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Glycoprotein methods protocols - biotechnology
MUC1 in Insect Cells 47147139Expression of MUC1 in Insect CellsUsing Recombinant BaculovirusPawel Ciborowski and Olivera J. Finn1. IntroductionMUC1 mucin undergoes multistep posttranslational modifications before it isfinally expressed on the apical surface of mammalian ductal epithelial cells. Two earlyprecursor proteins are both N-glycosylated and differ in molecular weight owing to aproteolytic cleavage of a 20-kDa fragment. Proteolytically modified form is trans-ported to the Golgi, where it undergoes extensive, although not complete, O-gly-cosylation on serine and threonine residues within the tandem repeat (TR) region.MUC1 is then transported to the cell surface. For additional glycosylation andsialylation, surface MUC1 is internalized and directed to trans-Golgi compartments.Mature form is again transported to the cell surface (1).MUC1 expressed by malignant epithelial cells such as breast and pancreatic adeno-carcinomas is underglycosylated (aberrantly glycosylated), which makes it structur-ally and antigenically distinct from that expressed by normal cells (2). As such, it maybe an excellent target for immunotherapy. One of the ways to utilize tumor-specificforms of this molecule is as immunogens. Purifying these forms from tumor cells isnot feasible because it is a labor-intensive process that gives low yields. A much moredesirable approach is purification of a recombinant molecule from an appropriateexpression system. Recombinant MUC1 expressed in a convenient prokaryotic sys-tem that does not glycosylate proteins, such as Escherichia coli, undergoes rapid andrandom proteolytic degradation. To obtain underglycosylated recombinant tumorlikeforms of MUC1 in mammalian cells through expression vectors such as vaccinia virus,retroviral vectors, and plasmid vectors requires a prolonged treatment of infected ortransfected cells with toxic and expensive inhibitors of O-linked glycosylation (3,4).Furthermore, vaccinia and retroviral constructs spontaneously recombine out most TRsthat characterize the major portion and the most immunogenic portion of MUC1 (5).We explored the baculovirus system that allows expression of MUC1 mucin inSpodoptera frugiperda Clone 9 (Sf-9) insect cells. We found that these cells, whenFrom:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 472 Ciborowski and Finninfected with a MUC1 recombinant baculovirus, produce fully glycosylated, full-size(no deletion of TRs) molecules that are expressed on the cell surface (6). Moreover,under specific starvation growth conditions that we determined empirically, Sf-9 cellscan also produce underglycosylated MUC1, similar to the MUC1 produced by tumorcells. The state of glycosylation of various forms can be evaluated by their migrationin sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels andreactivity with different anti-MUC1 antibodies in Western blot analysis (6,7). In thischapter, we present the techniques of expression of MUC1 mucin using threebaculoviral vectors: pBlueBacIII, pFastBac, and pIE1-4. Additional vectors are com-mercially available and, as one can expect, more will emerge on the market in thefuture. In our opinion, they provide an ideal expression system to study different formsof MUC1 protein, their function, and utility.2. Materials2.1. Cloning Reagents1. Vectors: pBlueBacIII was purchased from Invitrogen, San Diego, CA (see Note 1);pFastBac was purchased from Gibco, Life Technologies, Grand Island, NY; and pIE1-4was purchased from Novagen, Madison, WI.2. Competent E. coli cells such as MAX Efficiency DH5α™ Competent Cells and MAXEfficiency DH10Bac™ Competent Cells were obtained from Gibco-BRL.3. Restriction enzymes, agarose, ligase, and other reagents for cloning may be obtained fromany supplier of molecular biology reagents. Wizard™ Minipreps and Wizard™ Megaprepswere obtained from Promega, Madison, WI. Cationic liposomes InsecticinPlus™ wereobtained from Invitrogen, but can be also obtained from other commercial sources.BluoGal and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sigma,St. Louis, MO; X-gal was purchased from Boehringer Mannheim, Indianapolis, IN; andSeaPlaque agarose was purchased