From the
The cDNA encoding the NH
Mucosal epithelia of the human body, including the linings of
the digestive, respiratory, and urogenital systems, comprise a vast
surface permanently vulnerable to attack by outside pathogens.
Protection of mucosal surfaces against colonization and possible
invasion by pathogenic microorganisms is mediated by a special class of
antibodies known as secretory immunoglobulin A (sIgA)
The IgA moiety is produced by local mucosal
and glandular plasma cells. Two immunoglobulin A (IgA) antibodies are
dimerized via J chain in a tail to tail arrangement; the addition of J
chain occurs in plasma cells just before secretion (Parkhouse and Della
Corte, 1973; McCune et al., 1981). To interact with pathogens
on the luminal side of the mucosa, the dimeric IgA (dIgA) antibodies
have to be transported across the epithelium barrier by the
poly-immunoglobulin receptor (pIgR). The receptor, a type I
transmembrane protein, is expressed by the epithelial cells on the
basolateral side of the epithelia lining mucosal and glandular
surfaces. Upon binding to the receptor, the receptor-ligand complex is
internalized and transcytosed (Schaerer et al., 1991); during
transport or at the apical cell surface, the pIgR is cleaved and the
extracellular portion of the molecule, termed secretory component (SC),
is released free or bound to IgA (for review, see Mestecky et
al.(1991), Kraehenbuhl and Neutra (1992b), and Neutra et
al.(1994)). Studies on the stoichiometry of SC-IgA association
suggested that either one or two molecules of SC are contained per sIgA
complex (Kühn and Kraehenbuhl, 1982).
The initial binding of SC
to dimeric IgA in vitro is noncovalent, and interchain
disulfide bridges are formed in a second step (Lindh and Björk,
1977; Garcia-Pardo et al. 1979; Eiffert et al.,
1984). The noncovalent interaction has been shown to be mediated by the
amino-terminal domain of rabbit SC (Frutiger et al., 1986) and
human SC (Bakos et al., 1991). Binding of SC to IgA confers
resistance to proteolytic enzymes, including Pronase, papain, trypsin,
and pepsin (Underdown and Dorrington, 1974; Lindh, 1975). Brown et
al. (1970) have shown that sIgA in duodenal secretions is more
resistant than monomeric IgA to the action of proteases. Recently it
was reported that human SC was able to compete the binding of
Helicobacter pylori to a gastric epthelial receptor by virtue
of fucosyl residues associated with SC carbohydrate side chains (Falk
et al., 1993; Boren et al., 1993).
Despite the
discovery of SC almost three decades ago, little is known about the
properties the molecule confers to dimeric IgA antibodies, except for
resistance to proteolysis. In order to analyze the molecular role of SC
on IgA stability and determine its contribution to immune protection,
both monoclonal IgA antibodies and the same antibodies reassociated
with SC have to be available. Although protocols have been established
for the production of monoclonal dimeric IgA antibodies (Lee et
al., 1994), their combination to SC has been hampered by the
difficulty to produce sufficient amounts of recombinant SC. The aim of
this study was therefore to produce milligram quantities of hSC using
recombinant virus expression systems and insect or mammalian cells.
We report here that recombinant hSC (a) is produced and
secreted with high efficiency by both insect and mammalian cells,
(b) is glycosylated by both insect and mammalian cells, yet to
a different extent, (c) is readily isolated from serum-free
culture medium by lectin or ion-chelate affinity chromatographies, and
(d) binds to dimeric IgA, indicating that the molecule is
properly folded and retains its biological activity. This is the first
report on the production of biologically active recombinant hSC, that
can now be used to dissect the properties it confers to dimeric IgA of
defined specificity in in vitro systems (Hirt et al.,
1993) or upon mucosal administration.
Spodoptera frugiperda (Sf9) insect cells
(ECACC 89070101) were cultured at 28 °C in TC100 medium (Life
Technologies, Inc.) containing 10% fetal calf serum or in SF-900 II
medium (Life Technologies, Inc.) in the absence of serum. Cells grown
as monolayers in T-flasks were passaged at confluency. Cells in
suspension were grown as shaker cultures at 125 rpm in Erlenmeyer
flasks and were diluted with fresh medium to 5
On-line formulae not verified for accuracy
The resulting construct was referred to as pBS-hSC. The same
strategy using the oligonucleotides,served to generate pBS-hSC:6xHis. This sequence encoding the cleavage
site for Factor Xa, six consecutive histidines and a stop codon was
inserted downstream of the codon for Glu
In order to
insert SC sequences into the vaccinia transfer vector p11K (Bertholet
et al., 1986), the cDNAs were excised from pBS-hSC and
pBS-hSC:6xHis by EcoRI/XbaI double digestion, Klenow
filled-in, and blunt-end ligated into BamHI/EcoRI cut
and Klenow filled p11K. Clones carrying the insert in either
orientation were recovered for the generation of recombinant vaccinia
viruses. The constructs were called p11K-hSC, p11K-hSC
Human SC was also cloned
into the vaccinia transfer vector pHGS-1 containing the TAAATG element
including the translation start codon from the vaccinia 11K promoter
(Bertholet et al., 1985). The 5` end of the gene was modified
using a ClaI/AccI fragment generated by recombinant
PCR (Higushi, 1990), using oligonucleotides
5`-AATAATTTCGCGCGGCCCATTTATAGCATAGAAA-3` and 5`-TTTCTATGCTATAAATG
GGCCGCGCGAAATTATT-3` as ``inside'' primers and
oligonucleotides 5`-CCATCGATG AAGGACAGTTCTTTCCAG-3` and
5`-GGGGTACCGGTCACCGTTCTGCCCAGGTCC-3` as ``outside'' primers.
The PCR products were sequenced by the method of Sanger et al. (1977). Plasmids pHGS1-hSC and pHGS1-hSC:6xHis were constructed
via a four piece ligation including: (a) vector pHGS-1
ClaI/EcoRI
Cloning of hSC into the baculovirus insertion vector pVL1392
(Invitrogen) was carried out by ligating the
EcoRI/XbaI fragments containing the modified cDNAs,
excised from pBS-hSC or pBS-hSC:6xHis, into the corresponding
restriction sites of pVL1392. The resulting constructs were termed
pVL1392-hSC and pVL1392-hSC:6xHis, respectively.
