Disulfide bonds are the most common structural, post-translational
modification found in proteins. Antibodies contain up to 25 disulfide bonds
depending on type, with scFv fragments containing two disulfides and Fab
fragments containing five or six disulfide bonds. The production of antibody
fragments that contain native disulfide bonds can be challenging, especially on
a large scale. The protein needs to be targeted to prokaryotic periplasm or the
eukaryotic endoplasmic reticulum. These compartments are specialised for
disulfide bond formation, but both compartments have limitations.
Results
Here we show that the introduction into the cytoplasm of a catalyst
of disulfide bond formation and a catalyst of disulfide bond isomerization
allows the efficient formation of natively folded scFv and Fab antibody
fragments in the cytoplasm of Escherichia
coli with intact reducing pathways. Eleven scFv and eleven Fab
fragments were screened and ten of each were obtained in yields of >5 mg/L
from deep-well plates. Production of eight of the scFv and all ten of the Fab
showed a strong dependence on the addition of the folding factors. Yields of
purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to
42 mg/L were obtained. Purified fragments showed circular dichroism spectra
consistent with being natively folded and were biologically active.
Conclusions
Our results show that the efficient production of soluble,
biologically active scFv and Fab antibody fragments in the cytoplasm of
E. coli is not only possible, but facile.
The required components can be easily transferred between different E. coli strains.
Background
Antibody fragments, in particular scFv and Fab fragments
(Fig. 1) have a wide range of uses in
both academia and industry, including a critical role in diagnostics and an
increasing role in therapeutics. While the recombinant production of full length
antibodies is virtually exclusively performed in mammalian cell culture (for reviews
see [1, 2]), in particular in Chinese hamster ovary (CHO) cells due to
the requirement for post-translational modifications such as disulfide bond
formation and N-glycosylation, a wider range of production platforms are used for
scFv and Fab fragments which contain only disulfide bonds. These include production
in the periplasm of prokaryotes, such as Escherichia
coli (for example [3]),
in the endoplasmic reticulum of eukaryotes including yeast, insect and mammalian
cell culture (for examples [4,
5]) and in cell free expression
systems (for example [6]). The lack of a
clear frontrunner for a production system for antibody fragments reflects the fact
that all have advantages and disadvantages.
Fig. 1
Schematic representation of antibodies fragments. Single
chain (scFv) and Fab antibody fragments of types produced here are
shown along with the position of the intra- and inter-molecular
disulfide bonds
Production in E. coli has a number of
advantages over other systems, including low cost, rapid growth, high biomass,
easily scalable cultivation and clear regulation for therapeutic protein production.
The primary disadvantage of E. coli for antibody
fragment production comes from the fact that production of the folded state can only
occur in the periplasm as this is the only cellular compartment in E. coli in which catalysed formation of native
disulfide bonds naturally occurs. The disadvantages of periplasmic production are
twofold. Firstly the volume of the periplasm is much smaller than that of the
cytoplasm, being typically cited as 8–16 % of the cell volume [7]. Secondly, the capacity secretion apparatus
from the cytoplasm to the periplasm is easily overloaded, though this can be
mitigated by reducing expression levels [8]. Both of these result in general lower levels of production of
proteins in the periplasm compared with production in the cytoplasm.
To overcome these disadvantages we recently developed a system for the
efficient production of disulfide bond containing proteins in the cytoplasm of
E. coli, known as CyDisCo. CyDisCo is based
on coexpression of a catalyst of disulfide bond formation, usually a sulfhydryl
oxidase such as Erv1p [9, 10] but alternatively an inverted DsbB or VKOR
[11], plus a catalyst of disulfide
bond isomerization—either DsbC or PDI. CyDisCo has been used in house to produce
more than 200 proteins, with the most optimal combination to date using Erv1p and
PDI as the catalysts of native disulfide formation.