from FMC BioProducts, Rockland, ME.2.2. Cells, Media, and Antibodies1. The insect cell line Sf-9 can be obtained from American Type Culture Collection(Rockville, MD) or from other suppliers such as Invitrogen, San Diego, CA.2. Hink’s TNM-FH Insect Medium can be obtained from several sources such as JRH Bio-sciences, Lenexa, KS. Penicillin, streptomycin, fungizone, and geneticin can be obtainedfrom Gibco. Fetal bovine serum (FBS) was from Gibco-BRL.3. Anti-MUC1 antibodies used in this study are not commercially available. Monoclonalantibodies (MAbs) used for Western blot and flow cytometry analysis are listed inTable 1. The TD-4 MUC1 Workshop (see ref. 7, pp. 1–152) provides the most up-to-datelist of anti-MUC1 antibodies, their specific reactivities, and their sources.4. Tissue culture flasks, plates, roller bottles, and disposable plastic tubes of various sizescan be obtained from various sources, e.g., Sarsted, Falcon, etc. Any 27°C incubator canbe used, although one with a water jacket is recommended.2.3. Western BlotAll reagents and equipment for PAGE and Western blot, except nitrocellulose, werepurchased from Bio-Rad, Hercules, CA. Other suppliers can also be used. Nitrocellu-lose BioBlot-NC was purchased from Corning Costar, Corning, NY. Chemilumines- MUC1 in Insect Cells 473cence Western blotting detection kit was purchased from Amersham, Buckinghamshire,England.3. Methods3.1. Vector ConstructionThe cDNAs coding for MUC1 of various lengths owing to various numbers of TRswere obtained from previously made plasmid constructs. Plasmid expression vectorsencoding MUC1 with 22 repeats (22TRMUC1) and two repeats (2TRMUC1) weremade in our laboratory (3). The cDNAs can be isolated from the plasmid vectors asHindIII cassettes (Fig. 1). Plasmid expression vectors containing MUC1 cDNA with42 TRs (42TRMUC1) and MUC1 cDNA without TRs (TR–MUC1), both BamHI cas-settes, were obtained from Dr. A. Hollingsworth, University of Nebraska, Omaha.3.1.1. Cloning into pBlueBacIII Transfer VectorAn example we will use for cloning of the 3.2-kbp cDNA MUC1 with 22 TRs(22TRMUC1) and the 1.8-kbp cDNA MUC1 with 2 TRs (2TRMUC1). The resultingpBlueBacIII-22TR-MUC1 recombinant transfer plasmid is used for inserting MUC1cDNA into the genome of the wild-type Autographa californica Multiple NuclearPolyhedrosis Virus (wtAcMNPV), as described under Subheading 3.3.1.1. Digest pBlueBacIII transfer vector with HindIII or BamHI and treat with calf intestinephosphatase (CIP) to protect against self-ligation using standard methodology (see Note1 and 2).2. Prepare MUC1 cDNA cassette by HindIII digestion.3. Purify fragments by electrophoresis in 0.7% agarose.4. Ligate the cDNA cassette into the pBlueBacIII vector using T4 DNA ligase at 16°C over-night.Table 1MUC1 Specific AntibodiesaAntibody Isotype SpecificitySM-3 IgG1 APDTRPb, underglycosylatedcVU-4-H5 IgG1 PDTRPAP, underglycosylatedcVU-3-C6 IgG1 PDTRPAP, all formsBC-3 IgM APDTR, all formsBC-2 IgG1 APDTR, all form232A1 IgG Proteolytic cleavage siteaFor more details about antibodies see ISOBM TD-4 International Workshopon Monoclonal Antibodies against MUC1, Tumor Biology, 1998, 19 (Suppl. 1),1–152.bSingle letter code for amino acids. A, alanine; P, proline; T, threonine; D,glutamic acid; R, arginine.cTumor specific, recognizing underglycosylated but not fully glycosylatedMUC1. 474 Ciborowski and Finn5. Transform the ligated construct into E. coli MAX Efficiency DH5α Competent Cells fol-lowing the protocol provided by the manufacturer.6. Select recombinants using Luria agar with 10 µg/mL of ampicillin (8).7. Amplify ampicillin-resistant clones in 5-mL Luria broth/ampicillin (8) cultures. Use 1.5–3.0 mL of the culture to isolate plasmid DNA using WizardMinipreps.8. Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert.3.1.2. Cloning into pFastBac Transfer VectorAs an example, we will use cloning of 4.6-kbp cDNA with 42 TRs (42TRMUC1).Fragment of MUC1 cDNA coding for transmembrane and cytoplasmic domains wasreplaced with a sequence linking the outer membrane portion of MUC1 withglycosylphosphatidylinositol (GPI) anchor of human decay accelerating factor. Thisnew construct (42TRMUC1-GPI) was made in our laboratory and remains as a BamHIcassette (Alter, M., unpublished data). The resulting pFastBac-42TRMUC1-GPIrecombinant transfer plasmid is used for inserting MUC1 cDNA into the genome ofthe wtAcMNPV, as described under Subheading 3.3.2.1. Linearize pFastBac transfer vector with BamHI digestion and protect it with CIP againstself-ligation using standard methodology.2. Cut out the 42TRMUC1-GPI cDNA cassette by BamHI digestion.3. Purify a fragment of the correct size by electrophoresis in 0.7% agarose.4. Ligate the cDNA cassette into the vector using T4 DNA ligase at 16°C for overnight.5. Transform E. coli MAX Efficiency DH5αCompetent Cells with the ligated construct.6. Select recombinants using Luria agar with 10 µg/mL of ampicillin.7. Select ampicillin-resistant clones, and amplify and purify plasmid DNA using Wizard Minipreps.8. Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert.Fig. 1. MUC1 cDNA expression plasmid. The MUC1 cDNA is downstream from transla-tional start codon. Constructs with 2 or 22 TRs that were made in our laboratory are containedin the HindIII. MUC1 in Insect Cells 4753.1.3. Cloning into the Episomal Transfer Vector pIE1-4The vector pIEI-4 is used to provide stable expression of a cloned gene from thebaculovirus ie1 promoter. Cells are cotransfected with pIE1-neo providing neomycinselection marker expressed from ie1 promoter. As an example, we will use cloning of1.4-kbp MUC1 cDNA that lacks TRs (TR–MUC1). The resulting pIE1-4TR–MUC1-GPI recombinant transfer plasmid is used for cotransfection of Sf-9 cells with pIE-neo, as described under Subheading 3.5.1. Linearize the pIE1-4 transfer vector with BamHI and protect it with CIP against self-ligation using standard methodology (see Note 3).2. Prepare TR–MUC1 cDNA cassette by BamHI digestion.3. Purify the desired fragment by electrophoresis in 0.7% agarose.4. Ligate the cDNA cassette into the vector using T4 DNA ligase at 16°C for overnight.5. Transform E. coli MAX Efficiency DH5α Competent Cells with the ligated construct.6. Select recombinants using Luria agar with 10 µg/mL of ampicillin. Amplify ampicillin-resistant clones and purify plasmid DNA using WizardMinipreps.7. Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert.3.2. Conditions for Culturing the Sf-9 CellsSf-9 insect cells are cultured in Hink’s TNM-FH Insect Medium supplemented with5 or 10% FBS and penicillin/streptomycin/fungizone at the concentrations of 100U/mL, 100 µg/mL, and 2.5 µg/mL, respectively. Cells are grown as a monolayer at27°C. For small scale growth, 75-cm2vented flasks are used (Costar, Cambridge, MA).Typically 5 × 105cells and 20 mL of medium are used to start the culture of this size.For larger-scale growth, roller bottles are used. Cultures are usually started at the celldensity of 106cells/mL. During the logarithmic phase of growth, cells typically doubleevery 24 h. Therefore, equal amounts of fresh medium are added each day to the rollerbottle for up to 300 mL total volume. Figure 2 shows the kinetics of growth in atypical roller bottle culture (see Note 4).3.3. Production of Recombinant Virus by Cotransfection with Viraland Recombinant Transfer Vector DNAsThe wtAcMNPV viral DNA and the recombinant transfer vector DNA are shuttledinto Sf-9 cells by cationic liposomes. Within the cells, transfer vector DNA and viralDNAs recombine, incorporating the gene of interest into the viral genome. Dependingon the transfer vectors different protocols can be used to make recombinant virus. Twoprotocols are given next.3.3.1. Using pBlueBacIII VectorWhen using pBlueBacIII vectors, recombination leads to the replacement of theviral polyhedrin gene (phenotypically occ+) with part of the transfer vector containinglacZ gene and gene of interest. Therefore, the selection is based on the phenotypicobservation—lack of occlusion bodies (occ-) and expression of β-galactosidase(lacZ+).1. Seed Sf-9 cells in a 6-well plate (106cells/well) prior to the cotransfection, and rock themgently side-to-side for 1 h at room temperature to evenly distribute and attach the cells. 476 Ciborowski and Finn2. Remove nonattached cells and medium, gently wash the adherent monolayer once withserum-free medium, cover with 2 mL of serum free medium, and incubate for 30 min atroom temperature.3. Prepare five independent transfection mixtures. Mix 100, 200, 500, and 750 ng, or 1 µg ofthe recombinant pBlueBacIII transfer plasmid, respectively, with 500 ng of linearizedAcMNPV DNA, 40 mL Insectin-Plus Liposomes, and 1 ml of Hink’s TNM-FH InsectMedium.4. Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min.5. Remove serum-free medium from the cells, cover cell monolayer with one of the transfec-tion mixtures, swirl to mix, and incubate for 4 h at room temperature with slow rocking.6. Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% FBS) to eachwell, wrap plates with clear plastic wrap, and incubate at 27°C for 48 h.7. Take 100 µL of the culture supernatant that contains viruses produced by the transfected cellsfrom each well and screen by plaque assay for the presence of double recombinants (occ-,lacZ+). Transfer the remaining medium to sterile microcentrifuge tubes and store at 4°C.3.3.2. Using pFastBac Transfer VectorpFastBac transfer vector is a part of the Bac-To-Bac™ Baculovirus ExpressionSystem developed by Gibco. In the first step, competent MAX Efficiency DH10BacE. coli cells are transformed with pFastBac donor plasmid with a gene of interest. Thecompetent DH10Bac E. coli cells contain baculovirus shuttle vector (bacmid) and aFig. 2. Growth of SF-9 cells in a typical roller bottle culture. Infection was on d 4 and nonew medium was added afterward. On d 5 all cells were expressing β-galactosidase (see Sub-heading 3.6.1. for details). Cells were usually harvested after 72 h. MUC1 in Insect Cells 477helper plasmid. Bacmid propagates in E. coli and, besides viral DNA, contains severalother elements such as attachment sites for transposon Tn7 and an open reading frameof lacZαpeptide. Recombination between pFastBac transfer vector and bacmid occurswithin bacterial cells by transposition, with the aid of helper plasmid. Another featureof this system is that insertion of the gene of interest into the viral genome causesdisruption of lacZ gene and E. coli containing recombinant bacmid grow as whitecolonies in the presence of Bluo-gal and IPTG. This feature makes the system easy touse and eliminates posttransfection isolation of recombinant viruses.1. Transform E. coli MAX Efficiency DH10Bac Competent Cells that contain Bacmid DNAand helper plasmid. After transformation with recombinant pFastBac-MUC1-42TR-GPI,the transposition occurs inside E. coli cells disrupting LacZ gene within Bacmid DNA.2. Select white growing colonies from Luria agar supplemented with kanamycin (50 µg/mL),tetracycline (10 µg/mL), gentamicin (7 µg/mL), BluoGal (100 µg/mL), and IPTG (40 µg/mL)to make a larger amount of the recombinant Bacmid DNA.3. Amplify selected clones and purify Bacmid DNA using Wizard Minipreps.4. Seed 106Sf-9 cells/well in a 6-well plate immediately prior to transfection, and rock themgently side-to-side for 1 h at room temperature to evenly distribute and attach the cells.Alternatively, seed 2.5 × 105/well and grow them until desired density, usually 1 to 2 d.5. Remove nonattached cells and medium, wash gently the cell monolayer once with serumfree medium, cover with 2 mL of serum-free medium, and incubate for 30 min at roomtemperature.6. Prepare three independent transfection mixtures: 100 to 200 ng of Bacmid DNA, mixedwith 20, 40, or 60 µL Insectin-PlusLiposomes, and 1 mL of Hink’s TNM-FH InsectMedium.7. Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min.8. Remove serum-free medium from the wells, cover cell monolayers with the transfectionmixture, swirl to mix, and incubate for 4 h at room temperature with slow rocking.9. Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% of FBS) to eachwell, wrap with clear plastic wrap, and incubated at 27°C for 48 h.10. Transfer the culture supernatants that contain viruses produced by the transfected cells tosterile microtubes and store at 4°C.3.4. Isolation of Recombinant BaculovirusThe culture supernatants from cells cotransfected with the pBlueBacIII as a transfervector contain a mixture of recombinant and wild-type viruses. To isolate recombi-nant viruses, a plaque assay is performed followed by an end-point dilution round ofpurification.3.4.1. Plaque Assay1. Seed Sf-9 cells in 6-cm dishes with 2 × 106cells/dish, and rock them gently side-to-sidefor 1 h at room temperature to evenly distribute and attach the cells.2. Grow