The engineered hSC gene was introduced into the
polyhedrin gene of Autographa californica multiple nuclear
polyhedrosis virus (baculovirus), essentially as described by Summers
and Smith(1988), with modifications. Briefly, wild-type baculovirus DNA
and transfer plasmids containing the hSC DNA under control of the
polyhedrin promoter were cotransfected into Sf9 cells using cationic
liposomes (Felgner et al., 1987): 1 ml of TC100 medium without
serum containing 1 µg of baculovirus DNA prepared as described
(Piwnica-Worms, 1990), 10 µg of pVL1392-hSC or pVL1392-hSC:6xHis,
and 30 µl of DOTAP transfection reagent (Boehringer Mannheim) were
vigorously mixed for 15 s, incubated at room temperature for 15 min,
and added to 2
HeLaS3 cells grown to a
density of 6
Baculovirus inocula for infection of Sf9 cells were obtained from
cell culture supernatants recovered at least 4 days post-infection at
m.o.i.'s larger than 1 with master stock virus. Sf9 cells at
densities of 1
Following infection of insect cells with recombinant baculovirus,
expression of the corresponding recombinant protein is known to follow
slower kinetics when compared to vaccinia virus (Summers and Smith,
1988). Similar slow kinetics of hSC secretion were observed in Sf9
cells infected with pVL1392-hSC:6xHis recombinant baculovirus
(Fig. 2C). Suspension or monolayer cultures were
infected at estimated m.o.i.s of 1 and 5, and aliquots of culture
medium were removed at 0 through 5 days post-infection in 24-h
intervals and analyzed by immunoblotting. Suspension cultures start to
accumulate recombinant protein in the supernatant 2 days
post-infection. As compared to monolayer cultures, production in
suspension yielded higher amounts of hSC.
On immunoblots,
recombinant hSC produced from recombinant vaccinia virus was detected
in as little as 2.5 µl of cell culture supernatant, whereas the
baculovirus product was revealed in less than 1 µl of supernatant.
The protein expressed by each of the systems was quantitated
densitometrically by comparing the signals obtained from increasing
amounts of culture supernatant with known quantities of an sIgA
standard on the linear part of the response curve of the Western blot
(data not shown). The concentration of hSC recovered in culture
supernatants from the vaccinia virus/mammalian cell system ranged from
5 to 10 mg/liter, while the baculovirus/insect cell system yielded up
to 50 mg/liter.
Secretory IgA, the major immunoglobulin class in mucosal and
glandular secretions, consists of one dimeric IgA unit and two
additional polypeptide chains, J chain and SC. The heavy, light, and J
chains are synthesized and dimeric IgA is assembled in plasma cells,
whereas SC is contributed by the epithelial cells of mucosal and
glandular tissues. To produce large amounts of sIgA for passive oral
(mucosal) immunization (Apter et al., 1993a, 1993b; Lee et
al., 1994), it is necessary to synthesize both SC and dimeric IgA
in different cells and subsequently properly associate the two
components. While large amounts of dIgA can be produced by and purified
from hybridoma clones, preparation of complete sIgA has been so far
prevented by the lack of efficient production systems for SC. As a
first step toward in vitro reconstitution of sIgA antibody
molecules, we evaluated two viral expression systems for the production
of human SC that retains its capacity to bind and stabilize dimeric IgA
molecules.
The insect cell-based baculovirus system is well suited
for high-level expression of heterologous genes (Miller, 1993).
Appropriate folding, assembly, and targeting of recombinant proteins by
insect cells, as well as their capacity to perform many of the
post-translational modifications of higher eukaryotes, usually allowed
the recovery of large amounts of biologically active product. Complex
molecular structures such as murine immunoglobulin heterodimers have
been successfully produced utilizing this system (Putlitz, 1990).
Vaccinia virus has been used to express foreign genes in mammalian
cells for more than 10 years (Mackett et al., 1982; Panicali
and Paoletti, 1982). Due to the extended host range of vaccinia, the
heterologous gene contained in one single virus recombinant can be
expressed in almost any mammalian cell type. Therefore, infection with
recombinant vaccinia virus usually generates high amounts of a
mammalian protein that most closely resembles its natural counterpart
in terms of structure and function.
We first established that
recombinant SC was produced and transported along the secretion pathway
in both systems. The levels of expression for all constructs were
indistinguishable and within the expected range, i.e. 5-10 mg of protein/liter of culture medium for the vaccinia
system, and 50 mg of protein/liter of culture medium for the
baculovirus system. In addition, the use of translation initiation
sites of different origins in the vaccinia system did not have any
visible effect either. Thus, subtle modifications in the engineered
cDNA were very well accommodated by the cellular machinery responsible
for transcription, translation, maturation, and secretion of
recombinant hSC.
Human SC purified from milk is heavily glycosylated
with four N-linked sugar side chains accounting for over 20%
of its molecular weight (Mizoguchi et al., 1982). A major
disadvantage of the baculovirus system compared to the mammalian
cell-based system is its limited ability to terminally glycosylate
glycoproteins (Miller, 1993, and references therein). Our results with
endoglycosidase treatment indicate that complex-type carbohydrate
contributes approximately 10 and 19-24 kDa to the apparent
molecular mass of hSC produced in insect and mammalian cells,
respectively. hSC from purified sIgA antibodies behaves in a very
similar fashion as the recombinant proteins in terms of migration on
SDS-PAGE, as well as toward treatment with endoglycosidases, indicating
a strong structural relationship between the natural and overproduced
species. Recombinant SC produced by insect and mammalian cells were
both able to efficiently bind to dimeric IgA, suggesting that slight
differences in glycosylation did not affect the interaction, as
previously reported by Bakos et al.(1991, 1994). Indeed, these
authors showed that deglycosylated SC bound with equal or higher
affinity to polymeric IgAs. Whether glycosylation affects the kinetics
of SC-IgA association and play a role in protection will require
further biochemical and immunological investigations.
