MUC1 in Insect Cells 471
471
39
Expression of MUC1 in Insect Cells
Using Recombinant Baculovirus
Pawel Ciborowski and Olivera J. Finn
1. Introduction
MUC1 mucin undergoes multistep posttranslational modifications before it is
finally expressed on the apical surface of mammalian ductal epithelial cells. Two early
precursor proteins are both N-glycosylated and differ in molecular weight owing to a
proteolytic 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 and
sialylation, 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 may
be an excellent target for immunotherapy. One of the ways to utilize tumor-specific
forms of this molecule is as immunogens. Purifying these forms from tumor cells is
not feasible because it is a labor-intensive process that gives low yields. A much more
desirable approach is purification of a recombinant molecule from an appropriate
expression system. Recombinant MUC1 expressed in a convenient prokaryotic sys-
tem that does not glycosylate proteins, such as Escherichia coli, undergoes rapid and
random proteolytic degradation. To obtain underglycosylated recombinant tumorlike
forms of MUC1 in mammalian cells through expression vectors such as vaccinia virus,
retroviral vectors, and plasmid vectors requires a prolonged treatment of infected or
transfected cells with toxic and expensive inhibitors of O-linked glycosylation (3,4).
Furthermore, vaccinia and retroviral constructs spontaneously recombine out most TRs
that characterize the major portion and the most immunogenic portion of MUC1 (5).

We explored the baculovirus system that allows expression of MUC1 mucin in
Spodoptera frugiperda Clone 9 (Sf-9) insect cells. We found that these cells, when
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
472 Ciborowski and Finn
infected 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 cells
can also produce underglycosylated MUC1, similar to the MUC1 produced by tumor
cells. The state of glycosylation of various forms can be evaluated by their migration
in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and
reactivity with different anti-MUC1 antibodies in Western blot analysis (6,7). In this
chapter, we present the techniques of expression of MUC1 mucin using three
baculoviral vectors: pBlueBacIII, pFastBac, and pIE1-4. Additional vectors are com-
mercially available and, as one can expect, more will emerge on the market in the
future. In our opinion, they provide an ideal expression system to study different forms
of MUC1 protein, their function, and utility.
2. Materials
2.1. Cloning Reagents
1. Vectors: pBlueBacIII was purchased from Invitrogen, San Diego, CA (see Note 1);
pFastBac was purchased from Gibco, Life Technologies, Grand Island, NY; and pIE1-4
was purchased from Novagen, Madison, WI.
2. Competent E. coli cells such as MAX Efficiency DH5α™ Competent Cells and MAX
Efficiency DH10Bac™ Competent Cells were obtained from Gibco-BRL.
3. Restriction enzymes, agarose, ligase, and other reagents for cloning may be obtained from
any supplier of molecular biology reagents. Wizard™ Minipreps and Wizard™ Megapreps
were obtained from Promega, Madison, WI. Cationic liposomes InsecticinPlus™ were
obtained 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; and
SeaPlaque agarose was purchased from FMC BioProducts, Rockland, ME.
2.2. Cells, Media, and Antibodies
1. 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 obtained
from Gibco. Fetal bovine serum (FBS) was from Gibco-BRL.
3. Anti-MUC1 antibodies used in this study are not commercially available. Monoclonal
antibodies (MAbs) used for Western blot and flow cytometry analysis are listed in
Table 1. The TD-4 MUC1 Workshop (see ref. 7, pp. 1–152) provides the most up-to-date
list of anti-MUC1 antibodies, their specific reactivities, and their sources.
4. Tissue culture flasks, plates, roller bottles, and disposable plastic tubes of various sizes
can be obtained from various sources, e.g., Sarsted, Falcon, etc. Any 27°C incubator can
be used, although one with a water jacket is recommended.
2.3. Western Blot
All reagents and equipment for PAGE and Western blot, except nitrocellulose, were
purchased 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 473
cence Western blotting detection kit was purchased from Amersham, Buckinghamshire,
England.
3. Methods
3.1. Vector Construction
The cDNAs coding for MUC1 of various lengths owing to various numbers of TRs
were obtained from previously made plasmid constructs. Plasmid expression vectors
encoding MUC1 with 22 repeats (22TRMUC1) and two repeats (2TRMUC1) were
made in our laboratory (3). The cDNAs can be isolated from the plasmid vectors as

HindIII cassettes (Fig. 1). Plasmid expression vectors containing MUC1 cDNA with
42 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 pBlue
Bac
III Transfer Vector
An 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 resulting
pBlueBacIII-22TR-MUC1 recombinant transfer plasmid is used for inserting MUC1
cDNA into the genome of the wild-type Autographa californica Multiple Nuclear
Polyhedrosis Virus (wtAcMNPV), as described under Subheading 3.3.1.
1. Digest pBlueBacIII transfer vector with HindIII or BamHI and treat with calf intestine
phosphatase (CIP) to protect against self-ligation using standard methodology (see Note
1 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 1
MUC1 Specific Antibodies
a
Antibody Isotype Specificity
SM-3 IgG1 APDTRP
b
, underglycosylated
c
VU-4-H5 IgG1 PDTRPAP, underglycosylated
c

