Alan D Murdoch and Renato V Iozzo

1. Introduction

For molecules with such extensive posttranslational modifications, it may not be immediately obvious why one would wish to express the protein cores of proteoglycans in prokaryotic systems, where none of this modification can occur. However, there are several cases where this is not a problem, and some where there is a positive advantage to expressing a core protein with none of the normal eukaryotic modifications.

Bacterially expressed proteoglycan core proteins have been used successfully to raise polyclonal antisera (1,2) and as both immunization and screening agents in monoclonal antibody production, notably in the production of domain specific monoclonals (3,4). Bacterial fusion proteins have also been used to probe core protein domain-carbohydrate interactions (5), as substrate for modification enzymes such as core pro-tein-UDP-xylose xylosyltransferase (6,7), and to gain at least some structural and functional information about important extracellular matrix molecules (8,9).

Bacterial fusion systems can be divided roughly into two main groups depending on the size of the vector-encoded fusion partner. Those with small tags such as polyhistidine (6 x His), which allow purification on immobilized metal ions (e.g., Qiagen, Clontech), FLAG (Sigma), or c-myc peptide tags (e.g., Invitrogen), which allow detection and purification with specific antibodies, and the small (4-kDa) calmodulin-binding peptide, which allows purification on calmodulin resin (Stratagene), have the advantage that the tag can be left on the protein of interest with minimal or no interference with the structure or function. Larger tags such as glutathione S-transferase, protein A, maltose-binding protein, or cellulose-binding domain (Amersham Pharmacia Biotech, Novagen, New England Biolabs) may help with fusion proteins that have solubility problems, but the large fusion partner can present its own problems. There are many methods available for enzymatic or autocata-lytic cleavage of fusion partners, but in many cases, such as monoclonal antibody production or binding studies, it is not necessary to remove the fusion partner, as

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

experimental design can control for the presence of the partner. The choice of fusion partner may be largely empirical, since not all proteins will express in the first system of choice, and several may need to be tested before satisfactory expression levels are achieved.

The pMAL system from New England Biolabs fuses the protein of interest to maltose-binding protein (MBP), the product of the Escherichia coli malE gene (10,11). MBP is normally secreted into the bacterial periplasmic space; the pMAL-p2 vector can be used to take advantage of this and produce secreted periplasmic fusion proteins. The pMAL-c2 vector has the signal peptide sequence of the malE gene removed and can be used for high-level cytoplasmic expression of fusion proteins. In both cases, the fusions can be purified by virtue of the affinity of MBP for maltose using a column of immobilized amylose (a maltose polymer). In this chapter we present details of pilot-scale experiments for cytoplasmic and periplasmic expression of MBP fusion proteins, with an example of cytoplasmic expression, and a protocol for affinity purification. Further details about the system can be found in the pMAL system handbook (available on the NEB website at http://www.neb.com). In addition, since many fusion proteins will form insoluble inclusion bodies in many of the common E.coli strains, we include a convenient method for solubilization of protein from these inclusion bodies in a form suitable for affinity purification. 2. Materials

1. pMAL-c2 and pMAL-p2 vectors, components of the pMAL system. General molecular biology supplies: restriction endonucleases, T4 DNA ligase, etc., facilities for running horizontal agarose gels, water baths, DNA sequencing facilities. PCR reagents, including proofreading polymerases such as Pfu or Tli polymerases if necessary. SDS-PAGE gel equipment, columns, pumps, and fraction collector for affinity chromatography.

2. Luria-Bertani (LB) growth medium: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl per liter. Adjust pH to 7 and autoclave.

3. Glucose. 1 M glucose stock: Dissolve 18 g of glucose and make up to 100 mL. Filter sterilize and add 10 mL to cooled sterile LB for rich growth medium (see Note 1).

4. Ampicillin 1000 x stock: Dissolve 0.5 g of ampicillin in 5 mL of H2O. Filter sterilize into aliquots and store at -20° C.

5. X-gal: Dissolve at 40 mg/mL in dimethylformamide. IPTG 1 M stock: Dissolve 2.38 g of IPTG in 10 mL of H2O, filter sterilize into aliquots. Store both X-gal and IPTG at -20°C.

6. Column buffer: 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA. Dissolve 2.42 g of Tris base, and 11.7 g of NaCl per liter and add 2 mL of 0.5 M EDTA stock. Adjust pH to 7.4. In addition, the column buffer used in the example shown in Fig. 1 also contained 1 mM sodium azide (1 mL of 1 M stock per liter) and 10 mM p-mercaptoethanol (0.7 mL/L).

