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J. Biol. Chem., Vol. 275, Issue 45, 35129-35136, November 10, 2000
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From the Centre for Rheumatology, Bloomsbury Rheumatology Unit, the
Received for publication, March 7, 2000, and in revised form, July 11, 2000
The Fabs of three human autoantibodies (B3/33H11,
anti-DNA; UK4, anti-phospholipid) and six related hybrids have been
cloned, expressed in Escherichia coli, and purified to
homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot
analysis of the recombinant Fab demonstrated the purified Fab to be of
correct size and in assembled form. Protein expression levels of up to
5-9 mg per liter of culture were achievable. A sensitive and reliable
comparative anti-DNA enzyme-linked immunosorbent assay,
involving a defined biotinylated 35-mer oligonucleotide in its single-
or double-stranded form, is also described. Crithidia assay
and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent
assay analyses demonstrated convincing binding of the recombinant Fab
proteins to DNA/cardiolipin, confirming the expression of functional
molecule. The comparative DNA/cardiolipin binding analyses of the nine
Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin
(heavy, UK4) activity lies predominantly on one of the two chains.
However, a compatible partner chain is necessary for optimum antigen
binding activity of the antibody.
Systemic lupus erythematosus
(SLE)1 is an autoimmune
rheumatic disease affecting principally women during childbearing
years, between 40 and 200 per 100,000 women (depending upon ethnic
group) in the UK. Virtually all individuals with SLE have joint and/or skin involvement, and between 30 and 70% of the patients have kidney,
heart, lung, and central nervous system involvement (1). Anti-dsDNA
antibodies are serological markers of SLE often reflecting disease
activity (2, 3) and pathogenesis (4). These antibodies have been eluted
from kidneys of both patients with SLE and various mouse models.
Anti-phospholipid antibodies, especially anti-cardiolipin, are
associated with recurrent thrombosis, miscarriages, and
thrombocytopenia as part of the primary anti-phospholipid antibody
syndrome (5). Despite considerable improvement in the overall outcome,
lupus continues to cause considerable morbidity and mortality.
By using hybridoma technology we and others (6-8) have produced some
human anti-DNA and anti-cardiolipin autoantibodies and analyzed their
effects in SCID mice (9). Interestingly, although the anti-DNA
antibodies studied appeared to be similar in that most bound dsDNA in
ELISA and in Crithidia assays, they exhibited distinctive
patterns of tissue binding and differing abilities to cause proteinuria
(9) and pathogenicity including early histological features of lupus
nephritis (10).
The structural basis for antigen specificity and pathogenicity of these
antibodies is poorly understood (11). Such an understanding, however,
would be of considerable value in the development of therapies that can
inhibit or disrupt protein-nucleic acid interactions (12). Computer
modeling of some of these antibodies has highlighted possible modes of
interaction with DNA (13). However, these models are of limited
accuracy. A full understanding of the binding specificities can only be
achieved by experimental determination of detailed three-dimensional
structure of these antibodies alone and of their complexes with
specific DNA antigens. However, a prerequisite of such a study is the
ability to produce reasonable amounts (in excess of 5-10 mg) of the
antibody protein.
Our initial studies have therefore been focused on the cloning and
overexpression of the Fab of a well characterized human anti-DNA
antibody B3 (14) in a heterologous cell expression system. We now
describe construction of novel vectors pAGP2 ( The technology further allowed us to construct hybrid Fabs by swapping
of the heavy (H) and light (L) chains of the three antibodies with
which to investigate the role of the two chains in dictating the
autoantigen specificity. The contribution of H and/or L chain to the
DNA/cardiolipin binding of an autoantibody has been the subject of
considerable debate. Although a number of studies of murine anti-DNA
antibodies have been reported (15-17), similar studies on human IgG
anti-DNA antibodies are scarce (18, 19) and provide only limited
information. Given that over 90% of murine serum L chains are kappa
(20), whereas approximately 40% of human serum L chains are lambda in
addition to the human lambda locus being more extensive than that of
the mouse (21), the information currently available on the L chain
contribution in an autoantigen reactivity of an autoantibody from the
mouse may not reliably represent similar L chain contribution in a
human autoantibody. The autoantibodies studied presently possess lambda light chains encoded by 2A2 gene segment known to be
most commonly used among humans (22) and also to exhibit pairing with
different heavy chains (23). Furthermore, the 2A2 gene
segment has no known mouse homologue (24). The autoantibodies studied
presently thus present a relevant set of antibodies to investigate.
Such an analysis of lupus autoantibodies might provide clues to the molecular basis for antigenic specificity that might in turn dictate pathogenicity. We report a quantitative assessment of the influence on
anti-DNA/cardiolipin binding behavior, conferred to a human IgG
antibody by its constituent H/L chain. This is an important step
forward toward understanding and in vitro analysis of what could be an in vivo event.
Cloning of B3 Fab
Three plasmid vectors (pAGP1, pAGP2, and pAWtac) and two
Escherichia coli strains (LM1035 and W3110) were obtained
from CellTech (Slough, UK) for E. coli cloning and
expression of the Fabs of kappa antibodies. The final expression vector
pAWtac is based on pCTR008 (25) involving the secretion of L and H
chains under the direction of OmpA signal sequence and involves
co-translational coupling between the cistrons (26). The cDNA
encoding the variable (V For the construction of pAGP2 vector with lambda constant (C The cloning scheme of the L (V Protein Expression, Detection, Purification, and Quantitation
The final expression vector pAWtac containing
V Cultures were spun (9,000 rpm; 30 min; 4 °C; Sorvall, DuPont)
following induction. Supernatant was saved for the detection of any
leaked Fab protein from the cells. The cell pellet was suspended in
ice-cold water (30 ml of distilled H2O per 1 liter of
culture), stirred for 30 min at 4 °C, and spun as described above.