Metal-chelate
affinity chromatography first described by Porath et al.(1975)
is based on the ability of certain amino acids (histidine, tryptophan,
and tyrosine) to act as electron donors for reversible binding to
transition metal ions immobilized on a solid support. The affinity of
histidine residues for immobilized Ni
ConA affinity chromatography represents a
second efficient single-step procedure to purify recombinant hSC. This
was facilitated by growing the cells in serum-free conditions. In
addition, it appears that glycoproteins secreted by infected cells
represents a small proportion of the large number of cellular and viral
proteins present in cell culture supernatants (Fig. 5). The
recovery of vaccinia-produced hSC is almost complete, whereas
baculovirus-expressed hSC is only partially yielded under identical
conditions, reflecting a stronger affinity for the lectin due to the
lower content of complex sugars in insect cells rendering more terminal
We
have developed a simple dot blot overlay assay to assess IgA binding of
recombinant hSC (Fig. 6A). The recombinant hSC protein
binds exclusively to dimeric IgA, and is produced in an active
conformation, as less than 5% is found not to be reassociated with IgA
(Fig. 6, B and C; Fig. 7A). The
specificity of binding is further illustrated in control experiments
showing that hSC does not bind to IgG (Fig. 6D). It is
worth mentioning at this point of the discussion that we only used
purified dimeric IgAs, and not simply the secretion products of
hybridoma cultures known to contain monomers, multimers, aggregates,
and serum proteins that could nonspecifically trap recombinant hSC in
the assay. In addition, the quantitative shift in the position of
elution of recombinant hSC upon binding to dimeric IgA observed in gel
filtration experiments (Fig. 7B) further validates the
filter binding assay as a fast, qualitative, and reliable
test. These binding data indicate that, (a) specific
recognition between recombinant hSC and dimeric IgA occurs in solution,
(b) confirm that all, or nearly all the recombinant hSC
protein is competent for binding to dimeric IgA, (c) argue in
favor of an 1:1 stoichiometry of association, as suggested by previous
studies (Kerr, 1990). The biochemical nature and kinetics of the
interaction of heterologous and homologous IgA with recombinant hSC
produced by the different expression systems and cell types is
currently being analyzed.
In conclusion, the baculo- and vaccinia
viral systems are suitable to provide high yields of recombinant hSC.
Efficient procedures allowed purification of the recombinant protein in
a single step. This material will be used on a preparative scale for
reassociation with mouse and human monoclonal IgA, as well as with
humanized recombinant antibodies of the A isotype. This approach will
allow investigation in vitro of the stabilizing effect of hSC
in sIgA complex against protease degradation as well as its
contribution to mucosal immune protection (Boren et al.,
1993). In addition, thanks to the availability of sufficient amounts of
SC, it will be possible to determine in animal models the
immunoprotective potential offered by dimeric IgA alone and
reconstituted with recombinant SC.
Serial dilutions of culture supernatants were assayed
by ELISA using purified sIgA as a standard. The linear part of the
response curve was used for quantification.
-terminal 589 amino acids of
the extracellular domain of the human polymeric immunoglobulin receptor
was inserted into transfer vectors to generate recombinant baculo- and
vaccinia viruses. Following infection of insect and mammalian cells,
respectively, the resulting truncated protein corresponding to human
secretory component (hSC) was secreted with high efficiency into
serum-free culture medium. The Sf9 insect cell/baculovirus system
yielded as much as 50 mg of hSC/liter of culture, while the mammalian
cells/vaccinia virus system produced up to 10 mg of protein/liter. The
M
of recombinant hSC varied depending on the cell
line in which it was expressed (70,000 in Sf9 cells and 85-95,000
in CV-1, TK- 143B and HeLa). These variations in M
resulted from different glycosylation patterns, as evidenced by
endoglycosidase digestion. Efficient single-step purification of the
recombinant protein was achieved either by concanavalin A affinity
chromatography or by Ni
-chelate affinity
chromatography, when a 6xHis tag was engineered to the carboxyl
terminus of hSC. Recombinant hSC retained the capacity to specifically
reassociate with dimeric IgA purified from hybridoma cells.
(
)
(Mestecky and McGhee, 1987; Brandtzaeg, 1989; Kraehenbuhl
and Neutra, 1992a). The antibodies are believed to act by agglutinating
potential invaders and facilitate their clearance by peristaltic or
mucociliary movement.
Cell Culture Conditions
African green monkey kidney CV-1 cells (ATCC CCL70) were
grown in Dulbecco's modified Eagle's medium supplemented
with 8% fetal calf serum containing 50 units/ml penicillin and 50
µg/ml streptomycin. The cells were cultivated to confluency in
175-cm flasks to a final concentration of 2
10
cells/flask in a 5% CO
atmosphere at 37
°C. Human TK
143B cells (ATCC CRL 8303) and human
HeLa cells (ATCC CCL 2) were grown under the same conditions. Human
HeLa S3 cells (ATCC CCL 2.2; a clonal derivative of the parent HeLa
cell line CCL2) were grown in spinner bottles at 37 °C, in
minimal essential medium for suspension culture supplemented with 8%
fetal calf serum, to a concentration of 6
10
cells/ml.
10
cells/ml twice a week.
Engineering of Human Secretory Component Sequences
and Cloning into Viral Transfer Vectors
Human pIgR cDNA was obtained from Dr. Charlotte Kaetzel,
University of Cleveland, OH. Since Glu is suggested to
represent the authentic carboxyl terminus of human SC (Krajci et
al., 1989; corresponding to Glu
according to their
numbering), we introduced a stop codon immediately downstream of this
position in the recombinant protein. An EcoRI-BamHI
fragment containing the hSC Kozak (1986) sequence, the hSC ATG
initiation codon, the signal sequence for secretion, and the five
extracellular Ig-like domains (Mostov et al., 1984), including
Asp
, was subcloned into pBluescriptII KS+
(Stratagene). The sequence corresponding to Pro
to
Glu
, including a stop codon and an XbaI
restriction site, was subsequently introduced 3` to the BamHI
site as a double-stranded oligonucleotide,
.
(reverse orientation), p11K-hSC:6xHis, and
p11K-hSC:6xHis
, respectively.
; (b) the
ClaI/AccI PCR fragment containing the modified 5` end
of the hSC gene; (c) the AccI/KpnI fragment
from pCB6-hpIgR containing the central portion of the gene; and
(d) the KpnI/XbaI
fragment
from pBS-hSC or pBS-hSC:6xHis containing the respective 3` ends.
Generation of Vaccinia and Baculovirus Recombinants
The hSC cDNA under control of the 11K promoter was
incorporated into the genome of wild-type vaccinia virus strain WR
(ATCC VR1354) by homologous recombination into the thymidine kinase
(TK) gene. Briefly, subconfluent CV-1 cell monolayers were infected
with the temperature-sensitive vaccinia virus ts7 (Drillien and
Spehner, 1983) and transfected with WR vaccinia virus DNA and the p11K-
or pHGS1-hSC constructs according to Bertholet et al.(1985).
Thymidine kinase-negative recombinant viruses were selected from the
progeny virus by two rounds of plaque purification on TK 143B cells in the presence of 75 µg/ml bromodeoxyuridine.
