David J McQuillan Neung Seon Seo Anne M Hocking and Camille I McQuillan

1. Introduction

1.1. Only Mammalian Cells Will Make a Proteoglycan

Proteoglycans are molecules that comprise a core protein to which at least one glycosaminoglycan (GAG) chain is attached, and a consideration of recombinant proteoglycan expression must operate under this simple definition. There are numerous systems currently available to express proteoglycans (and other complex glycoconju-gates), but this chapter will focus on a system adopted by us to express proteoglycans and other extracellular matrix proteins in mammalian cells (1-5).

With few reported exceptions, proteoglycans are exclusively products of eukary-otic organisms, generally of mammalian origin. Although there is ample evidence that core protein homologs exist throughout evolution in many members of the phy-logenetic tree, the capacity to synthesize glycosaminoglycans (chondroitin/ dermatan, keratan, and heparin/heparan sulfates) appears to be exclusive to higher organisms. Hence, when embarking on a strategy to express heterologous proteoglycan genes, the host cell must possess the appropriate machinery (glycosyltransferases, sulfotransferases, epimerases, deacetylases, etc.) to synthesize and modify a GAG chain onto a recognizable acceptor site in the core protein substrate. In general, this requirement is satisfied by most mammalian cells. Although the capacity for GAG synthesis by different cells and tissues will vary, phenotypically stable cell lines will have the posttranslational machinery required for synthesis of some form of GAG.

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. lozzo © Humana Press Inc., Totowa, NJ

Structural and functional studies of proteoglycan domains can be facilitated by the isolation and purification of native proteoglycan. Most current procedures for isolation of proteoglycans from tissue require the use of denaturing solvents. An alternative method for the generation of native proteoglycan is the use of a recombinant expression system. Due to the extensive posttranslational modifications of proteoglycans, an expression system capable of complex modifications, especially addition of glycosami-noglycan chains, is essential. Prokaryotic expression systems are capable of generating high yields of protein, but these products lack the posttranslational modifications that may regulate folding, solubility, and biological activity. For example, in Escherichia coli expression systems, biglycan and decorin are synthesized as core proteins devoid of glycosaminoglycan chains and ^-linked oligosaccharides. The recombinant core protein is often insoluble, requiring the use of denaturing solvents for efficient extraction from inclusion bodies (6-9). The baculoviral expression system is also capable of high-level production of processed protein. However, conflicting data makes it difficult to assess whether Spodoptera frugiperda (Sf21) cells have the appropriate posttranslational machinery for effective processing of a proteoglycan. Perlecan domains expressed using the baculoviral system have been reported to be substituted with chondroitin sulfate (10), but, decorin produced in Sf21 cells was secreted devoid of glycosaminoglycan chains (11).

Recombinant proteoglycans expressed in stably transfected mammalian cells are likely to be folded and glycosylated correctly, but effective purification of the recombinant proteoglycan often involves the use of denaturing solvents. Therefore, it is also important to consider the method of purification of the proteoglycan subsequent to its expression so that it will be representative of the molecule found in vivo. This is most easily achieved by generation of a fusion protein in which the molecule of interest is expressed in complex with a tag (peptide or protein) that can be isolated by a high-affinity, nondenaturing purification procedure. Again, a variety of examples are available, including lectin domains, peptide-antibody pairs, protein-protein ligand complexes, and metal chelating peptides. In our laboratory we have utilized several fusion protein systems but, like numerous other investigators, have found many advantages in the divalent cation-binding properties of the hexa-histidine tag (12).

1.2. Vaccinia Virus as an Expression Vector in Mammalian Cells—Expression by All Cells Achieved with Natures Own Transfection Agent

Introduction of naked DNA into mammalian cells is generally inefficient, despite the increasing number of reagents available from vendors purporting to yield "high-efficiency" expression. In general, the use of calcium phosphate, DEAE-dextran, cationic lipid formulations, or electroporation yield less that 20% transfection efficiency (depending on the cell line) and can be both cumbersome and expensive. It has also been estimated that of the DNA that finds its way into a cell, only 10% will transit to the nucleus and be available for transcription. Therefore, a system whereby efficiency of uptake of DNA is optimized and circumvention of inefficiency of nuclear processing is achieved is likely to be optimal for overexpression of heterologous genes. The use of vaccinia virus as an expression vector satisfies these requirements.