The supernatant was filtered (0.22-µm filters; Millipore, Bedford,
MA) and stored as a periplasmic fraction at 4 °C. The pellet was
suspended in TE buffer (100 mM Tris·Cl, 10 mM
EDTA; 1 ml of buffer per 25 ml of culture), sonicated, and spun (15,000 rpm; 1 h; 4 °C; Sorvall, DuPont). The supernatant was filtered (0.22-µm filters, Millipore, Bedford, MA) and stored at 4 °C as cell extract.
Dot Blot, SDS-PAGE, and Western Blot Analyses of the Recombinant
Fab--
Samples of supernatant, periplasm, and cell extract were
applied on the nitrocellulose membrane (Millipore; Bedford, MA). The
membrane was then blocked (skimmed milk, 20 min), probed with goat
anti-human lambda antibody ( Protein Purification--
Proteins concentrated using 60%
ammonium sulfate were dialyzed against 20 mM sodium
phosphate buffer, pH 7, and loaded on a protein G (protein G-Sepharose
4 Fast Flow; Amersham Pharmacia Biotech, Bucks, UK) column. The bound
Fab protein was eluted with 100 mM glycine buffer, pH 2.7. Protein G-purified Fab protein was dialyzed against 10 mM
sodium phosphate buffer, pH 7.3, and loaded on a heparin
(heparin-Sepharose CL-6B; Amersham Pharmacia Biotech) column. The bound
Fab protein was eluted with a 100-800 mM NaCl salt
gradient. The purity of the purified Fab protein was assessed on
SDS-PAGE gels under reducing and non-reducing conditions.
Quantitative Assessment of the Expression of the Recombinant
Fab--
Protein concentration of the purified Fab was determined by
measuring the absorbance at 280 nm (27) using UV-2401 PC (UV-visible recording spectrophotometer, Shimadzu Corp., Japan). The level of
expression in individual periplasmic batches was assessed using an
enzyme-linked immunosorbent assay (ELISA). Briefly, the polystyrene plate was coated with a monoclonal antibody to human lambda chain (Mo Functional Assessment of the Recombinant Fab
Periplasmic extracts were tested in ELISA for calf thymus dsDNA
binding activities of the recombinant Fabs as described previously (29). The recombinant Fab protein was also evaluated for its binding
ability to dsDNA polar body of Crithidia lucilae as
described elsewhere (30) (Fig. 2b).
Cloning of 33H11 and UK4
The cloning scheme currently used involves the use of unique
restriction sites identified to be naturally present in the framework 1 (5' end) and J (3' end) regions of the L and H chain variable domains
and has the advantage of providing a non-PCR strategy thus avoiding the
inclusion of mutations and the time-consuming sequencing steps.
Furthermore, the proposed scheme involves the construction of ompA
leader (5' end) and constant (3' end) regions on the two ends of the
variable domain as opposed to replacing one variable domain with the
other keeping ompA leader and constant regions intact. This strategy
thus eliminates the possibility of the variable domain of one antibody
not being replaced by that of the other due simply to the inefficient
digestion of the DNA by the restriction enzymes employed resulting
either in no digestion or in the self-ligation of the singly cut DNA.
Furthermore, the proposed cloning strategy involves the use of
carefully selected restriction enzymes providing efficient digestion of
DNA and releases DNA fragments reliably distinguishable and separable
on the gel. The sequences encoding for V Cloning of the Light Chain--
All the three autoantibodies B3,
33H11, and UK4 are lambda antibodies and possess similar sequences up
to 72 bp at 5' end of V Construction of the Vector BluntZeo--
The vector
was constructed by deletion of an 899-bp FspI fragment
containing BsaI site, from PCR-Blunt followed by
self-ligation of the vector. The FspI fragment also
possessed a part of the open reading frame of the kanamycin resistance
gene thus resulting into the loss of kanamycin resistance by the novel
vector. However, the presence also of the zeocin resistance gene
allowed selection required during cloning. The novel vector had the
advantage of being small (2.6 kilobase pair) and allowing the release
of reliably distinguishable and conveniently separable fragments for
gel purification following the digestions by the unique restriction
enzymes selected for L chain cloning. Although the C
The scheme presently employed for the cloning of the L chains has been
summarized in Fig. 3a and can
be efficiently used for swapping of V Cloning of the Heavy Chain--
B3, 33H11, and UK4 each
possesses similar sequences up to 66 bp at 5' end of VH.
This region also contains two unique restriction sites PvuII
at position 10 and PpuMI at position 48. Furthermore, JH region of all the three antibodies possesses a unique
restriction site BstEII at position 346 at the 3' end. Since
the region between PpuMI and BstEII represents
all the differences exhibited by the three antibodies in their
VH domains, it provided an opportunity of convenient
cloning of the H chains in a non-PCR manner involving these naturally
occurring restriction sites in the VH region. It was
achievable by simply replacing the pre-existing
PvuII/PpuMI-BstEII fragment of
B3VH, earlier cloned in pAGP1 by that of 33H11 or UK4.