Here we addressed the question whether the CyDisCo system could be used
to efficiently make scFv and Fab antibody fragments in the cytoplasm of E. coli. Specifically, we wanted to know whether this
system was generic rather than specific i.e. if it would enable production of a wide
range of antibody types. Accordingly we wished to have representatives of (i)
different classes of antibodies (IgG1,
IgG2, IgG4,
IgA1, IgE and IgM were chosen); (ii) antibodies from
different organisms (human, mouse and humanized were chosen); (iii) representatives
of well-known and widely used antibody drugs (Humira, Herceptin and Tysabri were
chosen); (iv) representatives of antibodies that arose from Finnish academia with
potential use in diagnostics (Maa48, K2 and 3211 were chosen). Where possible we
also wanted structural information on the antibodies to be available such that any
differences observed in the efficiency of production could potentially be linked
back to differences in structure. The results indicated that more than 90 % of the
scFv and Fab fragments tested could be produced in the cytoplasm and that they were
correctly folded and biologically active.
Results and discussion
Systematic screening of antibody and antibody fragment production in the
cytoplasm of E. coli
Antibodies contain a large number of disulfide bonds in their
native state e.g. an IgG1 contains 19 disulfide bonds.
However, they generally have a regular pattern with one intra-molecular
disulfide per domain plus inter-molecular disulfide bonds which link the heavy
and light chains together and the two heavy chains together. Exceptions to this
pattern exist, for example in IgA and IgE there is an additional disulfide bond
in the first constant domain of the heavy chain (Fig. 1). The low number of disulfide bonds suggests that it should
be possible to make natively folded scFv (two disulfides) and Fab antibody
fragments (five disulfides, except IgA and IgE which have six disulfides) using
CyDisCo in the cytoplasm of E. coli. These
are especially attractive targets for production in the cytoplasm as they lack
the consensus N-glycosylation sites found in the Fc region of antibodies.
We therefore decided to systematically screen the production of
scFv and Fab antibody-fragments in the cytoplasm of E.coli using the CyDisCo system. Eleven antibodies were chosen
for this screen, three human IgG1 (Humira; PDB
structures 3WD5, 4NYL [12,
13]; Maa48 [14] and K2 [15]), two humanized IgG1 (Avastin; PDB
structures 1BJ1, 1CZ8 [16,
17] and Herceptin; PDB
structures 1FVC, 1N8Z, 4HKZ [18–20]), a mouse
IgG1 (3211; An anti-BNP antibody, Veijola,
Vuolteenaho and Takkinen, unpublished observations), a mouse
IgG2 (PDB structure 1IGT [21]), a humanized
IgG4 (Tysabri; PDB structure 4IRZ [22]), a human IgA1
(PDB structure: 3M8O [23]), a human
IgE (PDB structure: 2R56 [24]) and
a human IgM (PDB structure: 1QLR [25]). Each of the 11 scFv and Fab fragments derived from
these antibodies were expressed from otherwise identical vectors and expressed
and purified under identical conditions i.e. not optimized for individual
proteins, such that antibody specific differences could be observed.
Previously it has been reported that some antibody fragments can be
expressed in ΔtrxB/Δgor strains of E. coli (for
examples [26, 27]) in which the reducing pathways have
been removed, but which have no active pathways for disulfide bond formation
[28]. This suggests that some
antibody fragments may require little or no assistance from catalysts of
disulfide bond formation to reach a soluble state. To examine this possible
effect all 22 antibody constructs were expressed with and without CyDisCo
components in 24 deep well plates using the KEIO collection parental K12
E. coli strain. This strain has the
cytoplasmic disulfide bond reducing pathways intact. All expression tests were
conducted at least in quadruplicate.
Examination of the soluble lysates by SDS-PAGE followed by
Coomassie staining allowed the direct visualization of antibody fragment
production in high yields for many of the constructs with CyDisCo present (data
not shown). Since densitometric analysis of yields from lysates is prone to
error e.g. due to co-migrating E.coli
proteins, the 22 constructs expressed in the presence and absence of CyDisCo
components were purified by IMAC from 3 mls of culture grown in 24 deep well
plates. Since disulfide bond isomerization can occur in SDS if a free thiol
group is present, the purified proteins were treated with N-ethylmalemide (NEM)
to block free thiols before being analysed by reducing and non-reducing
SDS–PAGE. The results (Fig. 2;
Table 1) indicate that 10 out of the
11 scFv and 10 out of the 11 Fab were expressed in sufficient yield to be
visible in Coomassie stained gels (limit of detection circa 3 mg/L yield) when
CyDisCo components were present. In contrast only two scFv, Herceptin and
Tysabri, showed high-level CyDisCo independent production and no Fab fragments
were purified in the absence of CyDisCo.