cells at 27°C to approx 80% confluency. Alternatively, seed more cells, and after 2to 3 h during which the cells attach, the plates are ready for plaque assay.3. Make a serial 10-fold dilution of the medium harvested from transfected cells, in fullHink’s TNM-FH Insect Medium. Dilutions should range from 10–1 to 10–5.4. Remove the medium from the wells and add 1 mL of the culture supernatant containing amixture of wild-type and recombinant viruses (viral inoculum) to the side of the dish, and 478 Ciborowski and Finntilt the dish slowly to cover evenly all cells. It is important to do this gently in order not todisturb the attached cells. Incubate at room temperature for 1 h.5. Melt the required volume of 3% SeaPlaque agarose, cool down to 45°C, and keep in awater bath. Prewarm to 37°C an equal volume of Hink’s TNM-FH Insect Medium towhich was added 120 µg/mL of X-gal.6. Remove the viral inoculum by tilting the plate and aspirating from the edge.7. Mix warm agar with medium and overlay dishes with 5 ml of this medium. Leave leveleduntil agarose sets.8. Incubate at 27°C until blue plaques develop, usually 5–7 d.9. Using a sterile Pasteur pipet, pick isolated plaques with the recombinant virus (blue plaqueswithout occlusion bodies), transfer to tubes containing 2 to 3 mL of Hink’s TNM-FH InsectMedium, and vortex for 30 s. Allow viral particles to diffuse from agar for another hour atroom temperature. This could be used for screening and further purification.3.5. Production of an Episomal Recombinant Vectorfor Stable Expression3.5.1. Using pIE1-4 Vector1. Seed Sf-9 cells in a 6-well plate (106cells/well) prior to the cotransfection, and rock themgently side-to-side for 1 h at room temperature to evenly distribute and attach the cells.2. Remove nonattached cells and medium, gently wash the adherent monolayer once withserum-free medium, cover with 2 mL of serum free medium, and incubate for 30 min atroom temperature.3. Prepare five independent transfection mixtures. Mix 3 µg of recombinant transfer plas-mid pIE1-4-TR–-MUC1; 400 ng of pIE-neo plasmid DNA, and 20, 40, 60, 80, or 100 µLof Insectin-Plus liposomes. For mocktransfection, use 20 µL of Insectin-Plus liposomes.4. Vortex transfection mixtures vigorously for 10 s and incubate at room temperature for 30 min.5. Remove serum-free medium from the wells, cover cell monolayer with the transfectionmixture, swirl to mix, and incubate for 4 h at room temperature with slow rocking.6. Add 2 mL of complete Hink’s TNM-FH Insect Medium (containing 10% FBS) to eachwell, wrap the plates with clear plastic wrap, and incubate at 27°C for 48 h.7. Replace medium after 48 h with new medium containing 600 µg/mL of neomycin andgrow cells for another 7 d.8. After 7 d cells can be tested for MUC1 expression by flow cytometry and Western blot(see Notes 5 and 6).3.6. Infection of Sf-9 Cells and MUC1 Production3.6.1. Flask Cultures1. Grow cells to approx 100% confluency.2. Aspirate all medium and cover the cell monolayer with the minimal volume of a viralstock at 3 multiplicity of infection (see Note 7). This is usually 4 to 5 mL/75-cm2 flask.3. Rock the flask for 1 h at room temperature.4. Add 20 mL of serum-free medium and incubate at 27°C for a desired time. To controlyield of infection with recombinant virus carrying lacZ gene, aliquot a small sample ofthe culture (cells and supernatant) into a 1.5-mL microtube containing 1 µL of X-Gal at aconcentration of 40 mg/mL, and incubate for 30 to 60 min at room temperature. After thattime, infected cells should exhibit blue color when observed microscopically owing toβ-galactosidase expression, and the culture supernatant should turn blue. If recombinantvirus does not carry and express lacZ gene, other signs of infection such as swollen nuclei MUC1 in Insect Cells 479can be used to assess the efficiency of infection. Typically more than 80% of cells areinfected within the first day.3.6.2. Roller Bottle Cultures1. Start a roller bottle culture with Sf-9 cells in 20 mL of in Hink’s TNM-FH Insect Medium,at a density of 106cells/mL. The cell number usually doubles once every 24 h. Expandcells by adding every day an equal amount of fresh medium: 20 mL on d 2, 40 mL on d 3,80 mL on d 4, and so forth, up to half the desired final