Since PpuMI and BstEII both generate sticky ends,
the use of PpuMI was preferred over PvuII that
generates blunt ends. Furthermore, the novel vector
BluntZeo was chosen for the cloning also of the H chains of
33H11 and UK4, for two reasons. First, it did not possess the
restriction sites selected. Second, it allowed the release of
distinguishable and conveniently separable fragments for gel
purification following the digestions by the selected unique
restriction enzymes.
The scheme for the cloning of the H chain fragment (VH and
CH1 domains) has been summarized in Fig. 3b.
Given the proximity of the restriction sites PvuII or
BsgI to the 5' end, the use of these enzymes would allow the
scheme to be used efficiently for swapping of the VH
encoded by the same gene segment or different gene segments within a
gene family or by different gene families, using the cloning vectors
described. The occasional change of a residue upstream of these
restriction sites can be eliminated by PCR cloning of a VH
encoded by the allele of interest via restriction sites NruI
(5' end) and ApaI (3' end) in our vector V5 (Fig.
3b) using the primers described earlier for the cloning of
VH in pAGP1 following necessary modifications incorporating
residue changes. The scheme (Fig. 3b) can then be followed
for non-PCR cloning of other H chains encoded by the same locus via
their transfer to our vector Bluntzeo using appropriate
restriction sites (Fig. 3b).
Construction of the Final Expression Vector for 33H11 and UK4 and
the Hybrids--
The L (ompA-V Expression, Detection, and Functional Assessment of the Fabs
The final expression vectors for the eight constructs were
transferred to the expression strain W3110. The induction experiments, preparation of periplasm, dot blot, SDS-PAGE gel, Western blot analysis, and the functional assessment by anti-DNA antibody ELISA of
the expressed Fabs were conducted as earlier described for B3 Fab.
Anti-cardiolipin activity of the nine Fabs was determined using an
anti-cardiolipin antibody ELISA as described previously (8).
Periplasmic extracts prepared from E. coli with or without IPTG induction for individual constructs were tested in a sandwich ELISA for the binding of the recombinant Fabs to anti-human lambda antibody as earlier described for the quantitative ELISA assay of B3
Fab. The binding of the recombinant Fabs to DNA and cardiolipin was
compared using a novel ELISA immunoassay as described below.
Anti-DNA Activity--
Biotinylated or nonbiotinylated 35-mer
oligonucleotides
(5'-biotin-CCGAATCAGTTCACTTCCAGCCCAGGTATTTAGCC-3' and its
nonbiotinylated complementary strand) were synthesized. These
oligonucleotides are known to bind to B3 and have been used previously
for the evaluation of the affinity of DNA-binding proteins to DNA (31, 32), on BIAcore (surface plasmon resonance). The polystyrene plates
were coated (4 °C, overnight) with streptavidin (5 µg/ml, one-half
of the wells) and Mo Anti-cardiolipin Activity--
One-third of the wells of
polystyrene plate were coated with cardiolipin (50 µg/ml in ethanol,
dried overnight, 4 °C) and one-third of the wells with Mo
Binding of the Fabs to the oligonucleotide, cardiolipin, or Mo Dot blot analysis demonstrated the presence of the recombinant Fab
in the periplasm, supernatant, and cell extracts from IPTG-induced cells but not in those from uninduced cells. The expression of the Fab
was confirmed by Western blot analysis. Fab protein precipitated at
60% saturation of ammonium sulfate and, following elution from the
protein G column, resolved as about 45-kDa band on SDS-PAGE gels along
with some contaminating bands. Protein G-purified Fab protein loaded on
heparin column could be efficiently eluted with 200 mM NaCl
and resolved as a clean band at around 45 kDa on SDS-PAGE gels under
non-reducing conditions (Fig. 2a). Bands at approximately 45 kDa (unreduced, Fab) and 22 kDa (reduced, free H and
Molecular Cloning and Expression of the Fabs of Human
Autoantibodies in Escherichia coli
DETERMINATION OF THE HEAVY OR LIGHT CHAIN CONTRIBUTION TO THE
ANTI-DNA/-CARDIOLIPIN ACTIVITY OF THE Fab*
§¶,,,,
,, and Department of Medicine and the § Department of
Biochemistry and Molecular Biology, University College London,
London W1P 9PG, CellTech Therapeutics, 216 Bath Road,
Slough SL1 4EN, Berkshire, and the ** Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and
Bluntzeo that allow cloning of virtually any human lambda
antibody. We also describe a novel cloning scheme that could be used
for the cloning of many lambda antibodies belonging to
V
1 and V2 gene families known to
represent a number of pathogenic autoantibodies in a PCR and/or non-PCR
manner. The cloning scheme allowed us to clone rapidly two other well
characterized antibodies 33H11 (anti-DNA, see Ref. 6) and UK4
(anti-cardiolipin, see Ref. 8) in a non-PCR manner.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/VH) domains of L
and H chains of the B3 antibody (14) were amplified by PCR (95 °C, 2 min; 62 °C, 2 min; 72 °C, 30 s; 30 cycles) using Vent DNA
polymerase (New England Biolabs, Hitchin, UK) for cloning into the
above described vectors. However, the original CellTech vector pAGP2
contains L chain with kappa constant region. Since B3 is a lambda
antibody, the prerequisite for its cloning was to replace the kappa
constant with lambda constant region in pAGP2.
)
region, C region on plasmid pLN10 was a kind gift from Dr. Ray Field
of Cambridge Antibody Technology, Cambridge, UK. Construction of the
vector pAGP2 () has been summarized in Fig.
