- m I IYI■ I^IHI
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.
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.
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