7. Cold osmotic shock wash buffer: 30 mM Tris-HCl, pH 8.0, 20% sucrose. Dissolve 3.63 g of Tris base and 200 g of sucrose per liter and adjust pH to 8.0.

8. For the cold osmotic shock: 5 mM MgSO4. Dissolve 60 mg of anhydrous MgSO4 in 100 mL of H2O.

9. Anti-MBP serum, component of the pMAL system.

10. Amylose resin, component of the pMAL system.

11. Inclusion body lysis buffer: 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA. Dissolve 3.03 g of Tris base, and 2.92 g of NaCl and add 1 mL of 0.5 M EDTA for 500 mL of buffer and adjust the pH to 8.

12. Inclusion body wash buffer: as per lysis buffer above, but add 10 mL of 0.5 M EDTA and 2.5 mL of Triton X-100 per 500 mL.

13. Inclusion-body solubilization buffer: Lysis buffer containing 8 M urea and 0.1 mM PMSF. Dissolve 48 g of urea by adding 50 mL of H2O and then making the solution up to 100 mL. Deionize by adding 1 g of mixed-bed resin TMD-8 (Sigma) and stir for 1 h. Filter the beads out of the solution through a sintered glass filter. Add to the solution 0.61 g of Tris base, 0.58 g of NaCl, 0.2 mL of 0.5 M EDTA and adjust pH to 8. Add PMSF just before use to 0.1 mM (see Note 2).

14. Alkaline pH buffer: 50 mM KH2PO4, pH 10.7, 50 mM NaCl, 1 mM EDTA. Dissolve 3.4 g of KH2PO4 and 1.46 g of NaCl and add 1 mL of 0.5 M EDTA per 500 mL. Adjust the pH to 10.7.

3. Methods

3.1. Subcloning and Screening

1. Subclone the cDNA encoding the gene or open reading frame of interest into both the pMAL-c2 and pMAL-p2 vectors for small-scale expression. The translational reading frame of the insert must be the same as the malE gene from the vector. If no vector-derived amino acids are wanted in a factor Xa-cleaved protein, then the open reading frame should be cloned into the XmnI site in the vector. Bear in mind that the first encoded amino acid in this case should not be proline or arginine, since this will inhibit factor Xa cleavage. Otherwise, the remaining restriction sites can be used, preferably for directional cloning. If using the PCR to generate insert, proofreading polymerases will produce suitable blunt-ended fragments for XmnI cloning, and downstream primers should include a translational stop codon. A number of cloning strategies are also presented in the pMAL system manual.

2. Ligate 25-50 ng of suitably digested pMAL-c2 and -p2 with 1-4 times the molar amount of insert in a standard ligation. For directionally cloned inserts, include a vector-only control reaction.

3. Make competent TB1 or other E. coli strain (or purchase ready competent cells). We have found the CaCl2 method works well for TB1.

4. Mix the ligation reaction with 50 ^L of competent cells, incubate on ice for 30 min, and heat shock at 42°C for the length of time suitable for the strain used (2 min for TB1).

5. Recover the cells by adding 100-200 ^L of LB and incubating at 37°C for 30 min. Spread all of the transformation on 2 or 3 LB plates containing 100 ^g/mL of ampicillin and incubate overnight at 37°C.

6. If the insert was directionally cloned into the vector, and the vector control plate has few or no colonies, pick colonies into 3 mL of LB with 100 mg/mL of ampicillin and shake overnight at 37°C for plasmid minipreps (see Note 3).

7. If directional cloning was not possible and blue/white screening is necessary, either replica plate the colonies onto a fresh plate containing 100 ^g/mL amp, 80 ^g/mL of X-gal, and 0.1 mM IPTG, or pick colonies with sterile toothpicks and stab or patch them onto an LB amp plate and an LB amp/X-gal/IPTG plate and incubate at 37°C overnight. White colonies on the X-gal plate show which colonies should be picked from the LB amp plate for overnight liquid culture in 3 mL of LB with 100 ^g/mL amp (see Note 3). Do not pick colonies directly from plates containing IPTG.