Vaccinia virus, a member of the poxvirus family, has a number of unique properties that has made it very popular for a number of years as a vector for the expression of foreign genes (13). Vaccinia replicates entirely within the cytoplasm of eukaryotic cells and encodes a complete transcription system, including RNA polymerase, capping and methy-lating enzymes, and poly A polymerase. As a eukaryotic expression vector, vaccinia virus has a number of useful characteristics, such as a large capacity to incorporate foreign DNA (> 20 kb), retention of infectivity, a wide host range, high level of protein synthesis, and appropriate transport, processing, and posttranslational modification of proteins.

Detailed methods for the propagation, manipulation, and construction of recombinant vaccinia viruses will not be provided here. The reader is referred to several excellent reviews where this information is readily available (13,14). However, the protocols used in our laboratory for the design, construction, and selection of recombinant viruses used to express proteoglycans will be provided in the following sections. The primary disadvantage of the use of vaccinia virus is that it has virulence for humans and animals. Laboratory workers who work with viral cultures (or other infective materials) should always observe appropriate biosafety guidelines and adhere to published infection control procedures. In the United States, National Institutes of Health guidelines recommend that workers likely to come in direct contact with vaccinia virus be vaccinated with smallpox vaccine every 3-10 yr. Vaccination with smallpox vaccine is generally a simple and safe procedure, but investigators are advised to become well informed of all potential hazards before embarking on the use of this expression system.

1.3. Vaccinia Virus/T7 Phage Expression System—A Powerful System for Overexpression of Heterologous Genes

The vaccinia/T7 phage eukaryotic expression system was developed by Moss and co-workers (15,16) to exploit the high transcriptase activity, stringent promoter specificity, and excellent processivity of the RNA polymerase from the T7 bacteriophage. The main steps involved in the expression of proteoglycans using this system are outlined in Fig. 1. Eukaryotic cells infected with the recombinant virus, vTF7-3 (available from the ATTC, accession number VR-2153) express high cytoplasmic levels of T7 bacteriophage RNA polymerase (17). These cells are co-infected with a recombinant virus containing a cDNA under the control of the T7 promoter. This expression construct also includes an encephalomyocarditis virus (EMC) untranslated region (UTR) that facilitates cap-independent ribosome binding and hence increases translation efficiency up to 10-fold (15). Transcription of the PG cDNA are driven by phageT7 RNA polymerase in the cytoplasm, and the resultant high levels of PG mRNA are then available to the host cell machinery. Targeting to the secretory pathway by a signal sequence, and subsequent processing including addition of N- and O-linked oligosaccharides, GAGs, sulfation, phosphorylation, folding, and secretion, should all occur appropriately.

1.4. Expression Vectors—The Cam Series Works in Vitro and In Vivo

We have modified the basic vaccinia/T7 cloning and expression plasmid, pTM1 (15) to facilitate targeted secretion and nondenaturing purification of recombinant proteoglycans.

Phage Virus

Fig. 1. Schematic representation of vaccinia virus/T7 phage expression system. The major steps of this powerful eukaryotic recombinant protein expression system are shown. Vaccinia virus mediates the initial steps 1 and 2. Step 1, co-infection of mammalian cells by a recombinant virus containing the gene encoding phage T7 RNA ploymerase under control of the early/ late vaccinia virus promotor; and a recombinant virus containing the gene encoding the target protein under control of the phage T7 promotor. Step 2, cytoplasmic expression of T7 RNA

Fig. 1. Schematic representation of vaccinia virus/T7 phage expression system. The major steps of this powerful eukaryotic recombinant protein expression system are shown. Vaccinia virus mediates the initial steps 1 and 2. Step 1, co-infection of mammalian cells by a recombinant virus containing the gene encoding phage T7 RNA ploymerase under control of the early/ late vaccinia virus promotor; and a recombinant virus containing the gene encoding the target protein under control of the phage T7 promotor. Step 2, cytoplasmic expression of T7 RNA

The important domains of these vectors are shown in Fig. 2. The most versatile of the series is pT-cam1 (see Fig. 2a, b), and comprises (1) a bacteriophage T7 promoter; (2) an ATG start codon at a precise distance downstream of an encephalomyocarditis virus ribosome-binding site (EMC UTR); (3) a canine insulin signal sequence, for targeting to the secretory pathway; (4) a hexa-histidine (6 x His) sequence, for nondenaturing purification by metal-chelating affinity chromatography; (5) a factor Xa recognition cleavage sequence, for removal of the histidine tag after purification; (6) a versatile multicloning site, for in-frame insertion of the cDNA of interest (preferably devoid of endogenous signal sequence); and (7) stop codons in three reading frames upstream of a polyadenylation signal.