1a.
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Fig. 1.
a, construction of pAGP2( )
vector for the cloning of human lambda light chain. C
and B3 V were amplified separately in the first round of
PCR, with restriction sites incorporated into the primers
(V: NruI at the 5' end and BstEII
at 3' end; C: BstEII at the 5' end and
EcoR 1 at 3' end) using the following primers:
5'-GGGGGTCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAAGCTCAGTCTGCCCTGACTCAGCCTGCCTCCGT-3'
(5' primer) and
5'-CGGGAACAGGGTGACCGAGGGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGT-3' (3'
primer) for V, and
5'-CAGCCCAAGGCTGCCCCCTCGGTCACCCTGTTCCCG-3' (5' primer) and
5'-CCCCCGAATTCCTATGAACATTCTGTAGGGGCCAC-3' (3' primer) for
C. The V 3' primer also incorporated 36 nucleotides from the 5' end of the C sequence. These
overlapping amplified fragments were then used in a 4-molecule
recursive PCR reaction (47) along with the V 5' and
C 3' primers to assemble the complete lambda L chain
sequence. The final product (in summary:
NruI-V-BstEII-C-EcoRI)
was purified using PCR purification kit (Qiagen, Dorking, UK), cloned
into pCR-Blunt (Invitrogen, San Diego), and sequenced using ABI Prism
dRhodamine Terminator Cycle Sequencing kit (PE Applied Biosystems,
Warrington, Cheshire, UK). The lambda L chain was then inserted into
pAGP2 via NruI and EcoRI sites replacing the
pre-existing kappa L chain. b, cloning scheme of
V-C and VH-CH1 of
human lambda antibodies in the final expression vector pAWtac. The
VH domain was PCR-amplified building in part of the
ompA signal sequence including NruI site at 5' end
(5' primer) and ApaI site at 3' end (3' primer), using the
following primers:
5'-GGGGGTCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAAGCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGG-3'
(5' primer) and 5'-CCCCCGGGCCCTTGGTGGAAGCTGAGGAGACGGTGACCAGGGTTCC-3'
(3' primer). The PCR product was purified, cloned into pCR-Blunt,
and sequenced as above described. The VH domain was then
transferred to pAGP 1 as NruI and ApaI fragment
building in complete ompA leader sequence and the VH
domain. The scheme involves three steps as follows: (i) Cloning of
V into pAGP2 () as an
NruI-BstEII fragment and that of VH
into pAGP1 as an NruI-ApaI fragment; (ii)
subsequent transfer of V-C to pAWtac as a
XhoI-EcoRI fragment; and (iii) transfer of
VH-CH1 as an EcoRI fragment to
pAWtac containing V-C. kb,
kilobase pair.
-C) and H
(VH-CH1) chain variable and constant domains
into the final expression vector pAWtac via our new vector pAGP2 ()
and CellTech vector pAGP1 has been summarized in Fig. 1b.
This scheme allows cloning of any lambda antibody Fab fragment and its
overexpression in E. coli.
-C and VH-CH1
was transferred to the expression strain W3110. Single colonies were
picked from the transformants, and 50-ml cultures prepared in 250-ml
shake flasks (overnight, 30 or 25 °C). Fresh 1.5-liter cultures in
5-liter flasks were then set up using the overnight culture to
inoculate the medium to an A600 of 0.08. Cultures were induced with 1 mM isopropyl
-D-thiogalactoside (IPTG) at an
A600 of 0.5 and allowed to grow for a further
4-16 h. High Biomass Medium (13) or 2TY containing 30 µg/ml
chloramphenicol wad used to prepare cultures. Muslin cloth was used on
the flasks to allow better aeration. A600 was
recorded at periodic intervals before and after induction and of the
uninduced control cultures.
-H) conjugated to alkaline phosphatase (1 h), and developed using 5-bromo-4-chloro-4-indolyl phosphate/nitro blue tetrazolium substrate. Proteins were transferred to nitrocellulose membrane (27) following SDS-PAGE (28) (Fig. 2a) and probed as described
above for dot blot analysis.
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Fig. 2.
a, SDS-PAGE profile of the recombinant
B3 Fab protein. The Fab protein resolves in around 45-kDa
region. Lane 1, marker; lane 2 (unreduced), and
lane 3 (reduced), cell extract; lane 4,
flow-through from the heparin column; lane 5, protein
G/heparin-purified Fab protein. b, binding of the purified
recombinant B3 Fab protein to DNA in situ in
Crithidia assay.
-H, 1:1000; 4 °C, overnight) and blocked (PBS plus 10%
Marvel skimmed milk, 1 h, 37 °C). A range of concentrations of
the purified Fab (from 2,500 ng/ml, standard) and the periplasmic
samples was applied (1 h, 37 °C) in doubling dilutions. Binding of
the recombinant Fab was detected using goat -H conjugated to
alkaline phosphatase and p-nitrophenyl phosphate as
substrate. The quantity of the Fab present in the periplasmic samples
was determined from the standard curve constructed using the OD values
obtained for the standard Fab protein samples.
or
VH of 33H11 and UK4 antibodies have been cloned in
eukaryotic expression vectors pLN10 (V) and pG1D1
(VH) for their expression as IgG in our laboratory. V or VH were transferred from pLN10 and
pG1D1 to pAWtac using the cloning scheme described below.
. This region also contains a
unique restriction site BsaI at position 42. Furthermore,
the J region of all the three antibodies possesses a
unique restriction site AvrII at position 328 at the 3' end.