8. Prepare miniprep DNA from the overnight cultures. Cut the DNA with suitable restriction enzyme(s) and run on agarose gels to check for insert. Plasmids that appear to contain insert should be sequenced to check for appropriate insert sequence and that the reading frame across the MBP/fusion partner junction has been retained. Glycerol stocks

Fig. 1. Purification scheme for a maltose-binding protein/perlecan domain II fusion protein. A cDNA encoding the entire low-density lipoprotein-like region (domain II) of the basement membrane heparan sulfate proteoglycan perlecan was subcloned into the pMAL-c2 vector (3) and a pilot-scale expression was performed as described under Subheading 3.1. Samples were run on a 10% SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, molecular weight markers. Lane 2, uninduced crude total lysate. Lane 3, induced crude total lysate. Lane 4, crude soluble extract. Lane 5, crude insoluble material. Lane 6, amylose resin sample: material from the crude soluble extract that bound to the amylose resin in the small batch binding procedure detailed under Subheading 3.1.6.

should be prepared for archival purposes and cultures restreaked on LB plates containing 100 ^g/mL ampicillin for small-scale expression testing.

9. Grow a small (5-mL) culture from a single bacterial colony in LB + glucose + ampicillin, shaking with good aeration at 37°C, to an A600 of ~0.5. Take a 1-mL sample and pellet the cells in a microfuge (top speed, 1 min). Aspirate the supernatant, resuspend the pellet in 50 ^L of SDS-PAGE sample buffer and store at -20°C until needed (uninduced sample).

10. Add IPTG to the remaining 4 mL to 0.3 mM, and continue shaking at 37°C for 2 h. Remove a 0.5-mL sample, microfuge as before and resuspend in 100-^L SDS-PAGE sample buffer (induced sample).

11. Boil the uninduced and induced samples for 5 min and run 20-^L samples on an SDS-PAGE gel of a suitable percentage for the expected fusion protein size and check for an induced band of the correct size following staining with Coomassie blue (see Fig.1, lanes 2 and 3).

3.2. Pilot-Scale Expression—Cytoplasmic Expression

1. Grow an overnight culture of cells (5 mL) in LB + glucose + amp from a single colony of a stock identified as expressing the fusion protein (above).

2. Add 0.8 mL of overnight culture to 80 mL (1/100) of fresh growth medium and grow as before to an A600 of ~0.5. Take an uninduced sample as before, add IPTG to a final concentration of 0.3 mM, and shake for a further 2 h (see Note 4). Take an induced sample as before and centrifuge the remaining culture at 4000 g for 10 min. Resuspend the pellet in 10 mL of column buffer (see Note 5).

3. Freeze the suspension at -20°C (-70/80°C can also be used). Thaw in cold water.

4. Sonicate the cells to disrupt them and release protein. Prevent the suspension from overheating by using an ice-water bath and sonicating in short pulses. Protein release can be monitored with the Bradford assay. Sonicate until no more protein is being released.

5. Centrifuge the sonicated suspension at 9000 g for 20 min. Remove the supernatant (crude soluble extract) and store on ice. Resuspend the pellet in 10 mL of column buffer (crude insoluble material).

6. Add approx 200 ^L of settled amylose resin as supplied and microfuge briefly. Remove supernatant and wash the resin twice by resuspension in 1.5 mL of column buffer and microfuging. Resuspend the resin in 200 ^L of column buffer and add 50 ^L of resus-pended slurry to 50 ^L of the crude soluble extract. Incubate on ice for 15 min, microfuge for 1 min, wash pellet with 1 mL of column buffer, microfuge again, and resuspend the resin pellet in 50 ^L of SDS-PAGE sample buffer.

7. Take 5 ^L of the crude soluble extract and the crude insoluble material and add 5 ^L 2x SDS-PAGE sample buffer to each. Boil these and the uninduced, induced, and amylose resin samples for 5 min. Microfuge the tubes to pellet the amylose resin and run 20 ^L of the supernatant along with 20 ^L of the uninduced and induced total lysate samples and all of the crude soluble and insoluble samples on an SDS-PAGE gel (see Fig.1).

3.3. Pilot-Scale Expression—Periplasmic Expression

1. Proceed as above to grow and induce a culture of cells. After centrifuging for 10 min at 4000 g (see Subheading 3.1., step 2) resuspend the pellet in 10 mL of 30 mM Tris-HCl, pH 8.0; 20% sucrose.

2. Add EDTA to a final concentration of 1 mM (20 ^L of 0.5 M EDTA to 10 mL) and incubate at room temperature with shaking for 5-10 min.

3. Centrifuge at 8000 g at 4°C for 10 min. Decant all the supernatant and resuspend the pellet in 10 mL of ice-cold 5 mM MgSO4. Shake for 10 min in an ice-water bath.

4. Centrifuge again at 8000 g for 10 min at 4°C. The supernatant is the cold osmotic shock extract. Samples of this extract (10 ^L plus 10 ^L 2x SDS-PAGE sample buffer) can be run on SDS-PAGE gels. Depending on the level of expression, immunoblotting with the anti-MBP serum supplied with the kit may be necessary.