Fig. 2
Purification of antibodies and antibody fragments
expressed in DWP in the cytoplasm of E.
coli. Representative Coomasie stained
non-reducing SDS-PAGE analysis of NEM treated IMAC purified
antibody fragments in the cytoplasm of a K12 E. coli strain with (+) and without
(−) expression of CyDisCo components Erv1p and PDI. Expression
in 24 deep well plates, EnPressoB media at 30 °C. a scFv. The position of a Herceptin
scFv disulfide linked dimer is marked with an arrow; b Fab. The position of the Fab dimer
and light chain and heavy chain monomers are marked. In both
panels the order is molecular weight markers (1) Humira
(IgG1), (2) Maa48
(IgG1), (3) K2
(IgG1), (4) Avastin
(IgG1 humanized), (5) Herceptin
(IgG1 humanized), (6) 3211
(IgG1 mouse), (7) 1IGT
(IgG2 mouse), (8) Tysabri
(IgG4 humanized), (9) 3M8O (IgA1),
(10) 2R56 (IgE) and (11) 1QLR (IgM). All antibodies are human
unless otherwise indicated. In both panels an E. coli protein
which is occasionally seen co-eluting is marked with *.
Treatment with NEM results in a slight smearing and laddering of
the protein band. This is not seen in the absence of NEM (see
Fig. 3a as an
example)
All of the scFv produced were monomeric, with the exception of a
very faint band of disulfide linked dimer for the Herceptin scFv present in some
replicates. In contrast the Fab were predominantly a disulfide linked dimer of
the heavy and light chains, though some monomeric light and heavy chains i.e.
not disulfide linked, could be observed e.g. for Herceptin and Tysabri. When
small amounts of monomeric purified heavy and light chains were observed in the
non-reducing gel analysis the apparent ratio was always 1:1 suggesting that
dimers of the heavy and light chain were purified, but that some of these lacked
the inter-chain disulfide bond and so ran as monomers on non-reducing
SDS–PAGE.
Shake flask scale production, purification and analysis of antibody
fragments
Small scale production in 24 deep well plates (DWP) allowed
preliminary screening of the production of antibody and antibody fragments, but
it did not produce sufficient protein for more detailed analysis. To examine in
more detail the proteins produced using CyDisCo five scFv (Herceptin and Tysabri
which showed CyDisCo independence, and 3211, 3M8O and 2R56 which showed CyDisCo
dependence for production) and four Fab (Maa48, 3211, 3M8O and 1QLR) were chosen
for production in shake flasks. Based on the results from DWP expression,
Herceptin and Tysabri scFv were produced with and without CyDisCo, while the
other seven antibody fragments were produced only with CyDisCo present to
catalyse native disulfide bond formation. The proteins were purified by IMAC and
the quality analysed by SDS–PAGE. Similar patterns were observed as observed in
DWP expression (see Fig. 3a as example).
The purified proteins were quantified using absorbance at 280 nm
(Table 2). While the yields of scFv
were in line with estimates from SDS–PAGE of lysates, the yields of the Fab
fragments were on average two-fold lower than expected, suggesting that the
standard IMAC protocol or the placement of the hexahistidine tag at the end of
the heavy chain was non-optimal. The yields in mg/L obtained for the nine
antibody fragments tested in shake flask scale were on average 103 % of those
obtained in DWP (compare Table 1 with
Table 2) demonstrating the ease of
going from screening scale to a scale suitable for structural or functional
studies using CyDisCo in EnPresso B media.