Since the region between BsaI and AvrII
represents all the differences exhibited by the three antibodies in
their V domains, it was opportune to exploit the natural presence of these restriction sites for the cloning of their L chains.
Although it was achievable by simply replacing the pre-existing BsaI-AvrII fragment of B3 V
earlier cloned in pAGP2 () by that of 33H11 or UK4, most of the
available cloning vectors also contained BsaI and/or
AvrII site rendering them unsuitable for the proposed
purposes. One of the vectors PCR-Blunt used in our laboratory does not
have AvrII site, however, possesses a BsaI site
in the open reading frame of the ccdB lethal gene. We
therefore constructed a new vector BluntZeo by deleting the
BsaI site as described below.
region of the antibodies also possessed a BsaI site, the
cloning scheme described below efficiently overcame the posed problem
by virtue of the fact that the construction of C at 3'
end of the V did not involve the use of
BsaI.
of the antibodies
encoded by the same gene segment. However, swapping of V
encoded by different gene segments (loci) within the two lambda gene
families may result in the change of 1 or 2 (V2) or up
to 4 (V1) amino acid residues upstream of the
restrictions sites BsaI/BstEII in the framework 1 region. In order to eliminate such changes if required, we recommend
PCR cloning of a V encoded by the allele of interest via
restriction sites NruI (5' end) and BstEII (3'
end) in our vector V2 (Fig. 3a) using the primers described
earlier for the construction of our vector pAGP2 () following
necessary modifications incorporating residue changes. The described
scheme (Fig. 3a) can then be followed for a quick non-PCR
cloning of other L chains encoded by the same locus via their transfer
to our vector Bluntzeo using appropriate restriction sites
(Fig. 3a, III).
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Fig. 3.
Scheme used for the cloning of human lambda
antibodies 33H11 and UK4. a, cloning of light chain.
The PCR-amplified V and C domains of B3 L
chain were transferred as an EcoRI fragment from PCR-Blunt
earlier constructed containing the sequenced domains, to
BluntZeo (construct V1). The ompA signal
sequence was then transferred as a KpnI fragment from pAGP2
() to the 5' end of the V in BluntZeo
(construct V2). Simultaneously, V of 33H11 or
UK4 cloned in pLN10 was transferred to BluntZeo as
HindIII-BamHI fragment (construct V3).
The constructs V2 and V3 allowed the construction of the final and
pAGP2 ( version) equivalent (Fig. 1) construct V4 by virtue of the
presence of both donor (construct V2) or recipient
(construct V3) antibody fragments on the same vector
(BluntZeo) as follows. ompA signal sequence
(BsaI-DraI) and subsequently C
(AvrII-SfiI) were transferred from V2 and inserted at the 5'
and 3' ends, respectively, of the 33H11 or UK4 V present
in V3. b, cloning of heavy chain. The variable and constant
domains of B3 along with the ompA signal sequence
(ompA-VH-CH1), were transferred as an
EcoRI fragment from pAGP1 (Fig. 1) to BluntZeo
(construct V5). Simultaneously, VH of 33H11 or
UK4 cloned in pG1D1 for their expression as IgG molecule in eukaryotic
expression system was transferred to BluntZeo as
HindIII-BamHI fragment (construct V6).
The constructs V5 and V6 allowed the construction of the final and
pAGP1 equivalent (Fig. 1b) construct V8 via V7 by virtue of
the presence of both donor (construct V5) or recipient
(construct V6) H chain fragments on the same vector
(BluntZeo) as follows. ompA signal sequence
(PpuMI-DraI) and subsequently CH1
(BstEII-SfiI) were transferred from V5 and
inserted at the 5' and 3' ends, respectively, of the 33H11 or UK4
VH present in V6. However, the CH1 domain also
possesses a BstEII site which resulted in the release of a
219-bp fragment following digestion with BstEII. This 219-bp
fragment released previously was inserted back in the intermediate
vector V7 to achieve the construction of vector V8 containing the
required ompA-VH-CH1 fragment of 33H11 or UK4.
Construction of the new generation of the vector V5 involving the
elimination of BstEII site in the H chain constant domain is
currently underway. This vector will eliminate the cloning step
currently required for the construction of the vector V8 from V7.
kb, kilobase pair.
-C) and
subsequently the H (ompA-VH-CH1) chain variable
and constant domains along with the ompA signal sequence were inserted
into the final expression vector pAWtac via our new vectors V4 (pAGP2
equivalent) and V8 (pAGP1 equivalent) employing the scheme
described in Fig. 1b. In brief, the L chains of the three
antibodies (B3, 33H11, or UK4) were singly inserted into pAWtac as
XhoI-EcoRI fragments thus constructing the three intermediate vectors, one for each antibody L chain: pAWtac-L (B3),
pAWtac-L (33H11), and pAWtac-L (UK4). The three H chains were finally
singly inserted in each of the three constructs as EcoRI
fragments, thus allowing the construction of the two-antibody (in
addition to the earlier cloned B3) and six-hybrid final expression vectors. The key to the legends used in the figures is as follows: letters B, R, and U denote B3, 33H11, and UK4 respectively; the first
of the two letters name of the Fab construct denotes the L chain and
the second of the letters the H chain.