The other vectors shown are essentially modifications of pT-cam1, for situations where all the elements of the parent vector are not required. If one has an alternative method of purification (e.g., antibody affinity column or interaction with hyaluronan), pT-cam2 does not have the hexa-histidine tag nor the factor Xa cleavage sequence. If potential bioactive domains are predicted to be located in the vicinity of the N-terminus, pT-cam3 has the hexa-histidine tag located at the C-terminus of the secreted protein. Our experience indicates that the insulin signal sequence can enhance expression of some proteins compared to the endogenous sequence, but pT-cam4 is designed for insertion of the cDNA inclusive of endogenous signal sequence with the hexa-histidine tag at the C-terminus. In many instances, removal of the small hexa-histidine sequence following purification is likely to be no advantage and potentially cumbersome, and in these circumstances pT-cam5, which is devoid of the factor Xa cleavage sequence, may be the vector of choice. All vectors and complete sequences are available upon request from the authors. 2. Materials

1. Cell lines, shown in Table 1.

2. Wild-type vaccinia virus, WR (ATCC # VR-119), titer should be about 5 x 109 pfu/mL.

3. DMEM alone (DMEM SF), containing 2.5% FCS (DMEM 2.5%), containing 10% FCS (DMEM 10%).

4. Lipofectin™ transfection kit (GIBCO-BRL #18292-011) or other efficient transfection reagents.

5. Plasmid containing cDNA (pT-cam series) at 1 ^g/^L.

6. Sterile polystyrene tubes.

7. Bromodeoxyuridine (BrdU) 200-fold concentrated stock solution, 5 mg/mL in water, filter sterilized.

8. 143B (TK-) cells in log phase, passaged at least once in the presence of BrdU.

9. All culture media for the plaque assays should be supplemented with 25 ^g/mL BrdU.

10. Neutral red solution, 3.33 mg/mL, tissue culture grade (Gibco-BRL #15330-079).

11. Low-melting-point agarose (Gibco-BRL #15517-014), 2% solution in PBS, autoclave and mix well. Can be stored at 45 °C for 1-2 wk.

polymerase and transcription of mRNA encoding the target protein. The host mammalian cell machinery is utilized for steps 3 and 4. Step 3, ribosome directed translation of the target protein mRNA that is enhanced by inclusion of a ribosome "landing pad" (UTR); targeting to the rough endoplasmic reticulum by specific signal sequence; signal sequence cleavage; and addition of N-linked oligosaccharides to the nascent chain. Step 4, posttranslational modifications as appropriate for the target protein, including processing of N-linked oligosaccharides, addition of 0-linked oligosaccharides, synthesis of glycosaminoglycan chains, and finally secretion of a proteoglycan.

Fig. 2. pT-cam series of expression vectors. (A) Main structural features of the pT-cam series of vectors. tk-L and tk-R, thymidine kinase flanking sequences to facilitate insertion of the expression cassette via homologous recombination into wild-type vaccinia virus genome; EMC, encephalomyocarditis untranslated region (UTR) that allows for cap-independent ribo-some binding; INS, insulin signal sequence; 6xHis, hexa-histidine sequence; Xa, factor Xa cleavage site. (B) cDNA and protein sequences of pT-cam series expression cassettes, showing unique restriction sites available in the multicloning site (MCS), and positioning of other structural features in relation to the MCS.

Fig. 2. pT-cam series of expression vectors. (A) Main structural features of the pT-cam series of vectors. tk-L and tk-R, thymidine kinase flanking sequences to facilitate insertion of the expression cassette via homologous recombination into wild-type vaccinia virus genome; EMC, encephalomyocarditis untranslated region (UTR) that allows for cap-independent ribo-some binding; INS, insulin signal sequence; 6xHis, hexa-histidine sequence; Xa, factor Xa cleavage site. (B) cDNA and protein sequences of pT-cam series expression cassettes, showing unique restriction sites available in the multicloning site (MCS), and positioning of other structural features in relation to the MCS.

Table 1

Cell Lines Used for Homologous Recombination, Plaque Assays, Amplification, Titration, and Protein Expression

Cell line (origin, morphology) Culture medium" ATTC catalog number

CV-1 (monkey kidney cell, DMEM + 2.5% FCSfc fibroblastic) 5.0% FCS CCL-70

(human osteosarcoma, MEM + 5% FCS CRL-8303

fibroblastic) 10% FCS

HeLa

(human adeno-carcinoma, DMEM + 10% FCS CCL-2

epithelial)

UMR-106

(rat osteosarcoma, DMEM + 10% FCS CRL-1661

fibroblastic)

HT-1080C

(human fibrosarcoma, DMEM + 10% FCS CCL-121

epithelial)

inclusion of antibiotics is optional, except in the case of the initial transfection for the homologous recombination. If using Lipofectin, then antibiotics should be omitted, as it can inhibit transfection efficiency.