Fig. 3
Analysis of antibody fragments produced using CyDisCo in
the cytoplasm of E. coli
grown in shake flasks. a
Coomassie stained non-reducing SDS-PAGE analysis of IMAC
purified 3211 (IgG1 mouse) scFv
(lane 1) and Fab
(lane 2) antibody
fragments. b Far UV circular
dichroism spectra of IMAC purified 3211 scFv and Fab fragments.
c 3211 scFv and Fab binding
to recombinant NTproBNP1-76; d
Maa48 Fab binding to native and modified LDL; e Maa48 Fab binding to native and
modified BSA
To confirm that the proteins obtained were correctly folded far UV
circular dichroism (CD) was performed. All of the proteins showed a CD spectra
with a minima around 217 nm (Fig. 3b),
consistent with the β-sheet found in the Ig fold.
To further confirm that the antibody fragments obtained were
natively folded, a selection of them were tested for their ability to bind to
their reported antigens. The scFv and Fab of 3211 bound to a peptide fragment of
BNP, the antigen it was raised against (Fig. 3c). Due to the availability of suitable substrates, the
binding specificity of the Fab fragment of Maa48 was analysed in more detail.
Maa48 Fab bound to malondialdehyde-acetaldehyde (MAA)-modified antigen, but not
to malondialdehyde (MDA)-modified antigen or to the non-modified antigen
(Fig. 3d, e). In addition, Maa48 Fab
did not bind to copper oxidized LDL or carbamylated LDL. These results match the
published specificity of Maa48 (also known as Fab-pre; [14]) and imply that antibody fragments
produced using CyDisCo in the cytoplasm of E.
coli retain biological activity and specificity.
Analysis of Herceptin and Tysabri scFv
Both Herceptin and Tysabri were unusual in that the scFv were
solubly expressed in high yields in the absence of CyDisCo components i.e. under
conditions in which disulfide bond formation should not occur. While the
purified yields in the absence of CyDisCo were up to one-third lower of that
obtained in the presence of CyDisCo components, the high level produced suggests
either that the proteins are soluble and proteolytically stable in the absence
of disulfide bond formation or that disufide bond formation occurs via an
alternative route for these scFv. To examine the redox state of these the IMAC
purified scFv produced in the presence and absence of CyDisCo were
analysed.
Herceptin scFv produced using CyDisCo showed a small mobility shift
in SDS–PAGE in the presence and absence of β-mercaptoethanol (data not shown),
indicating that it contained at least one intra-molecular disulfide bond. In
contrast the same scFv produced in the absence of CyDisCo showed no mobility
shift. The absence of disulfide bonds in both Herceptin and Tysabri scFv when
produced in the absence of CyDisCo was confirmed by electrospray mass
spectrometry (Table 3). Similarly both
scFv were confirmed to contain two disulfide bonds when made in the presence of
CyDisCo.
Table 3 Molecular mass of scFv fragments produced with and
without CyDisCo
By using the CyDisCo system for disulfide bond formation we were able
to generate high yields of folded, biologically active, antibody fragments (scFv and
Fab) in the cytoplasm of E. coli with a >90 %
success rate. The direct, systematic, side by side comparison of eleven different
antibodies of eight different types with no protein dependent optimization
demonstrates the flexibility of the system. The use of CyDisCo for the production of
proteins containing up to nine disulfide bonds [10], suggests that it could also be used to produce antibody
fragments with engineered disulfide bonds to increase stability [29].
Of the 22 constructs tested only two, the scFv of Herceptin and
Tysabri, were produced in a soluble state in the absence of CyDisCo, the rest being
produced as inclusion bodies. Sequence analysis of the eleven scFv (see Additional
file 1 for an alignment) showed no
consensus in sequence either at a global or at a local level to explain why these
two alone were able to fold to a soluble stable state in the absence of disulfide
bond formation. Similarly no consensus was observed when comparing the available
structures of the variable domains of these two antibodies in comparison with those
antibodies whose scFv were only produced in a soluble state when CyDisCo components
were present.
In vitro studies of antibody folding strongly suggest the involvement
of multiple protein folding factors beyond those catalysing disulfide bond formation
(reviewed in [30, 31]). In particular cis-prolyl isomerization is a rate limiting step that requires the
action of a peptidyl prolyl cis–trans isomerase (PPI) and the first constant domain of
the heavy chain (CH1) requires the action of the molecular
chaperone BiP, a HSP70 family member, to retain it in a folding competent state
until the heavy and light chain associate. Our system adds neither of these factors
and yet yields of purified protein of up to 250 mg/L of folded scFv and 42 mg/L of
folded Fab are obtained from shake flasks without protein specific optimization.