volume of the culture (see Note 8).2. Infect cells with the viral stock. There are two procedures for infection of a roller bottleculture. In the first, the culture is infected by adding the viral stock directly to the bottleand supplementing the culture with an equal amount of fresh medium 2 to 3 h after infec-tion. The second method is to collect cells by centrifugation, resuspend in minimal vol-ume of medium, usually one-tenth of the original, add the viral stock, and rock for 1 h at roomtemperature. Transfer infected cells back to the bottle, and add fresh medium in the amountequal to that prior to infection. We found both methods worked equally well. The only differ-ence is that in the first method, in order to accomplish complete infection of all insect cellswithin 24 h, cultures should be infected at 5 m.o.i. or higher. In the second method, althoughmore laborious, less viral stock is used and infection at 3 m.o.i. is sufficient.3.7. Starving Sf-9 Cells in Cultureto Obtain Underglycosylayted Forms of MUC13.7.1. Flask Cultures1. Grow cells to 100% confluency, and prolong the time of culture for another day or twowithout changing the medium in order to deplete most of the nutrients. At this time, thenumber of dead cells slightly increases.2. Aspirate all medium, clear by centrifugation and filtration through a 0.22-µm filter, and save.3. Cover the cell monolayer with a minimal volume of the viral stock, usually 4 to 5 mL/75-cm2flask.4. Rock the flask for 1 h at room temperature.5. Add the saved nutrients-depleted medium and incubate the culture at 27°C for a desiredtime. To control efficiency of infection with recombinant virus carrying lacZ gene, fol-low the procedure described under Subheading 3.6.2.3.7.2. Roller Bottle Cultures1. Start and expand a roller bottle culture as described under Subheading 3.6.2. Starve cells by growing at high density for 48 h without supplementing with freshmedium.3. Infect cells with a viral stock as described under Subheading 3.6. In starved cultures, ifcells are harvested for infection, nutrient-depleted medium should be collected, clearedby centrifugation and filtration through a 0.22-µm filter, and added back to the bottle.3.8. Analysis of Recombinant MUC1 Produced by Sf-9 CellsThe quickest method to test the expression of MUC1 in insect cells is byimmunostaining with specific MAbs. There is a large number of well characterizedMAbs against different peptide and sugar epitopes on MUC1. Western blot is themethod of choice for testing total expression, and flow cytometry is used for testingcell surface expression. The pattern of reactivity of baculovirus expressed MUC1 with 480 Ciborowski and Finnvarious anti-MUC1 MAbs does not reflect the degree of glycosylation of the recombi-nant product. Although we do not provide a detailed protocol for Western blot andflow cytometry in this chapter, some technical details could be found in the figurelegends (see Note 9).MUC1 is the only transmembrane mucin known to date. It is transported to theapical surface of the ductal epithelial cells and anchored in the cell membrane via itstransmembrane domain. MUC1 is removed from the cell surface by proteolytic cleav-age in the membrane proximal domain, or, to lesser extent, it is internalized anddegraded in the phagolysosomes. In insect cells, MUC1 also undergoes complex pro-cess of posttranslational modification as in mammalian cells. It is also transported tothe surface of Sf-9 cells (6).A single, large band with an apparent molecular weight above the 221 kDa, repre-sents the only form of MUC1 expressed by insect cells when they are grown in fullysupported medium containing 10% FBS. However, when cells are starved for at leasttwo days prior to infection and then grown in nutrient-depleated medium, the majorityFig. 3. Different forms of MUC1 expressed in Sf-9 cells using recombinant baculovirus.Form designated as (A) is a protein precursor which undergoes a proteolytic modifictionyeilding a 20 kDa smaller protein (B). Form (C) has not been characterized yet, and form (D) isMUC1 released from the cell surface. [...]