-H (1:1000; one-fourth of the wells). The
biotinylated oligonucleotide was applied to the streptavidin-coated wells (5 µg/ml, 1 h, 37 °C). An immobilized double-stranded
oligonucleotide was generated by annealing the nonbiotinylated
oligonucleotide (5'-GGCTAAATACCTGGGCTGGAAGTGAACTGATTCGG-3', 20 µg/ml)
to the immobilized biotinylated oligonucleotide in situ, in
half of the wells coated with the first strand of the oligonucleotide.
Hybridization was performed in 1 M sodium chloride, 20 mM monosodium phosphate, 0.1 mM EDTA, pH 7 (1 h, 37 °C), as recommended by Amersham Pharmacia Biotech Biosensor AB
(Application Note 306, 1995). The plate was then blocked with 10%
fetal calf serum in PBS (1 h, 37 °C), and the periplasmic
preparations for the nine constructs with or without IPTG induction
were applied (1 h, 37 °C).
-H
(1:1000, 2 h following coating with cardiolipin, 37 °C). The
plate was blocked (PBS plus 10% fetal calf serum, 1 h, 37 °C),
and the periplasmic preparations for the nine constructs with or
without IPTG induction were applied (1 h, 37 °C).
- H
(total IgG/Fab capture) was detected as described above. The
DNA/cardiolipin specific binding is expressed in relation to that for
Mo-H as percent binding using Equation 1,
(Eq. 1)
All chemicals were purchased from Sigma, St. Louis, MO, unless
otherwise mentioned. Polystyrene plates (Immunolon type 1) were
purchased from Dynatech Labs. Mo
![(OD<SUB>Mo&agr;-H&lgr;</SUB>−background)]×100](/content/vol275/issue45/fulltext/35129/img002.gif)
-H was from Immunostics, London, UK.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chains) on
Western blots (Fig. 4) suggest the
purified Fab to be of correct size and in assembled form. Expression
levels of up to 5 mg of Fab protein per liter of culture were recorded
using the quantitative ELISA. Crithidia assay on the
purified Fab confirmed the binding of the recombinant B3 Fab protein to
DNA (Fig. 2b) comparable to that of the whole B3 IgG
molecule (data not shown), suggesting the molecule was functional.
View larger version (25K):
[in a new window]
Fig. 4.
Western blot analysis of the recombinant Fab
proteins demonstrating the binding of the assembled Fabs (approximately
45 kDa, unreduced, lane a) and of free light chains
(reduced Fab, lane b) to anti-human
antibody. Key: 1, BB;
2, BR; 3, BU; 4, RB; 5, UB;
6, UR; 7, RU; 8, RR; 9, UU.
Letters B, R, and U denote B3, 33H11, and UK4,
respectively; the first of the two-letters name of the Fab construct
denotes the light chain and the second of the letters denotes the heavy
chain.
In order to determine the role of the L and/or the H chain in conferring the anti-DNA and/or anti-cardiolipin activity to the molecule, two more antibodies 33H11 (anti-DNA) and UK4 (anti-cardiolipin) were cloned as described (Fig. 3). This allowed the construction of the six hybrid Fabs by swapping of the chains of the three antibodies B3, 33H11, and UK4. The expression of the Fabs was achieved by IPTG induction of the W3110 cells containing the final expression vector. All the recombinant Fabs resolved in around 45-kDa region in their unreduced forms (Fig. 4, lane a) on the Western blots, whereas upon reduction, the dissociated H and the L chains resolved in around 22 kDa region (Fig. 4, lane b) as expected. This observation suggests that all the expressed Fabs were in assembled form.
The recombinant Fabs exhibited variable binding to calf thymus dsDNA
(Fig. 5a) or cardiolipin (Fig.
5b) suggesting that the Fabs were functional and that
different H and L chains and their combinations had affected the
binding patterns of the Fabs. Relative anti-DNA activity of the nine
Fabs is summarized in Fig. 5c. The native H and L chain
combinations of the Fabs bound to ssDNA in the following decreasing
order: BB > RR > UU (Fig. 5c). Binding of BB to
dsDNA was comparable to its binding to ssDNA (Fig. 5c), whereas RR and UU exhibited weak binding to dsDNA. These observations are in line with the fact that B3 is predominantly an anti-dsDNA (13),
whereas UK4 is mainly an anti-cardiolipin antibody (8). However, 33H11
would appear to be a predominant binder of ssDNA in the assay system
used. The Fabs exhibited anti-DNA activities in the following
decreasing order of their percent binding to ssDNA: BR > RB 
BU BB > RU RR > UR UB > UU or to dsDNA: RU > BB BU > RB > BR > UB UU > UR
RR (Fig. 5c). B3 or 33H11 L chain in association with
any of the three H chains studied, except for 33H11 L chain in
combination with 33H11 H chain, conferred significant anti-ss/-dsDNA
antibody to the Fab.