6Fetal calf serum, or serum substitutes.

cAny other cell line that is likely to process proteoglycans appropriately can be substituted here, with the exception of CHO cells.

12. Proteinase K stock solution (20 mg/mL, Gibco-BRL #25530-031).

13. TE-saturated phenol.

14. Reagents or kit for standard PCR reaction.

15. Thermal cycler.

16. Crystal violet, 0.1% in 20% ethanol.

17. vTF7-3, recombinant vaccinia virus encoding phage T7 RNA polymerase.

18. Methionine and cysteine-free DMEM.

19. Trans-35S-label, a mixture of 35S-methionine and 35S-cysteine (ICN Biochemicals, #51006).

20. Sephadex G-50 (available from Pharmacia-Amersham Biotech).

21. Sepharose 6B conjugated to iminodiacetic acid functional group (slurry or prepacked 1-mL columns available from Pharmacia-Amersham Biotech).

22. Column buffers: For optimal recovery, column solvents (with the exception of the "charge buffer") should contain a detergent (either 0.1% or greater Triton X-100, or 0.2% or greater CHAPS).

a. Charge buffer: 100 mM NiCl2.6H2O.

b. Sample and loading buffer: 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0.

c. Wash buffer: 20 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0.

d. Elution #1: 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0.

e. Elution #2: 250 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0.

f. Strip buffer: 100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl, pH 8.0.

23. Sonicator, either probe or cup (e.g., Fisher Sonic Dismembranator 550).

24. Phase-contrast light microscope.

25. Biohazard containment.

3. Methods

3.1. In Vitro Transcription and Translation (see Note 1)

We routinely use the TnT® coupled reticulocyte lysate system available from Promega (#L4610), and essentially follow the manufacturer's instructions. In a typical cell-free transcription/translation reaction, we would use the following protocol.

1. Mix in an eppendorf tube for a final reaction volume of 50 ^L: 25 ^L rabbit reticulocyte lysate, 2 ^L TnT reaction buffer, 1 ^L T7 RNA polymerase, 1 ^L methionine-free amino acid mixture, 4 ^L Trans35S-label, a mixture of 35S-methionine and 35S-cysteine (ICN Biochemicals), 1 ^L RNAsin® ribonuclease inhibitor, 1 ^L plasmid, containing cDNA (1 ^g/^L CsCl-purified, or equivalent quality), and 15 ^L nuclease-free water

2. Incubate mixture at 30°C for 90 min.

3. Analyze reaction products by (a) direct application to SDS-PAGE; (b) immunoprecipita-tion followed by SDS-PAGE; or (c) purification on small metal chelating column followed by SDS-PAGE (reaction mix must be exchanged into appropriate binding buffer, e.g., PD-10 column from Amersham-Pharmacia Biotech).

4. Bands are visualized by standard autoradiography/fluorography. The predominant signal should be the target protein of interest migrating at the predicted size for the unsubstituted core protein.

5. If DNA sequencing does not show any errors, and cell-free translation generates a protein product of the predicted size, then one can confidently move to the next step of generating a recombinant virus from the plasmid.

3.2. Generating a Recombinant Virus

In this section, several protocols are outlined. The homologous recombination allows transfer of the cDNA under control of the T7 promoter to be inserted into the thymidine kinase (TK) locus of wild-type vaccinia virus. Disruption of the TK gene allows for selection of positive recombinants by culture in the presence of bromo-deoxyuridine, a lethal analog of deoxyuridine that is incorporated into replicating DNA by an active TK gene. Plaques are selected and undergo three rounds of purification to ensure clonal selection and remove contaminanting dormant wild-type virus. These procedures require standard cell culture ware and tissue culture facilities. As indicated above, these protocols assume a basic knowledge of virus manipulation. Extensive protocols relating to vaccinia virus are available elsewhere (13). Cells required are shown in Table 1, indicating the ATTC catalog number and the recommended culture medium. Specialized equipment includes a sonicator (either probe or cup, e.g., Fisher Sonic Dismembranator 550), phase-contrast light microscope, and biohazard containment.

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