... and C represent cells stained respectively with IgG-control, BC-3, or VU-4H5 primary antibody followed with fluorescein isothiocyanate (FITC)-labeled secondary antibody. Antibody BC-2, like antibody BC-3, recognizes MUC1 indepen- dent of its state of glycosylation, while antibody VU-4H5 recognizes underglycosylated, tumor-specific MUC1 similarly to SM-3. It has been recently reported by Wright (12) and... adherent monolayer once with serum-free medium, cover with 2 mL of serum free medium, and incubate for 30 min at room temperature. 3. Prepare five independent transfection mixtures. Mix 3 µg of recombinant transfer plas- mid pIE 1-4 -TR – -MUC1; 400 ng of pIE-neo plasmid DNA, and 20, 40, 60, 80, or 100 µL of Insectin-Plus liposomes. For mocktransfection, use 20 µL of Insectin-Plus liposomes. 4. Vortex transfection... (1989) Specific, MHC-unrestricted recognition of tumor associated mucins by human cytotoxic T cells. Proc. Natl. Acad. Sci. USA 86, 7159–7163. 3. Jerome, K. R., Bu, D., and Finn, O. J. (1992) Expression of tumor-associated epitopes on Epstein-Barr Virus-immortalized B-cells and Burkitt’s Lymphomas transfected with epi- thelial mucin complimentary DNA. Cancer Res. 52, 5985–5990. 4. Magarian-Blander, J., Domenech,... Hink’s TNM-FH Insect Medium, and vortex for 30 s. Allow viral particles to diffuse from agar for another hour at room temperature. This could be used for screening and further purification. 3.5. Production of an Episomal Recombinant Vector for Stable Expression 3.5.1. Using pIE 1-4 Vector 1. Seed Sf-9 cells in a 6-well plate (10 6 cells/well) prior to the cotransfection, and rock them gently side-to-side... Denko K.K., Tokyo, Japan), and Sephadex (CM- Sephadex from Pharmacia, Uppsala, Sweden), significantly decreasing yield of sepa- Fig. 4. Time course of MUC1 expression. Cell lysates (C) and supernatants (S) were ana- lyzed by SDS-PAGE in 7% gel under reducing conditions. After transblotting, one membrane was probed with BC-3 MAb (A), and the other was probed with SM-3 MAb (B). The arrows on the left indicate... and effective T-cell recognition of cells transfected with truncated human mucin cDNA. In: Specific Immuno- therapy of Cancer with Vaccines. Ann. NY Acad. Sci. 690, 231–243. 5. Bu, D., Domenech, N., Lewis, J., Taylor-Papadimitriou, J. and Finn, O. J. (1993) Recom- binant vaccinia mucin vector: in vitro analysis of expression of tumor-associated epitopes for antibody and human cytotoxic T-cell recognition.... undergoes a proteolytic modification yielding a 20 kDa smaller protein desig- nated as form (b). Form (c) has not been characterized yet, and form (d) is MUC 1- released from the cell surface (Fig. 4, lane 72 h/s). It is important to note that MUC1 is mostly cell associated and only a very small fraction of the low-molecular-weight form is found in the supernatant. It must be noted, however, that in... Baculovirus Expression Vec- tors. A Laboratory Manual. Oxford University Press, New York, pp. 124–138. 12. Hu, P. and Wright, S. E. (1998) Recombinant breast carcinoma-associated mucins expressed in a baculovirus system contain a tumor specific epitope. Immunotechnology 4, 97–105. 482 Ciborowski and Finn phy precolumn from Toso-Haas, Stuttgart, Germany), silica (HPLC size-exclusion column from Shodex,... not clear why insect cells do not O-glycosylate this recombinant product. GalNAc trans- ferases purified from insect cells are able to glycosylate in vitro a synthetic peptide corresponding to one tandem repeat but not the recombinant product. 3.9. Large-Scale Purification of Recombinant MUC1 The most commonly used method for purification of a protein from complex mix- tures is affinity chromatography... mucin effectively. Recom- binant MUC1 is found in the top layer containing lipids. The form of MUC1 that is shed from the cell surface and does not have transmembrane and cytoplasmic domains migrates to the middle of the CsCl gradient (approx 1.4 g/mL). MUC1 binds nonspecifically to various resins such as glass (high-performance liquid chromatogra- MUC1 in Insect Cells 485 MUC1/neo-positive population . over-night.Table 1MUC1 Specific AntibodiesaAntibody Isotype SpecificitySM-3 IgG1 APDTRPb, underglycosylatedcVU-4-H5 IgG1 PDTRPAP, underglycosylatedcVU-3-C6. µg of recombinant transfer plas-mid pIE 1-4 -TR–-MUC1; 400 ng of pIE-neo plasmid DNA, and 20, 40, 60, 80, or 100 µLof Insectin-Plus liposomes. For mocktransfection,