|
Relative anti-cardiolipin activities of the nine Fabs have been
summarized in Fig. 5d. The Fabs exhibited anti-cardiolipin activities in the following decreasing order of their percent binding:
RB > BB > RR > RU BU > BR > UU > UB UR. In contrast to BB or RR L chain, UU L chain conferred
significant anti-cardiolipin activity to the Fab only in the presence
of UU H chain. BB and RR though predominantly anti-DNA antibodies, also
exhibited significant anti-cardiolipin cross-reactivity. The percent
binding of the Fabs of the two antibodies to cardiolipin was,
surprisingly, relatively higher than the binding of UU previously shown
to be a predominant anti-cardiolipin antibody in its IgG form.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The cloning strategy and the vectors used in the present study have for the first time allowed overexpression of human anti-DNA antibody Fab fragments in a heterologous cell system. Expression levels of approximately 5 (B3) to 9 (UR) mg of Fab protein per liter of culture were achievable. Stollar (33) has recently reported an E. coli expression of scFv antibody fragments ranging from 30 µg to 15 mg per liter of culture. However, Fab fragments remain the favorite for their crystallization which is the long term objective of the present studies. B3 recombinant Fab protein was purified to homogeneity. The initial step to purify Fab protein was an ammonium sulfate precipitation at 60% saturation. The presence of the CH1 domain in the Fab facilitated the use of protein G as an intermediate purification step. Like many DNA-binding proteins, the anti-DNA B3 Fab protein exhibited heparin-binding properties in our pilot studies. Interestingly, such an interaction for antibodies recognizing nucleosomes (DNA and histones) has been proposed as a mechanism allowing their binding to the kidney via heparan sulfate, a major glycosaminoglycan component of the glomerular basement membrane (34). The final purification step therefore involved purification of the partially purified Fab protein on a heparin affinity column, yielding pure Fab protein. SDS-PAGE and Western blot analysis of the purified recombinant Fab protein confirmed the molecule to be of correct size and in assembled form. The recombinant B3 Fab protein also bound to the 35-mer oligomer coupled to the sensor chip in its single- or double-stranded form on BIAcore.2 These observations, in addition to the binding of the Fab protein to Crithidia, provide confirmatory evidence in support of the expression of correctly folded and functionally active molecules.
The cloning scheme described in Fig. 3 allows swift cloning of the
antibodies primarily encoded by the gene segment following the cloning
of one antibody belonging to that locus. The novel cloning scheme
allowed us the flexibility of rapid cloning of 33H11 and UK4 antibodies
in a non-PCR manner and of constructing their hybrids. This cloning
scheme allows rapid transfer of antibody chains cloned, for example, in
Cambridge Antibody Technology eukaryotic expression vectors pLN10
(light) or pG1D1 (heavy) to CellTech prokaryotic expression vector
pAWtac via the newly constructed shuttle vectors pAGP2 (),
Bluntzeo, V2, V3 (light chain), V5, and V6 (heavy chain)
thus also providing a bridge between the two extensively used vectors
for antibody expression. The restriction sites used occur naturally in
the framework regions of the studied autoantibodies and are relatively conserved in the antibodies belonging to the following gene families: V1 (BstEII at position 52)/J1
(AvrII) and V2
(BsaI)/J2 (AvrII) (lambda light
chain) and in all seven VH gene families (VH,
BsgI and/or PvuII and/or PpuMI;
JH, BstEII) with the exception, however, of
VH2 and V1-03 gene segment of VH1
(heavy chain) family (35). Although we have created a BstEII
site at the 5' end of the lambda constant region present in pAGP2
(), the cloning scheme described overcomes the problem of the
presence of this additional BstEII site in the context of
the cloning of V1 as the insertion of the lambda
constant region at the 3' end of the variable domain does not involve
the use of BstEII. The described cloning scheme allowed us
to rapidly swap and make novel combinations of H and L chains with
which to investigate the contribution of the two chains to the antigen binding.
An extensive comparative study of conventional anti-DNA antibody assays led to the conclusion that more than one assay was required to confirm the anti-DNA activity of the antibodies reliably (36). These assays involve coating of complex DNA (e.g. calf thymus, salmon sperm, human placental, or others) either directly or via poly-L-lysine on UV-treated polystyrene microplates (37). However, these methods involve the risk of inconsistency in the amount of DNA coated on the plate, and the use of poly-L-lysine may result in high back ground levels. Immobilization of dsDNA on the solid phase may also expose determinants associated with ssDNA (18). Furthermore, the preparation of ssDNA from such complex DNA molecules may not guarantee complete elimination of determinants associated with dsDNA. Given the limited reliability of the conventional ELISAs in the quantitative assessment of the reactivity of anti-DNA antibodies, we utilized a defined biotinylated oligonucleotide as the antigen in an ELISA for the comparisons required presently. It has the advantage that the DNA is coated reliably in its single- or double-stranded form in reproducibly consistent quantities on streptavidin-coated polystyrene plates and significantly reduces background binding. Interestingly, most of the autoantibodies tested do not seem to exhibit cross-reactivity against streptavidin used to immobilize the oligonucleotide in the novel ELISA immunoassay which is probably the explanation for the observed low background levels. A comparison of the sensitivity of the two immunoassays revealed that the novel ELISA is able to detect a sticky antibody exhibiting high background level (over 70%) in conventional assays even when present in concentrations as low as 78 ng/ml. The novel ELISA thus allowed us to assess the anti-DNA activity of RR antibody (Fig. 5c) which was earlier undetectable in conventional immunoassay (Fig. 5a). The sensitivity of the conventional assay for a reliable assessment of anti-DNA activity of an antibody was determined to be 200-300 ng/ml of immunoglobulin protein. The novel ELISA thus provides significant improvement and overcomes most of the drawbacks inherent in the conventional ELISA and has allowed us to compare the binding of all the Fab proteins studied to ss- or dsDNA. Such a quantitative analyses of autoantibodies would be difficult to carry out on a BIAcore system.