This suggests that intrinsic E. coli proteins
fulfil these roles. E. coli has six cytoplasmic
PPIs (the gene products of fkpB, fkbX, ppiB,
ppiC, slyD
and tig) and one cytoplasmic HSP70 family member
(the gene product of dnaK) and the most probably
explanation is that these fulfil the roles necessary for antibody fragment folding.
Only the catalysts of native disulfide bond formation need to be added.
The production levels of the scFv and Fab obtained varied by circa
60-fold for the 10 scFv and nearly 20-fold for the 10 Fab produced. No patterns were
observed based on antibody subtype—for example the best produced scFv were
IgG1 and IgG4 subtypes, while the
best and worse Fab were both IgG1 subtypes. In addition, no
patterns were observed based on sequence analysis (see Additional file 1: Figures S1 and S2) or on structural
analysis—based on the available structures for eight of the antibody fragments. In
general the levels of Fabs were lower than those of scFv (23 mg/L on average
compared with. 63 mg/L on average, but note the bias caused by the two scFv which do
not have a requirement for CyDisCo). However, there was no simple correlation
between the production levels of the scFv and that of the corresponding Fab, even
within a single antibody subtype. This is neatly exemplified by Humira and Maa48,
both human IgG1 subtypes, with the yields from DWP of the Fab
compared with the scFv being circa 6× lower for Humira, but circa 10× higher for
Maa48 (Table 1).
As we wanted to have a systematic side-by-side comparison of the
abilities of the system, no systematic protein specific optimization was performed.
As such this makes direct comparison with published data for other production
systems problematic as usually protein specific optimization is performed as there
only one or two target proteins rather than, as here, a wider ranging proof of
concept for a class of proteins. Other systems, such as expression in the
endoplasmic reticulum of CHO or yeast or periplasmic expression in E. coli have achieved yields in excess of 1 g/L for
antibodies and/or antibody fragments after optimization. Preliminary data using
CyDisCo suggests that with optimization higher yields may be obtained, with an
increase in yield of at least twofold having already been obtained for more than
half of the 22 constructs tested here. For example yields of more than 150 mg/L of
Maa48 Fab and more than 100 mg/L of 2R56 scFv have been obtained from DWP expression
during preliminary optimization. As per any heterologous protein expression such
optimization may need to include choice of vector, codon usage, translation
initiation, mRNA stability, relative expression levels of subunits in multi-subunit
complexes, bacterial strain, media and expression, induction and purification
conditions, etc. Further studies aimed towards increasing yields and being able to
make sequence based predictions of yields are ongoing. However, preliminary data
from these antibody fragments, combined with data from successful expression of more
than 100 other proteins using CyDisCo, within our research group suggests that one
of two effects may be limiting yields for specific antibody fragments: (1)
proteolytic stability i.e. that the protein is made, folds, but the folded state is
prone to proteolysis by cytoplasmic proteases; (2) solubility of the folding
intermediates or, less commonly, of the final folded state. While the solubility of
the final native state can be readily determined, or to some extent predicted based
on sequence, the solubility of folding intermediates is currently unpredictable.
However, testing a wider range of scFv to identify more examples like Herceptin and
Tysabri derived scFv which are able to reach a stable, soluble state in the absence
of disulfide bond formation may allow further elucidation of factors which increase
the solubility of folding intermediates of antibody fragments and hence increase
yields. Such data may also impact on yields obtained in other production systems. To
date no patterns have been identified to allow prediction of which factors require
optimization for any given protein, except that disruption of the reducing pathways
e.g. the use of a ΔtrxB/Δgor strain such as rosetta-gami, combined with CyDisCo components
expressed at high levels (such as from the plasmid used here) is usually deleterious
to the production of native disulfide bonds. This effect probably arises as the
system becomes over-oxidizing and is unable to catalyse isomerization of non-native
disulfides to the native state.
