant, large stocks of virus are made and can be aliquoted and stored at -70°C for extended periods. For generation of milligram amounts of protein, and to avoid significant batch-to-batch variation, it is recommended to make as much virus stock as is practical.

8. After generating up to eight putative positive recombinant viruses that have undergone at least three rounds of selection in the presence of BrdU, a simple proteoglycan/protein expression screen is done to identify genuine positives. There are a number of ways to screen for the protein of interest, including Western blotting with specific antibodies or even sensitive bioassays if available. However, in our laboratory we almost exclusively utilize biosynthetic labeling of the core protein and purification with the hexa-histidine tag, followed by predicted migration on SDS-PAGE.

9. Most cells will tolerate serum-free conditions for the period of labeling, and this has the advantage of reducing total protein in the harvested conditioned medium. However, for cell lines that are particularly sensitive to serum-free conditions, one can add minimal serum or some other form of defined medium.

10. The behavior of a particular protein is not easily predicted, and better performance can often be achieved by an empirical approach, testing different resins, different divalent cautions, and changes in solvent conditions. However, for the purposes of an initial screen, this method is appropriate.

References

1. Chan, D., Lamande, S. R., McQuillan, D. J., and Bateman, J. F. 1997. In vitro expression analysis of collagen biosynthesis and assembly. J. Biochem. Biophys. Meth. 36, 11-29.

2. Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J. and Bateman, J. F. 1996. Site-directed mutagenesis of human type X collagen. Expression of alpha1X) NC1, NC2, and helical mutations in vitro and in transfected cells. J. Biol. Chem. 271, 13,566-13,572.

3. Hocking, A. M., Strugnell, R., Ramamurthy, A. P., and McQuillan, D. J. 1996. Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity. J. Biol. Chem. 271, 19,571-19,577.

4. Krishnan, P., Hocking, A. M., Scholtz, J. M., Pace, C. N., Holik, K. K., and McQuillan, D. J. 1999. Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability. J. Biol. Chem. 274,10,945-10,950.

5. Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. 1996. Recombinant decorin glycoforms. Purification and structure. J. Biol. Chem. 271, 19,578-19,584.

6. Hering, T. M., Kollar, J., Huynh, T. D., and Varelas, J. B. 1996. Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion. Anal. Biochem. 240, 98-108.

7. Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D., Twardzik, D. R., Border, W. A., and Ruoslahti, E. 1994. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem. J. 302, 527-534.

8. Schonherr, E., Hausser, H., Beavan, L., and Kresse, H. 1995. Decorin-type I collagen interaction. Presence of separate core protein-binding domains. J. Biol. Chem. 270, 8877-8883.

9. Schonherr, E., Witsch-Prehm, P., Harrach, B., Robenek, H., Rauterberg, J., and Kresse, H. 1995. Interaction of biglycan with type I collagen. J. Biol. Chem. 270, 2776-2783.

10. Groffen, A. J., Buskens, C. A., Tryggvason, K., Veerkamp, J. H., Monnens, L. A., and van den Heuvel, L. P. 1996. Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells. Eur. J. Biochem. 241, 827-834.

11. Gu, J., Nakayama, Y., Nagai, K., and Wada, Y. 1997. The production and purification of functional decorin in a baculovirus system. Biochem. Biophys. Res. Commun. 232, 91-95.

12. Hochuli, E., Dobeli, H., and Schacher, A. 1987. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177-184.

13. Mackett, M., Smith, G. L., and Moss, B. 1985. The construction and characterization of vaccinia virus recombinants expressing foreign genes; in DNA Cloning, volume II (Glover, D. M., ed.), IRL, Oxford, UK, pp.191-212.

14. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. M., Seidman, J. G., Smith, J. A., and Struhl, K., eds. 1987. Current Protocols in Molecular Biology. Wiley, New York, NY.

15. Elroy-Stein, O., Fuerst, T. R., and Moss, B. 1989. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl. Acad. Sci. (USA) 86, 6126-6130.

16. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. (USA) 83, 8122-8126.

17. Fuerst, T. R., Earl, P. L., and Moss, B. 1987. Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. Mol. Cell. Biol. 7, 2538-2544.

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