All the Fabs containing the BB L chain (BB, BR, and BU) exhibited significant binding to ss- or dsDNA (Fig. 5c). The replacement of the UU L chain with that of BB resulted in a nearly 4-fold increase (BU), whereas substitution of the BB L chain with that of UU resulted in a >50% drop (UB), in the percent DNA binding of the hybrid Fabs suggesting that the B3 L chain plays an important role in conferring to Fab binding to DNA. RB exhibited nearly 3-fold (dsDNA) or nearly 25% (ssDNA) increase in the binding to DNA suggesting better compatibility of BB H and RR L chains. The replacement of UU L chain with that of RR resulted in nearly 3- (ssDNA) or 5- (dsDNA) fold increase in the DNA binding of RU. However, the substitution of the RR L chain with that of UU resulted either in a drop (ssDNA) or no effect (dsDNA) in the DNA binding of UR suggesting 33H11 L chain also possesses anti-ssDNA or anti-dsDNA activity. BB and RR exhibited significant anti-cardiolipin cross-reactivity even higher than the anti-cardiolipin activity of UU. The replacement of the UU H chain with that of BB or RR resulted in a significant drop in the binding of the hybrid Fab UB or UR suggesting that in the context of UU L chain, the anti-cardiolipin activity of UU predominantly lies on the H chain. A significant drop in the anti-cardiolipin activity of BR as compared with that of BB and a significant increase in the anti-cardiolipin activity of RB as compared with that of RR, in the presence of poor anti-cardiolipin activity of UR, suggests the anti-cardiolipin activity lies predominantly on RR light chain. A comparison of the binding activities of the hybrid Fabs BB, RB (good binders), and UB (poor binder), all containing BB H chain, would suggest that the BB L chain like the RR L chain, probably also has the potential to confer anti-cardiolipin activity when present in association with an appropriate H chain. BB and RR L chains earlier observed to have the potential of conferring anti-DNA activity would also appear to be involved in conferring anti-cardiolipin cross-reactivity to the Fab in the presence of an appropriate H chain.
The present results that anti-DNA activity of B3 lies on the L chain are supported by our computer modeling studies predicting the B3 L chain arginines (CDR-L1-27A, CDR-L2-54) to interact directly with the docked DNA (13). Mutation of B3 arginine at L1-27A to serine results in a significant loss in the anti-DNA activity of the mutant IgG compared with that of the wild type IgG.3 A number of studies using mouse models suggest that the L chain confers or modulates DNA binding (16, 17, 38, 39) or expands the specificity of (15, 16), or confers second cross-reactivity to (40), the autoantibody. Some studies have also implicated components of VH alone or those in combination with VL in conferring DNA binding activity (17, 41-44). However, such studies are only scarcely available on human IgG autoantibodies. The requirement of restrictive pairing of a heavy chain with lambda light chains (18) and the involvement of basic residues of CDR3 and L chain (19) have been suggested to confer DNA binding activity to human autoantibodies. No information is available in the literature on the L or H chain determination of anti-cardiolipin activity of an antibody. The present results would suggest that although the anti-DNA (L chain) or anti-cardiolipin (H chain) activity predominantly lies on one of the two chains, the partner chain probably also plays an important role in determining the final outcome of the pairing. Correct conformation of the assembled molecule required for antigen binding may not be achieved in the presence of an incompatible partner chain. Kieber-Emmons et al. (45) demonstrated that multiple L and H chains contain residues that can facilitate DNA binding, reaffirming the notion that there are multiple ways that different amino acids combine to form an antigen-binding pocket with affinity for dsDNA and ssDNA or cardiolipin. It was later shown that the DNA binding affinity of the autoantibody was in part dependent on the particular H/L chain pairing (46).
Our study describes the use of newly constructed vectors and provides a
technology allowing cloning in a PCR and/or non-PCR manner and
overexpression and purification of virtually any human lambda antibody.
The expression system described would, in particular, be useful for the
production of human anti-DNA antibodies known to be difficult for their
overexpression in a heterologous cell system, to a quantitative level
required for their functional or structural studies. The novel
comparative ELISA immunoassays described presently allow a reliable
evaluation of relative anti-DNA or anti-cardiolipin activities of the
autoantibodies in a controlled manner. The technology described
encourages further studies to determine whether the observed light
(anti-DNA/cardiolipin) or heavy (cardiolipin) chain involvement in
dictating antigen specificity is a general phenomenon among human autoantibodies.
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ACKNOWLEDGEMENTS |
|---|
We are grateful to CellTech, Slough, and to Dr. Ray Field, Cambridge Antibody Technology, Cambridge, for providing us with their vectors. We thank Thomas Winkler and Joachem Kalden for supplying us with 33H11.
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FOOTNOTES |
|---|
* This work was supported by the Arthritis Research Campaign, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Institute of Cancer Research, Chester Beatty Labs, 237 Fulham Rd., London SW3 6JB, UK. Tel.: 44 207 970 6050; Fax: 44 207 970 6051; E-mail: sanjeev@icr.ac.uk.
Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M001976200
2 S. Kumar, J. Kalsi, D. S. Latchman, D. A. Isenberg, and L. H. Pearl, unpublished observations.
3 A. Rahman, J. Haley, S. Kumar, and D. A. Isenberg, unpublished observations.
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ABBREVIATIONS |
|---|
The abbreviations used are:
SLE, systemic lupus
erythematosus;
PAGE, polyacrylamide gel electrophoresis;
ELISA, enzyme-linked immunosorbent assay;
PCR, polymerase chain reaction;
dsDNA, double-stranded DNA;
ssDNA, single-stranded DNA;
IPTG, isopropyl
-D-thiogalactoside;
bp, base pair;
PBS, phosphate-buffered saline;
H, heavy;
L, light.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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