To date CyDisCo works in all E. coli
strains tested and in all media tested, including minimal media in batch or
batch-fed fermentation (manuscript in preparation) and no deleterious effects have
been observed of CyDisCo component expression in production strains in any media
tested. Hence, despite the requirement for further optimization and scale up our
results opens up a wide range of possibilities for the production of therapeutic and
diagnostic proteins on both a laboratory and industrial scale.
Methods
Vector construction
Expression vectors (see Table 4 for vectors used in this study) were made by standard
molecular biology techniques.
Table 4 Details of the plasmid vectors used in this
study
Genes for the Erv1p, mature PDI along with the heavy and light
chains of the antibodies tested (lacking the N-terminal signal sequence) were
synthesized codon optimized for E .coli
expression (GenScript; Additional file 1: Figure S3). IgE and IgM heavy chains were synthesized
without the C-terminal region involved in oligomerization.
The expression vector used was a modified version of pET23 in which
the T7 promoter was replaced with Ptac promoter from previously modified (SpeI
site inserted) pMal-p2X [10] by
digesting the pMal-p2X with MscI/SpeI and ligating the fragment into MscI/XbaI
digested pET23. Synthetic multi-cloning sites for Fab fragments (EcoRV/XhoI) and
scFv fragments (EcoRV/CelII) were synthesised (GenScript) and ligated into this
vector backbone.
The variable domains of the light and heavy chain were amplified by
PCR from the synthetic genes and cloned into a synthetic multi-cloning site
using NdeI/KasI (light chain) and XhoI/BamHI (heavy chain) to generate a scFv
with a C-terminal hexahistidine-tag (Fig. 4). Since the vector backbone, tag and linker region were
constant any differences in scFv production comes from the variable
regions.
Fig. 4
Structure of the expression vectors used in this study.
a scFv vector. The spacer
region including the KasI and XhoI sites encodes for the
sequence
-Gly-Ala-Ser-(Gly4-Ser)3-Ser-
while the hexahistidine-tag including the BamHI site adds
Gly-Ser-His6. rbs = ribsome binding site. The initiating Met
is included in the NdeI site (CATATG); b Fab vector. This polycistronic vector includes
two ribosome binding sites (rbs) to initiate translation of the heavy and
light chain. The hexahistdine-tag including the BamHI site adds
Gly-Ser-His6
The truncated heavy chain for Fab production was amplified by PCR
from the synthetic gene and cloned XbaI/BamHI into a polycistronic vector with
the light chain (cloned NdeI/Hind III). These polycistronic vectors include two
ribosome binding sites (rbs) and make two proteins by co-expression from two
translation initiation sites (Fig. 4).
All heavy chain fragments included a C-terminal hexahistidine-tag. The tag was
placed on the heavy chain rather than the light chain as preliminary studies on
other Fab fragments had suggested that in some cases soluble light chain could
be generated and purified in the absence of heavy chain co-expression whereas
the opposite was not observed i.e. having the tag on the heavy chain was
designed to increase the quality of the final product.
A polycistronic expression construct for codon optimized Erv1p and
codon optimized mature PDI was made in modified pET23 as described previously
[9]. The polycistronic fragment
was transferred into the new vector with Ptac promoter by cloning XbaI/XhoI.
From there the fragment containing Ptac promoter, codon optimized Erv1p and
codon optimized PDI was cloned NsiI/AvrII into modified a pLysSBAD-vector
described previously [10] to
generate pMJS205 expression vector. A control construct identical to pMJS205,
except lacking the genes for Erv1p and PDI was made by removing the genes by
NdeI/SpeI digestion and ligating in short annealed primers with complementary
sticky ends.
All plasmid purification was performed using the Gen-Elute HP
Plasmid Miniprep Kit (Sigma Aldrich) and all purification from agarose gels was
performed using the Gel/PCR DNA Fragments Extraction Kit GeneAid), both
according to the manufacturers’ instructions.
All plasmids generated were sequenced to ensure there were no
errors in the cloned genes.