3.4. Affinity Chromatography

1. The maltose-binding protein will bind to the amylose resin in a variety of buffer systems at around pH 7 and in varying ionic strengths. Intracellular proteins extracted in column buffer are ready to apply to the resin, following dilution if necessary (see below). Proteins in cold osmotic shock extracts should be adjusted to 20 mM Tris-HCl, pH 7.4 by the addition of 1 M Tris-HCl pH 7.4. Proteins from cytoplasmic extracts that contain intramolecular disulfide bridges (such as the perlecan domain II fusion in Fig. 1) may need to be purified over the column in a reducing environment (e.g., 10 mM p-mercaptoethanol) to prevent loss from incorrect disulfide bonding and aggregation. A refolding protocol may then be necessary for the downstream application (see Note 6).

2. Pour an amylose resin column suitable for the amount of protein to be purified. The pMAL system handbook suggests a 2.5 x 10 cm column; we have found K9/15 and XK16/20 columns from Pharmacia and 1 x 10 cm econocolumns from Bio-Rad to be satisfactory. Column size and bed volume should be adjusted to the expected yield given a binding capacity of ~3 mg/mL packed resin. We have generally used 10-mL columns (~30 mg binding capacity is more than enough for many purposes, and larger bed volumes only need longer washing steps). Wash the column with 8 vol of column buffer.

3. Dilute the crude extract to a total protein content of 2.5 mg/mL or less with column buffer. Cold osmotic shock extracts may not need diluting. Load the extract onto the column at [10 x (diameter of column in cm)2] mL/h (~0.5 mL/min for an XK16 column).

4. Wash the column with 12 volumes of column buffer (or with an excess overnight).

5. Elute the bound fusion protein in column buffer containing 10 mM maltose, collecting fractions of one-fifth column size (usually 2-3 mL). Fusion protein will usually start to elute within the first column volume and should be quite a tight peak; collecting beyond 3 column volumes is usually not necessary.

6. Determine the protein content of fractions with the Bradford assay and pool the fractions containing protein.

7. Check the protein concentration of the pooled material and run samples on SDS-PAGE gels to verify the integrity of the protein.

8. Protein can be dialyzed, or concentrated and buffer-exchanged in Centricon concentrators (Amicon) if it is necessary for your application.

3.5. Inclusion Body Solubilization

1. This method is taken from Sambook et al. (12), based on a method of Marston (13) and uses a combination of 8 M urea and alkaline pH for solubilisation. Unless stated, all procedures should be carried out on ice or in a cold room.

2. Pellet up to 1 L of an induced culture of cells by centrifugation at 4000 g for 10 min at 4°C.

3. Decant the supernatant and weigh the pellet. Resuspend the pellet in 3 mL of lysis buffer per gram wet weight.

4. Add 8 ^L of 50 mM PMSF per gram of cells (see Note 2) and then 80 ^L of 10 mg/mL lysozyme per gram. Incubate for 20 min with occasional mixing.

5. Stir the lysate with a glass rod and, while stirring, add 4 mg of deoxycholate per gram of original cell weight. Move the lysate to a 37°C water bath and continue to stir. When the lysate becomes viscous, add 20 ^L of 1-mg/mL DNAse I per gram of cells, mix well, and incubate at room temperature.

6. Check the lysate periodically for viscosity. After approx 30 min, the mixture should be no longer viscous.

7. Centrifuge the lysate at 12,000 g for 15 min at 4°C.

8. Resuspend the pellet in 9 vol of inclusion body wash buffer and incubate at room temperature for 5 min (first wash).

10. Remove the supernatant and keep. The pellet can be rewashed several times. At each wash, save the supernatant and analyze small (10-^L) samples along with a small sample of the final pellet resuspended in 100 ^L of H2O by SDS-PAGE. If large amounts of the fusion protein are washing out of the pellet, the washes can be kept and pooled with the solubilised and dialyzed material from the pellet (below). The small amount of Triton X-100 in a diluted sample applied to an amylose column may interfere with MBP binding, but will have to be determined on an empirical basis.

11. Resuspend the pellet in 100 ^L of inclusion body solubilization buffer. Store at room temperature for 30 min.

12. Add the mixture to 9 vol of alkaline pH buffer. Incubate at room temperature for 30 min. Check the pH by removing very small samples to pH strips at regular intervals. If the pH begins to drop, add KOH to maintain it at 10.7.