Recombinant virus plaques were isolated, and positive clones were
identified by PCR screening with primers TKL+
(5`-CGGAACGGGACTATGGACGC-3`) and TKR-
(5`-GTCCCATCGAGTGCGGCTAC-3`) specifically hybridizing to regions in the
left and right portion of the TK gene flanking the insertion site.
Recombinant viruses were amplified by infecting TK
143B cell monolayers in the presence of 50 µg/ml
bromodeoxyuridine, and large stocks were prepared in CV-1 cells without
selection. Recombinant viruses were named after the DNA inserted into
the TK locus.
10
adherent Sf9 cells in a 60-mm
plate, previously washed with serum-free medium. After 4 h of
incubation on a rocking platform at room temperature, 1 ml of complete
TC100 medium was added and incubation continued at 28 °C in a
humidified environment. Five days later, the medium was harvested, and
recombinant virus was plaque-purified by optical screening for plaques
with occlusion negative phenotype (Summers and Smith, 1988). After two
rounds of purification, the presence of recombinant and the absence of
wild-type virus was confirmed by PCR as described (Malitschek and
Schartl, 1991), using the polyhedrin gene flanking primers
5`-TTTACTGTTTTCGTAACAGTTTTG-3` (forward) and 5`-CAACAACGCACAGAATCTAG-3`
(reverse).
Production of Recombinant hSC in Mammalian and Insect
Cells
Stationary phase CV-1, HeLa, or TK 143B
cells at a density of 2.0-2.5
10
cells/175
cm
T-flask in 20 ml of medium were washed with PBS
(Sambrook et al., 1989) and infected with recombinant vaccinia
virus at the indicated multiplicity of infection (m.o.i.). Trial
expression assays were performed with cells cultivated in 6-well dishes
in 1 ml of medium. Infected cells were cultured in Dulbecco's
modified Eagle's medium in the absence of serum and antibiotics.
To assay for secreted hSC, 20-50-µl aliquots of culture
medium were removed at defined intervals and replaced by fresh medium
in order to avoid changes in the starting culture volume. Culture
supernatants originating from cells grown in the absence of virus,
infected with wild-type virus, or infected with recombinant virus
carrying the engineered hSC DNA in reverse orientation with respect to
the promoter were included as controls.
10
cells/ml in suspension minimal
essential medium were transferred under sterile conditions to
centrifuge bottles, pelleted 120
g for 10 min at room
temperature, then washed twice with PBS and once with serum-free
suspension minimal essential medium. After resuspension at a density of
1.0
10
cells/ml, they were poured into a spinner
bottle, infected with an m.o.i. of 10 for 1 h, diluted to 4
10
cells/ml in serum-free suspension minimal essential
medium, and then incubated for 21-24 h. Culture supernatant was
harvested by centrifugation for 15 min at 120
g.
10
cells/ml in shaker cultures or 5
10
cells/25-cm
T-flask were infected at
estimated m.o.i.'s of 1 or 5 by addition of a corresponding
volume of virus inoculum directly to the cultures. Four days
post-infection (or as indicated), cells were centrifuged for 10 min at
600
g and the supernatants containing secreted
recombinant protein were harvested. Expression of secreted and
intracellular recombinant SC from mammalian or insect cells was
monitored by immunoblotting.
Affinity Purification of Recombinant Human Secretory
Component
Nickel Chelate Affinity
Chromatography
Protein recovered from mammalian cell
culture medium was dialyzed/concentrated (100-fold) in 20 mM
sodium phosphate, 500 mM NaCl, pH 7.8 (Ni binding buffer), in a Spectrum Micro-ProDiCon system model FS-15
using 25-kDa molecular weight cut-off membranes. This material was
applied to a Hitrap chelating matrix (Pharmacia Biotech; 1-ml bed
volume/ml of concentrated culture supernatant), charged with nickel
ions and equilibrated with Ni
binding buffer. Culture
supernatants originating from infected Sf9 cells were loaded three
times onto Ni
-nitrilotriacetic acid-agarose columns
(Qiagen; 300-µl bed volume for 1 ml of culture supernatant for
analytical, and 2-ml bed volume per 100 ml of supernatant for
preparative assays), equilibrated with Ni
binding
buffer. Columns were subsequently washed with 20 mM sodium
phosphate, 500 mM NaCl, pH 6.3 (Ni
washing
buffer), followed by the same buffer containing 20 mM
imidazole. Bound protein was eluted with washing buffer containing 100
mM imidazole and fractions were collected in siliconized
polypropylene tubes. Eluted proteins were subjected to SDS-PAGE, and
purity as well as lack of degradation were assayed by silver staining
and immunoblotting.
Concanavalin A Affinity
Chromatography
Mammalian cell culture supernatant were
dialyzed and concentrated as above, in 10 mM Tris-HCl, 150
mM NaCl, 1 mM CaCl, 1 mM
MnCl
(ConA-binding buffer). This material or unconcentrated
insect cell culture supernatant were passed over ConA-agarose beads
(Vector Laboratories, 500-µl bed volume per ml of loaded material),
equilibrated with ConA binding buffer. The columns were extensively
washed with ConA binding buffer, and bound material was eluted with the
same buffer containing 500 mM
methyl-
-D-mannopyranoside (Fluka). Collection and
subsequent analysis of fractions were carried out as above.
Enzymatic Deglycosylation
Recombinant hSC produced in various host cell types was
treated with the endoglycosidases PNGase F (EC 3.2.2.18 and 3.5.1.52)
or EndoH (EC 3.2.1.96; both Boehringer Mannheim) according to the
manufacturer's instructions. Briefly, 150 µl (insect) or 600
µl (mammalian) cell culture supernatant or, as controls,
appropriate amounts of human sIgA (Sigma) or purified nonglycosylated
hSC produced in bacteria, were extracted with a mixture of methanol and
chloroform and precipitated with methanol, as described (Wessel and
Flügge, 1984). The precipitates were solubilized and denatured by
boiling for 2 min in 50 µl of 1% SDS. 15 µl of this solution
were added to 135 µl of PNGase F buffer (20 mM sodium
phosphate, 50 mM EDTA, 10 mM sodium azide, 0.5%
Nonidet P-40, 0.1 M -mercaptoethanol, pH 7.2) for mock
treatment and PNGase F digestion, or to 135 µl of the same buffer,
but adjusted to pH 5.5, for EndoH digestion. The samples were again
boiled for 2 min, then 4 µl of PNGase F (0.2 unit/µl), EndoH (1
milliunit/µl), or H
O were added to the corresponding
tube. Digestions and control samples were incubated at 37 °C for 16
h, then extracted and precipitated using the methanol/chloroform
procedure. The resulting pellets were dissolved in sample buffer for
analysis by immunoblotting.