VU-3-C6 IgG1 PDTRPAP, all forms
BC-3 IgM APDTR, all forms
BC-2 IgG1 APDTR, all form
232A1 IgG Proteolytic cleavage site
a
For more details about antibodies see ISOBM TD-4 International Workshop
on Monoclonal Antibodies against MUC1, Tumor Biology, 1998, 19 (Suppl. 1),
1–152.
b
Single letter code for amino acids. A, alanine; P, proline; T, threonine; D,
glutamic acid; R, arginine.
c
Tumor specific, recognizing underglycosylated but not fully glycosylated
MUC1.
474 Ciborowski and Finn
5. 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 Wizard
Minipreps.
8. Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert.
3.1.2. Cloning into pFast
Bac
Transfer Vector
As 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 was
replaced with a sequence linking the outer membrane portion of MUC1 with
glycosylphosphatidylinositol (GPI) anchor of human decay accelerating factor. This
new construct (42TRMUC1-GPI) was made in our laboratory and remains as a BamHI

cassette (Alter, M., unpublished data). The resulting pFastBac-42TRMUC1-GPI
recombinant transfer plasmid is used for inserting MUC1 cDNA into the genome of
the wtAcMNPV, as described under Subheading 3.3.2.
1. Linearize pFastBac transfer vector with BamHI digestion and protect it with CIP against
self-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 DH5α
Competent 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 contained
in the HindIII.
MUC1 in Insect Cells 475
3.1.3. Cloning into the Episomal Transfer Vector pIE1-4
The vector pIEI-4 is used to provide stable expression of a cloned gene from the
baculovirus ie1 promoter. Cells are cotransfected with pIE1-neo providing neomycin
selection marker expressed from ie1 promoter. As an example, we will use cloning of
1.4-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 Wizard
Minipreps.
7. Analyze recombinant DNA by restriction enzyme digestion for orientation of the insert.
3.2. Conditions for Culturing the Sf-9 Cells
Sf-9 insect cells are cultured in Hink’s TNM-FH Insect Medium supplemented with
5 or 10% FBS and penicillin/streptomycin/fungizone at the concentrations of 100
U/mL, 100 µg/mL, and 2.5 µg/mL, respectively. Cells are grown as a monolayer at
27°C. For small scale growth, 75-cm
2
vented flasks are used (Costar, Cambridge, MA).
Typically 5 × 10
5
cells 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 cell
density of 10
6
cells/mL. During the logarithmic phase of growth, cells typically double
every 24 h. Therefore, equal amounts of fresh medium are added each day to the roller
bottle for up to 300 mL total volume. Figure 2 shows the kinetics of growth in a
typical roller bottle culture (see Note 4).
3.3. Production of Recombinant Virus by Cotransfection with Viral
and Recombinant Transfer Vector DNAs
The wtAcMNPV viral DNA and the recombinant transfer vector DNA are shuttled

into Sf-9 cells by cationic liposomes. Within the cells, transfer vector DNA and viral
DNAs recombine, incorporating the gene of interest into the viral genome. Depending
on the transfer vectors different protocols can be used to make recombinant virus. Two
protocols are given next.
3.3.1. Using pBlue
Bac
III Vector
When using pBlueBacIII vectors, recombination leads to the replacement of the
viral polyhedrin gene (phenotypically occ
+
) with part of the transfer vector containing
lacZ gene and gene of interest. Therefore, the selection is based on the phenotypic
observation—lack of occlusion bodies (occ
-
) and expression of β-galactosidase
(lacZ
+
).
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 for 1 h at room temperature to evenly distribute and attach the cells.
476 Ciborowski and Finn
2. Remove nonattached cells and medium, gently wash the 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 100, 200, 500, and 750 ng, or 1 µg of
the recombinant pBlueBacIII transfer plasmid, respectively, with 500 ng of linearized
AcMNPV DNA, 40 mL Insectin-Plus Liposomes, and 1 ml of Hink’s TNM-FH Insect
Medium.

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 each
well, 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 cells
from 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 pFast
Bac
Transfer Vector
pFastBac transfer vector is a part of the Bac-To-Bac™ Baculovirus Expression
System developed by Gibco. In the first step, competent MAX Efficiency DH10Bac
E. coli cells are transformed with pFastBac donor plasmid with a gene of interest. The
competent DH10Bac E. coli cells contain baculovirus shuttle vector (bacmid) and a
Fig. 2. Growth of SF-9 cells in a typical roller bottle culture. Infection was on d 4 and no
new 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.