13. Adjust the pH to 8.0 with HCl and incubate for a further 30 min at room temperature. It is important during these alterations in the pH that the temperature is not allowed to rise above room temperature.

14. Centrifuge at 12000 g for 15 min at room temperature. Decant the supernatant and store. Resuspend the pellet in 100 ^L of SDS-PAGE sample buffer. Run 10 ^L samples of supernatant and resuspended pellet on an SDS-PAGE gel to check the amount of solubilized material.

15. The supernatant should be dialysed into pMAL column buffer before affinity chromatography (above). We have found a single-step dialysis in the presence of p-mercaptoethanol to be satisfactory, but depending on the fusion partner and the downstream application a stepwise dialysis from 8 M urea or dropwise dilution into an excess of column buffer may be tried.

4. Notes

1. The glucose in the growth medium represses the expression of E. coli amylase. Expression of amylase can lead to problems with the affinity purification due to degradation of the amylose resin.

2. Some of the protocols listed here use PMSF as a serine protease inhibitor. In many cases this compound can now be replaced with an equimolar concentration of the much safer and water-soluble AEBSF (Calbiochem).

3. In our hands the pMAL-c2 and -p2 vectors are not as stable as other plasmids such as pBluescript. In order to generate plasmids containing insert with no obvious deletions or rearrangements of the vector, it is normally necessary to pick at least 10 colonies at a time for overnight liquid culture. More than one set of 10 may need to be screened by restriction digestion.

4. The conditions for IPTG induction may need to be optimized for the particular fusion. Lower or higher IPTG concentrations, induction for shorter or longer times, and induction at higher cell densities can all be tested. Problems with fusion protein solubility can also be improved by altering the growth and induction conditions—for example, expressing the protein at a lower temperature—but often it is easier to optimize for maximum expression and then solubilize the protein inclusions, especially if the application does not require native folded protein.

5. Adding additional protease inhibitors at this step may help with protein stability if this is a problem.

6. Many refolding protocols are available. A good selection to start from can be found in the pMAL handbook (at http://www.neb.com).

References

1. Dudhia, J., Davidson, C. M., Wells, T. M., Vynios, D. H., Hardingham, T. E., and Bayliss, M. T. (1996) Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage. Biochem. J. 313, 933-940.

2. Zimmerman, D. R., Dours-Zimmerman, M. T., Schubert, M., and Bruckner-Tuderman, L. (1994) Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis. J. Cell Biol. 124, 817-825.

3. Murdoch, A. D., Liu, B., Schwarting, R., Tuan, R. S., and Iozzo, R. V. (1994) Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J. Histochem. Cytochem. 42, 239-249.

4. Whitelock, J. M., Murdoch, A. D., Iozzo, R. V., and Underwood, P. A. (1996) The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J. Biol. Chem. 271, 10,079-10,086.

5. Ujita, M., Shinomura, T., Ito, K., Kitagawa, Y., and Kimata, K. (1994) Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chon-droitin sulfate proteoglycan. J. Biol. Chem. 269, 27,603-27,609.

6. Brinkmann, T., Weilke, C., and Kleesiek, K. (1997) Recognition of acceptor proteins by UDP-D-xylose proteoglycan core protein beta-D xylosyltransferase. J. Biol. Chem. 272, 11,171-11,175.

7. Weilke, C., Brinkmann, T., and Kleesiek, K. (1997) Determination of xylosyltransferase activity in serum with recombinant human bikunin as acceptor. Clin. Chem. 43, 45-51.

8. Williamson, R. A., Natalia, D., Gee, C. K., Murphy, G., Carr, M. D., and Freedman, R. B. (1996) Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies. Eur. J. Biochem. 241, 476-483.

9. Ruppert, R., Hoffman, E., and Sebald, W. (1996) Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur. J. Biochem. 237, 295-302.

10. Guan, C., Li, P., Riggs, P. D., and Inouye, H. (1987) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67, 21-30.

11. Maina, C. V., Riggs, P. D., Grandea, A. G. III, Slatko, B. E., Moran, L. S., Tagliamonte, J. A., McReynolds, L. A., and Guan, C. (1988) A vector to express and purify foreign proteins in Escherichia coli by fusion to, and separation from, maltose binding protein. Gene 74, 365-373.

12. Sambrook, J., Fritsch, E. F., and Maniatis, T., ed (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MA.

13. Marston, F. A. O., Lowe, P. A., Doel, M. T., Schoemaker, J. M., White, S., and Angal, S. (1984) Purification of calf prochymosin (prorennin) synthesised in Escherichia coli. Bio/ Technology 2, 800-804.

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