Immunoblotting
Proteins were subjected to PAGE on 6 or 8% resolving gels
under denaturing (0.1% SDS) and reducing (100 mM
dithiothreitol) conditions, together with prestained molecular weight
markers (Bio-Rad). Separated proteins were then transferred to
nitrocellulose or polyvinylidine difluoride membranes (Bio-Rad)
according to the manufacturer's recommendations. Nonspecific
binding sites were saturated for 1 h at room temperature by incubation
in a blocking buffer made of TBS (25 mM Tris-HCl, 137
mM NaCl, 2.7 mM KCl, pH 7.5), 5% non-fat dry milk,
and 0.05% Tween 20 (Bio-Rad). The membrane was probed for 1 h at room
temperature with either a rabbit antiserum against recombinant
hSC(
)
or a mouse monoclonal antibody to hSC
(Sigma), diluted 1:3,000. Bound antibodies were detected using
horseradish peroxidase-conjugated anti-rabbit or anti-mouse
immunoglobulin antibodies (Sigma) and the enhanced chemiluminescence
kit from Amersham.
Dot Blot Reassociation Assay
Dimeric IgA specific for mouse mammary tumor virus gp52
protein were purified from mouse hybridoma clone MB2 (Weltzin et
al., 1989) by size exclusion chromatography on a Sepharose CL-4B
column (2.2 100-cm; Pharmacia Biotech Inc.). Mouse IgG were
obtained from Sigma. Binding of recombinant hSC to dIgA antibodies was
tested as follows. 500 ng of mouse dIgA, or as a control 200 ng of
mouse IgG in 10 µl of PBS containing 6.25% glycerol, were spotted
onto 24-mm diameter nitrocellulose membranes (0.2-µm pore size;
Schleicher & Schuell) and allowed to air dry. Filters were then
transferred to 6-well tissue culture dishes and blocked with TBS
containing 5% non-fat dry milk and 0.05% Tween 20 for 1 h at room
temperature. After removal of the blocking solution, the dIgA-carrying
filters were overlaid with 100 ng of recombinant hSC protein (recovered
from crude culture supernatant, or purified by affinity chromatography)
in 1.5 ml of fresh blocking solution for 1 h at room temperature. After
three 10-min washes with TBS, 0.05% Tween 20, recombinant hSC bound to
the dIgA on the membranes was detected using monoclonal anti-hSC
antibodies (Sigma) under the conditions given for immunoblotting.
Similarily, samples of recombinant hSC containing 100 ng of protein
were immobilized onto nitrocellulose and overlaid with mouse dIgA or
IgG under the conditions given above. Detection of the bound component
was performed with anti-mouse IgA or anti-mouse IgG Fc
specific-monoclonal antibodies (Sigma), respectively.
ELISA
Two-antibody sandwich assay was performed according to Harlow
and Lane(1988). The first antibody was mouse monoclonal antibody to hSC
(Calbiochem) at a dilution of 1:500, the second antibody was rabbit
polyclonal antiserum to hSC used at a dilution of 1:1,000,
and detection was carried out using a goat anti-rabbit IgG peroxidase
conjugate antibody (Sigma; dilution of 1:1,000) with
1,2-phenylenediamine as the chromogene. These conditions were optimized
to detect as little as 0.25 ng of hSC in a native conformation.
High Pressure Gel Filtration Chromatography
In a total volume of 100 µl of PBS, varying amounts of
purified recombinant hSC were incubated with a constant amount of
dimeric IgA, yielding solutions of 0.5:1, 1:1, and 2:1 µM,
respectively. The solutions were incubated for 16 h at ambient room
temperature and then passed over a 1-cm 30-cm Superose 12 HR
10/30 column (Pharmacia) coupled to a FPLC system (Pharmacia) at 0.3
ml/min. Elution was effected with PBS, with continuous monitoring
performed at 278 nm. The presence of dimeric IgA and recombinant SC in
peak fractions was confirmed by Western blotting.
Construction of Recombinant Virus
In order to
determine the role of SC in IgA antibody stability and its contribution
to protection against microbial pathogens, we have produced recombinant
human SC that retains its capacity to bind dimeric IgA using two viral
expression systems. Since SC is generated by proteolytic cleavage of
the pIgR during transepithelial transport we introduced a stop codon in
the receptor's cDNA at a position corresponding to the putative
natural cleavage site. The 5` region encoding the hSC signal peptide
was preserved in order to direct the protein into the secretory pathway
and to subsequently recover the recombinant protein from the culture
medium. Finally, to facilitate the purification of the protein, a
histidine hexamer tag preceded by a factor Xa cleavage site was
inserted upstream of the artificial stop codon, encoding six
consecutive histidines preceeded by a Factor Xa cleavage site. The
resulting recombinant genes were subcloned into vaccinia and
baculovirus transfer vectors (Fig. 1).
Figure 1:
Schematic representation of cloning and
engineering of hSC sequence into baculovirus and vaccinia virus
expression vectors. The pVL1392 construct contains the hSC cDNA
fragment encoding amino acid Met to Glu
downstream of the baculovirus polyhedrin promoter (pPH).
A stop codon (End) or an artificial sequence coding for 6
histidines and a stop codon (6xHisEnd) was introduced 3` of
amino acid 589. The naturally occurring Kozak sequence in hSC was
conserved in the cloning process. Constructs p11K and pHGS1 contain the
same segment of hSC cDNA, with the presence or absence of codons for a
6xHis tail. Both constructs are under the control of the vaccinia virus
late 11K promoter (p11K). Construct p11K carries the hSC Kozak
sequence, while in construct pHGS1, the highly conserved vaccinia virus
promoter element TAAAT(G) is coupled to the initiation ATG codon as in
the natural situation. At the level of the junctions between viral and
hSC sequences, the hSC 5` region is shown in bold characters.
Sequences involved in homologous recombination are depicted as open
rectangles, promoters as dot-filled arrows, and the ss
box upstream of the hSC gene stands for signal
sequence.