Protein expression
For expression in EnPresso B media in 24 deep well plates,
E. coli strains containing expression
vectors were streaked out from glycerol stocks stored at −70 °C onto LB agar
plates containing 5 g/L glucose and suitable antibiotics to allow for selection
(100 μg/ml ampicillin for pET23 derivatives, 35 μg/ml chloramphenicol for pLysS
derivatives) and the plates incubated at 37 °C overnight. The next day one–three
colonies from these plates were used to inoculate 2 ml of LB media supplemented
with 2 g/L glucose, containing suitable antibiotics, and the cultures grown at
30 °C, 200 rpm (2.5 cm radius of gyration) in 24 deep well plates covered with
an oxygen permeable membrane for 6–8 h. These cultures were used to seed 24 deep
well plates containing 3 mls of EnPresso B media (Biosilta Oy; as manufacturer’s
instructions) per well containing suitable antibiotics and the cultures grown at
30 °C, 200 rpm (5 cm radius of gyration) in 24 deep well plates covered with an
oxygen permeable membrane for approximately 16 h. The cultures were then boosted
(as manufacturer’s instructions) and induced with 0.5 mM IPTG. Cultures were
harvested after a further 24 h of growth. Final OD600
values of the cultures were in the range 20–37. The cells were collected by
centrifugation and resuspended in 3 mls of 50 mM sodium phosphate pH 7.4,
20 μg/ml DNase, 0.1 mg/ml egg white lysozyme. After 10 min incubation the
resuspended cultures were frozen. Cells were lysed by freeze-thawing.
Protein expression in shake flasks was as per 24 deep well plates
except the media volume was 25 mls (250 ml flask) of EnPresso B media and
cultures were grown at 30 °C, 250 rpm (2.5 cm radius of gyration). Resuspension
was done in the same volume as the initial culture.
Protein purification and analysis
Purification of hexa-histidine tagged proteins was performed by
standard immobilized metal affinity chromatography using HisPur Cobalt Superflow
Agarose (Thermos Scientific) resin under native conditions following clearance
of the cell lysate by centrifugation (4000 rpm, 20 min, 4 °C) for 24 deep well
plate. For 3 ml cultures from 24 deep well plates IMAC was performed using
0.5 ml resin in small gravity feed columns. The resin was washed with 2 × 5 mls
of water, equilibrated with 2 × 5 mls of 50 mM phosphate buffer (pH 7.4). After
loading the sample the column was equilibrated with 5 ml of 50 mM phosphate
buffer (pH 7.4), washed with 4 × 5 mls of wash buffer (50 mM sodium phosphate,
5 mM imidazole, 0.3 M sodium chloride; pH 7.4) then 5 mls of 50 mM sodium
phosphate (pH 7.4) before elution with 3 × 0.7 mls of 50 mM sodium phosphate,
150 mM imidazole (pH 7.4). For 25 ml cultures the same protocol was used with
the following changes: 1.0 ml of resin; 6 × 5 mls of wash buffer; elution with
4 × 1 ml of buffer. Where needed, 2.5 ml of eluted sample was desalted into
50 mM sodium phosphate (pH 7.4) on PD-10 columns (GE Healthcare). Appropriate
samples were treated with 20 mM NEM for 20 min at room temperature prior to
making SDS–PAGE samples or mass spectrometry analysis.