The foreign DNA was
introduced into the double-stranded DNA genome of vaccinia or
baculovirus following published procedures (Mackett et al.,
1984; Summers and Smith, 1988). The foreign genes require viral
promotors to drive efficient transcription. The transfer plasmids p11K
and pHGS-1 contain the vaccinia late promoter P11K (Hänggi et
al., 1986) flanked by vaccinia TK sequences to direct
recombination into the TK locus of the viral genome. In the p11K
recombinants, the Kozak sequence (Kozak, 1986) was derived from the hSC
gene itself, while pHGS-1 recombinants contain viral transcriptional
and translational regulatory sequences originating from the 11K viral
gene. Recombinant hSC DNAs containing their own translation initiation
signals were subcloned into the baculovirus transfer vector pVL1392,
downstream of the promoter governing expression of the viral polyhedrin
gene. The presence of the hSC cDNA insert in the viral genomes, as well
as the absence of contaminating wild-type virus, was ascertained by PCR
utilizing hSC sequence- or transfer vector-specific primers,
respectively, that generated unique amplification products of the
expected sizes (data not shown).
Optimization of Recombinant Human Secretory Component
Expression
We first determined the kinetics of hSC production
after infection at a multiplicity of infection (m.o.i.) of 1 or 5. CV-1
cells were infected with recombinant vaccinia viruses p11K-hSC:6xHis
and pHGS1-hSC:6xHis. To facilitate subsequent purification of the
product, the cells were cultured in serum-free Dulbecco's
modified Eagle's medium during infection and the production
phase. Aliquots of culture supernatant were removed at 3, 6, 9, 20, and
23 h post-infection, separated by polyacrylamide gel electrophoresis,
and immunoblotted with hSC antiserum. The recombinant protein appeared
9 h after infection using the p11K-hSC:6xHis recombinant virus
(Fig. 2A). Increasing the m.o.i. from 1 to 5 accelerated
the production without increasing the final yield of hSC by 20 and 24 h
after infection (). Addition of serum to the culture medium
did not enhance SC production (data not shown).
Figure 2:
Time course of hSC expression in mammalian
and insect cells as a function of multiplicity of infection. Panel
A, cultures of CV-1 cell monolayers were infected either with
vaccinia virus recombinant p11K-hSC:6xHis (lanes 1-5) or
vaccinia virus recombinant pHGS1-hSC:6xHis (lanes 6-10)
at m.p.i. of 1 plaque forming unit/cell (top) and 5 plaque
forming units/cell (bottom). At 3, 6, 9, 20, and 23 h
post-infection, aliquots were taken up, separated by SDS-PAGE (8%
separating gel; reducing conditions), transferred to blotting membrane,
and hSC was detected immunochemically as described under
``Experimental Procedures.'' Molecular size markers are given
in kilodaltons. Panel B, refined time course analysis of hSC
expression in HeLa cells infected with vaccinia virus recombinant
p11K-hSC:6xHis at a m.o.i. of 5 plaque forming units/cell. Aliquots of
supernatant were collected 3, 6, 9, 12, 15, 27, 30, and 33 h
post-infection. Protein accumulation in the culture medium was
monitored using Western blot. Panel C, analysis of hSC
synthesis during recombinantbaculovirus infection. Sf9 cell grown in
suspension (lanes 1-6) or as monolayers (lanes
7-12) were infected with approximately 1 and 5 plaque
forming units/cell of baculovirus recombinant pVL1392-hSC:6xHis.
Culture medium of cells grown in suspension or as a monolayer were
taken up at day 0, 1, 2, 3, 4, and 5, and analyzed by
immunodetection.
The kinetics of hSC
production reflected that observed with other heterologous cDNAs driven
by the vaccinia late promoter (Hänggi et al., 1986). To
test whether the rate of hSC production and secretion observed in CV-1
cells was similar in another cell line, we infected HeLa cells at an
m.o.i. of 5 with the p11K-hSC:6xHis recombinant virus, and looked for
hSC production at shorter intervals. The appearance of hSC was assessed
by immunoblotting (Fig. 2B), and quantitative analysis
was based on ELISA (). Secreted recombinant hSC could be
observed 6 h after infection, starting to accumulate after 9 h.
Addition of Sequences to the COOH Terminus of hSC
Does Not Alter Expression
The cDNAs introduced into the viral
genomes contained initiation sites for translation derived either from
the hSC gene itself or, in the case of vaccinia pHGS-1 recombinants,
from the viral 11K gene. In addition, a 6xHis tag preceded by a Factor
Xa cleavage site were fused to the artificial carboxyl termini of the
recombinant proteins (Fig. 1). To determine whether these minor
differences in nucleotide and amino acid sequence altered hSC
expression, CV-1 cell monolayers were infected with the various
vaccinia virus recombinants at an m.o.i of 1. Twenty hours
post-infection, equal amounts of supernatant were collected, and the
presence of hSC protein was assessed by Western blotting. As shown in
Fig. 3A, comparable signal intensities on the Western
blot and similar patterns of migration of recombinant hSC production
were observed. With both the hSC and 11K regulatory sequences upstream
of the ATG codon, equivalent amounts of protein were synthesized as
assayed by ELISA (), reflecting comparable translation
initiation efficiencies. The addition of six histidyl residues together
with the target sequence for Factor Xa at the carboxyl terminus of the
protein did not alter the secretion of recombinant hSC using either
viral expression systems (Fig. 3, A and B). No
hSC production was detected when CV-1 cells were infected with a
vaccinia recombinant carrying the hSC gene in reverse orientation with
respect to the viral promoter. Accordingly, no recombinant protein was
produced by CV-1 or Sf9 infected with wild-type virus or by
non-infected cells (data not shown).
Figure 3:
Western blot analysis of engineered hSC
produced in CV-1 and Sf9 cells. Panel A, expression of hSC as
a function of engineered cDNA inserts. CV-1 cell monolayers were
infected either with recombinant vaccinia virus p11K-hSCEnd, vaccinia
virus recombinant p11K-hSC, two individual vaccinia virus
recombinants p11K-hSC:6xHis, recombinant vaccins virus pHGS1-hSC, three
individual vaccinia virus recombinants pHGS1-hSC:6xHis at a
multiplicity of 1 plaque forming unit/cell. 20 h post-infection, cell
culture media were collected and 20 µl of each supernatant were
analyzed by immunoblotting as described under ``Experimental
Procedures.'' Molecular size markers are given in kDa. Panel
B, expression of hSC carrying or lacking a 6xHis tag in Sf9 cells
infected for 4 days with baculovirus recombinant pVL1392-hSC or
pVL1392-hSC:6xHis, respectively. No expression of hSC occurs in Sf9
cells infected with wild type BV.
The Glycosylation Pattern of hSC Differs Depending on
the Cell Line and the Recombinant Viral Expression System
The
apparent molecular weight of secreted recombinant hSC varied
significantly depending on the host cell type used for expression
(Fig. 4A). When produced in insect cells which are
unable to generate fully glycosylated forms, the baculovirus
hSC product migrated faster than natural hSC on SDS gels. When
recovered from human HeLa or TK 143B cells, hSC
significantly and reproducibly migrated at a slightly higher
M
than natural human SC, or recombinant hSC
produced by monkey CV-1 cells. In order to correlate these size
differences with glycosylation, we examined the susceptibility of
recombinant hSC to the action of endoglycosidase PNGase F, which
removes high mannose, hybrid, and complex type N-linked
carbohydrates from glycoproteins (Tarentino and Plummer, 1994). Upon
PNGase F treatment, two bands were observed with recombinant hSC
produced in mammalian cells (Fig. 4B); a 66-kDa band
comigrating with the unglycosylated bacterial product, and a 60-kDa
band corresponding to the deglycosylated ``natural'' product.
In Sf9 insect cells, only the 60-kDa band could be detected. We explain
this digestion pattern as follows. Following transcytosis, the pIgR is
trimmed of both its transmembrane and cytoplasmic domains by a so far
unidentified protease activity, leading to the release of SC. The site
of protease cleavage has not been defined with dependable accuracy. Our
constructs are based on cleavage at Glu
(see``Experimental Procedures''), leading to the
synthesis of a recombinant protein with a calculated molecular mass of
66,026 daltons in the absence of any glycosylation. Assuming that the
band at 60-kDa represents the deglycosylated, naturally cleaved pIgR, a
portion of the hSC proteins expressed in mammalian cells and all of the
protein produced in insect cells have somehow been trimmed to the size
of natural hSC. On the other hand, a proportion of the mammalian
products remains intact and migrates at the expected position for a
fully deglycosylated recombinant hSC, such as the unglycosylated
recombinant bacterial product. Such proteolytic cleavage within the
COOH-terminal region of the recombinant protein would also remove the 6
x histidine tag, thus affecting the yield of hSC purified on
Ni
affinity resins (see below). Endoglycosidase H, an
enzyme that does not cleave complex type N-linked carbohydrate
(Elbein et al., 1982), did not induce any significant change
in the molecular weight of recombinant hSC (Fig. 4B),
suggesting that the bulk of carbohydrates N-linked to
recombinant hSC is of the complex type.
Figure 4:
Enzymatic deglycosylation of recombinant
hSC expressed in different cell lines. Panel A, whole cell
lysate from E. coli M15 strain transformed with expression
vector pQE9-hSC6xHisEnd (B), crude supernatants from
baculovirus recombinant pVL1392-hSC:6xHis-infected Sf9 cells
(Sf9), vaccinia virus recombinant p11K-hSC:6xHis-infected
HeLaS3 cells (S3), HeLa cells (H), TK 143B cells (TK), CV-1 cells (C), and standard
human secretory IgA (IgA) were separated on a reducing 8%
SDS-polyacrylamide gel, blotted to polyvinylidine difluoride membrane,
and location of hSC was identified using anti-hSC monoclonal antibody.
Note the difference in the patterns of migration of the overproduced
hSC proteins compared to the natural hSC. Panel B,
supernatants shown in panel A were incubated in the presence
of PNGase F (F), endoglycosydase H (H), or the
corresponding buffer alone (-). The resulting samples were
electrophoresed on 6% polyacrylamide gel, and processed for
immunodetection.
Purification of Recombinant Human Secretory Component
Recombinant hSC protein carrying a carboxyl-terminal 6xHis tag
produced by HeLaS3 or Sf9 cells maintained in serum-free medium was
purified from 20-h or 4-day post-infection supernatants, respectively,
by Ni-chelate affinity chromatography. Small volumes
of about 100-fold concentrated HeLa or unconcentrated Sf9 supernatant
were applied onto equilibrated in binding buffer at pH 8.0. In order to
remove unspecifically bound material, the column was washed stepwise
with washing buffer (pH 6.3), and subsequently with washing buffer
containing a subelution concentration (20 mM) of imidazole
competitor. Recombinant hSC was eluted at 100 mM imidazole,
indicating high binding specificity to the Ni
resin.
Purified hSC from Sf9 cells migrated on SDS-PAGE as a single band of
about 70 kDa (Fig. 5C, lane 4) with no degradation
products as revealed by immunodetection (Fig. 5C, lane
5) and the recovery was estimated to be 80-90% of the amount
of recombinant hSC loaded onto the column. The recovery of purified hSC
produced using the vaccinia system reached almost 100% with several
SC-unrelated proteins probably of viral origin contaminating the
preparation (Fig. 5A, lanes 4 and 5).
Figure 5:
Purification of recombinant hSC by
affinity chromatography. Analysis by SDS-PAGE (8% separating gel,
reducing conditions) of purified hSC produced either in HeLaS3 cells
infected with vaccinia virus recombinant p11K-hSC:6xHis (panels A and B), and in Sf9 cells infected with
baculovirus recombinant pVL1392-hSC:6xHis (panels C and
D). Panels A and C correspond to samples
resulting from affinity chromatography on Ni-chelate
column, whereas panels B and D contain fractions from
the ConA affinity column. Samples in panels A-D were
loaded in the following order: lane 1, cell culture
supernatant (SN); lane 2, column flow-through
(FT); lane 3, column fraction containing the first
wash (W); lanes 4 and 5, column fraction
containing the elution peak of hSC (E). Proteins were
visualized by silver staining (lanes 1-4) and
immunodetection (lane 5). Molecular size markers are given in
kilodaltons (kDa).