Protein analysis
Far-UV circular dichroism spectra were recorded on a Chirascan-
plus CD spectrophotometer. All scans were collected at 25 °C as an average of
four scans, using a cell with a path length of 0.1 cm, scan speed 2 nm/s, step
size 0.5 nm, a spectral band width of 1.0 nm. The maximal HT voltage was
750 V.
For determining binding of 3211 Fab and scFv to their ligand,
100 ng of recombinant NTproBNP1-76 [32] in 0.1 M sodium bicarbonate buffer pH 9.6 was coated per
well on a shaking ELISA plate over-night at 4 °C. The wells were emptied and
rinsed three times with 250 μl of 1xPBS (20 mM phosphate, 150 mM sodium
chloride, pH 7.4) containing 0.05 %v/v tween 20 and then incubated with 250 μl
of blocking buffer (0.2 % gelatin, 0.5 % BSA, 0.05 % v/v tween20 in 1xPBS, pH
7.4) for 20 min at room temperature. Then 100 μl samples containing 0–20 ng of
scFv or Fab, diluted in the blocking buffer were incubated for 2 h at room
temperature shaking. After removal of the sample and washing the wells six times
with 300 μl of 1xPBS containing 0.05 % v/v tween 20, 100 μl of alkaline
phosphatase labelled anti-HIS antibody (Sigma) diluted 1:10000 in blocking
buffer was added and the reactions incubated for 1 h at room temperature. After
removal of the detection antibody and washing the wells six times with 300 μl of
1xPBS containing 0.05 % tween 20, the 1 mg/ml pNPP-substrate solution (Sigma) in
0.2 M Tris, 5 mM MgCl2 was added and incubated for 30 min
at room temperature. The absorbance at 405 nm was measured with a Tecan Infinite
M1000PRO multilabel reader.
For determining the specificity of Maa48 Fab binding, the ligand
(MAA-LDL, MDA-LDL, copper oxidized-LDL, carbamylated-LDL, native LDL, MAA-BSA,
MDA-BSA or native BSA; sources) at 0–20 μg/ml concentration in PBS was bound to
an ELISA plate at 4 °C overnight. The antigens were prepared as described
[15]. The plate was washed
three times with 0.27 mM EDTA in PBS using an automated plate washer.
Nonspecific binding was blocked with 0.5 % fish gelatin and 0.27 mM EDTA in PBS
for 1 h at room temperature. Maa48 Fab (1 µg/ml) was incubated for 1 h at room
temperature. Alkaline phosphatase-conjugated anti-human IgG (Fab) (Sigma) was
used as a secondary antibody and LumiPhos 530 (Lumigen) as a substrate in the
assay [14]. The chemiluminescence
was measured as relative light units (RLU) with a Wallac Victor3 multilabel
reader (Perkin Elmer).
Masses of purified and desalted proteins, treated and not treated
with 20 mM NEM were measured by LCMS with an Aquity UPLC-system (Waters)
connected to a Synapt G1 Q-ToF –type mass spectrometer. The analytical column
was a BEH 300 C4, 2.1 ×100 mm (Waters) run at 0.4 ml/min using a gradient from
3 % acetonitrile in water/0.1 % formic acid to 70 % acetonitrile over 15 min.
Samples were acidified with trifluoracetic acid to about 0.5 % v/v and 5 µl of
the sample was injected. The mass spectrometer was operated in sensitivity mode
with lock mass corrected 1 s scans in continuous mode for m/z 400–2000.
Capillary voltage was 3.5 kV, cone voltage 30 V. Mass spectra were base line
subtracted and deconvoluted with MaxEnt1.
Abbreviations
DWP:
deep well plate
NEM:
N-ethyl maleimide
PDI:
protein disulfide isomerase
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AG, JV, YU, MS, CW and SH performed the research. LWR conceived and
coordinated the study. All authors were involved in experimental planning and have
read and approved the final manuscript.
Acknowledgements
This work was supported by the Academy of Finland, Sigrid Juselius
Foundation, Finnish Foundation for cardiovascular Research and Biocenter Oulu.
We would like to acknowledge the Biocenter Oulu proteomics and protein analysis
core facility for assistance with the mass spectrometry.
Competing interests
Patent applications for CyDisCo have been filed.
Author information
Author notes
Authors and Affiliations
Faculty of Biochemistry and Molecular Medicine and Biocenter
Oulu, University of Oulu, Oulu, Finland
Anna Gaciarz, Johanna Veijola, Yuko Uchida, Mirva J. Saaranen & Lloyd W. Ruddock
Department of Medical Microbiology and Immunology and Medical Research
Center, University of Oulu, Oulu, Finland
Chunguang Wang & Sohvi Hörkkö
Nordlab Oulu, Oulu University Hospital, Oulu, Finland
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Gaciarz, A., Veijola, J., Uchida, Y. et al. Systematic screening of soluble expression of
antibody fragments in the cytoplasm of E. coli
.
Microb Cell Fact15, 22 (2016). https://doi.org/10.1186/s12934-016-0419-5