This
drawback prompted us to evaluate a different affinity chromatography
protocol. Since recombinant hSC produced in mammalian and Sf9 cells is
glycosylated (Fig. 4), we explored the possibility of purifying
the recombinant protein on a lectin column. Dialyzed/concentrated CV-1
supernatant or unconcentrated Sf9 supernatant were loaded onto the ConA
column and, after extensive washing, recombinant hSC was eluted with
0.5 M methyl--D-mannopyranoside. Human SC
produced by the vaccinia/HeLaS3 system eluted as a single
M
85,000-90,000 band on silver-stained
SDS-PAGE (Fig. 5B, lane 4) or immunoblot
(Fig. 5B, lane 5). The recovery was over 90%. The
protein expressed in Sf9 cells was also efficiently purified
(Fig. 5D), but the elution was over a broader range of
fractions with a final recovery below 60%
Interaction Between Recombinant hSC and Dimeric
IgA
The IgA binding activity of recombinant SC was assessed by a
dot blot reassociation assay (DORA). The principle illustrated in
Fig. 6A is described under ``Experimental
Procedures.'' After immobilization of one of the interacting
partners on a nitrocellulose filter, the other component is added as an
overlay in a buffered solution in the presence of nonspecific protein
competitor. Association is determined by immunodetection of the
component in the overlay phase. Formation of a specific SCdIgA
complex was observed with hSC recovered from the supernatant of insect
and mammalian cells infected with the appropriate recombinant virus
(Fig. 6B, lanes 1-5 and
7-11). Both interacting partners retained their binding
capacity indicating that the functional structure of the immobilized
protein was preserved. This was further supported by the recognition of
recombinant SC by SC-specific antibodies (Fig. 6D, lane
3). No binding was detected in supernatants from cells infected
with recombinant vaccinia expressing SC in the reverse orientation
(p11K-hSC:6xHis
; Fig. 6B, lanes 6 and 12), with wild-type virus or from non-infected cells
(data not shown). In order to further demonstrate the specificity of
the interaction, we repeated the DORA with purified hSC produced in
three different cell lines (Fig. 6C). Consistently,
immobilized SC bound dimeric IgA (lanes 1-3) and
recombinant hSC bound to immobilized dimeric IgA (lanes
4-6). Recombinant SC did not bind IgG
(Fig. 6D, lanes 1 and 2). To determine
which proportion of recombinant hSC is capable of associating with
dimeric IgA, we analyzed by ELISA the hSC recovered in the overlay
after incubation with the nitrocellulose filters carrying the polymeric
immunoglobulins (Fig. 7A). No more than 5% of the
recombinant hSC protein input can be detected when an equimolar mixture
of the partners is mixed (compare lanes 1 and 3 with
lanes 2 and 4), reflecting both quantitative binding
to dimeric IgA and correct three-dimensional folding. In the absence of
dimeric IgA (lane 6), or when monomeric IgA was spotted
(lane 8), recombinant hSC remained free in the overlay. These
series of binding data demonstrate the usefulness of the DORA test for
rapid and sensitive assessment of SC
dIgA reassociation in
vitro.
Figure 6:
In vitro determination of binding
specificity between recombinant SC and dimeric IgA. Panel A,
cartoon representation of DORA. Details are given under
``Experimental Procedures.'' Panel B, binding of
mouse dimeric IgA (IgA) to human SC secreted in culture
supernatant (SN) of recombinant virus-infected cells
(lanes 1-6). Sf9 cells were infected with baculovirus
recombinant pVL1392-hSC:6xHis, and mammalian cells with vaccinia virus
recombinant p11K-hSC:6xHis. SN
corresponds to
supernatant of CV-1 cell infected with vaccinia virus recombinant
p11K-hSC:6xHis
. Lanes 7-12 show binding of
the same set of SC-containing SN to spotted dIgA. SC and dIgA were
present at a 1:1 molar ratio. S3, HeLaS3 cells;
anti-
-chain, affinity purified antibody against
the heavy chain of mouse IgA; anti-SC, monoclonal antibody
against human SC. Panel C, binding of dIgA to purified
recombinant SC produced in culture SN of Sf9, HeLaS3, and CV-1 cells
infected as for the experiments in panel B (lanes
1-3). Association between purified SC and immobilized dIgA
is shown in lanes 4-6. Panel D, lack of interaction
between purified recombinant SC produced in virus-infected Sf9 and
HeLaS3 cells and mouse IgG (lanes 1 and 2). Binding
of anti-SC antibody to spotted SC is not abolished after incubation
with IgG (lane 3). Anti-
-chain, affinity purified
antibody against the Fc domain of mouse IgG.
Figure 7:
In vitro determination of
binding efficiency between recombinant SC and dimeric IgA. Panel
A, over 95% of recombinant hSC input binds dimeric IgA
(dIgA). DORA was performed as described under
``Experimental Procedures,'' and unbound hSC in the
supernatant was analyzed by ELISA. Lanes 1,3,5, and 7, input hSC; lanes 2,4,6, and 8, hSC in the overlay after a 1-h incubation.
Lanes 1 and 2, hSC expressed in Sf9 cells and dIgA;
lanes 3 and 4, hSC expressed in HeLa cells and dIgA;
lanes 5 and 6, hSC expressed in HeLa cells and no
dIgA; lanes 7 and 8, hSC expressed in HeLa cells and
monomeric IgA (mIgA). The ELISA read-out corresponding to
unbound hSC is expressed as 100% on the vertical axis.
Panel B, high pressure gel filtration chromatography of
varying ratios of recombinant hSC to dIgA. The reactants were present
at a 0.5:1 molar ratio (-), a 1:1 molar ratio (- - -), and a 2:1
molar ratio () of hSC to dimeric IgA, respectively.
The position of the sIgA complex and free SC are indicated on the
top of the peaks.
SCdIgA complex formation was further demonstrated
by high pressure gel filtration chromatography experiments. Different
molar ratios of recombinant hSC and dimeric IgA were reacted at room
temperature for 16 h, and their products were resolved by size
fractionation (Fig. 7B). Observation of a shift in the
position of elution of hSC to that of dimeric IgA indicates that the
two proteins can recognize each other in this dynamic assay totally
performed in buffered solution. At a 0.5:1 ratio of hSC to IgA, only
the high molecular weight complex peak is present, indicating that the
reformation of hSC
IgA complex occurs, and accordingly, that
recombinant hSC is properly folded. At a 1:1 ratio, the hSC peak is
again totally shifted to the position of the high molecular mass
species. At a 2:1 ratio, the hSC
IgA complex peak remains the
same, but a free hSC peak is now observed.
ions allows
selective purification of proteins containing a stretch of at least six
consecutive histidines in surface-exposed regions of the molecule, such
as amino and carboxyl termini (Hochuli et al., 1987). Fusion
of six histidines adds only 720 daltons to the protein, and its
biological function and immunogenic properties are usually retained
(Janknecht et al., 1991; Parvin et al., 1992; Taussig
et al., 1993). Efficient single-step purification of
baculovirus-expressed hSC with dimeric IgA binding capability further
demonstrates the potential of this chromatography. In contrast, hSC
protein expressed with the vaccinia system could not be purified to
homogeneity as contaminating polypeptides were repeatedly co-eluted at
high imidazole concentration. Since the same pattern of contaminants
was observed with different cell lines, it is likely that those are of
vaccinia viral origin.
-D-mannose residues accessible to immobilized ConA.
Table:
Production of recombinant hSC using the vaccinia
virus system
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.