Here we describe the design and construction of an expanded in vitro VNAR library with more extensive synthetic CDR3 loop variations,and hypothesize that this library should contain bind
Trang 1Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70
Stewart D Nuttall1,2, Usha V Krishnan1,2, Larissa Doughty1, Kylie Pearson2,3, Michael T Ryan2,3,
Nicholas J Hoogenraad2,3, Meghan Hattarki1, Jennifer A Carmichael1,2, Robert A Irving1,2
and Peter J Hudson1,2
1
CSIRO Health Sciences and Nutrition, and2CRC for Diagnostics, Parkville, Victoria, Australia;3Department of Biochemistry,
La Trobe University, Bundoora, Victoria, Australia
The new antigen receptor (IgNAR) from sharks is a
disul-phide bonded dimer of two protein chains,each containing
one variable and five constant domains,and functions as an
antibody In order to assess the antigen-binding capabilities
of isolated IgNAR variable domains (VNAR),we have
con-structed an in vitro library incorporating synthetic CDR3
regions of 15–18 residues in length Screening of this library
against the 60 kDa cytosolic domain of the 70 kDa outer
membrane translocase receptor from human mitochondria
(Tom70) resulted in one dominant antigen-specific clone
(VNAR12F-11) after four rounds of in vitro selection VNAR
12F-11 was expressed into the Escherichia coli periplasm and
purified by anti-FLAG affinity chromatography at yields of
3 mgÆL)1 Purified protein eluted from gel filtration columns
as a single monomeric protein and CD spectrum analysis
indicated correct folding into the expected b-sheet
confor-mation Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd¼ 2.2 ± 0.31 · 10)9M )1) indi-cating that these VNARdomains can be efficiently displayed
as bacteriophage libraries,and selected against target anti-gens with an affinity and stability equivalent to that obtained for other single domain antibodies As an initial step in producing intrabody variants of 12F-11,the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed High affinity binding was only retained in the wild-type protein,which combined with our inability to affinity mature 12F-11,sug-gests that this particular VNARis critically dependent upon precise CDR loop conformations for its binding affinity Keywords: new antigen receptor; variable domain; peptide display; Tom70; mitochondrial import
Conventional antibodies recognize antigens through the
combination of six complementarity determining region
(CDR) loops displayed three each upon variable heavy (VH)
and variable light (VL) chain immunoglobulin domains [1]
These CDR loops vary in size and composition allowing
formation of a large number of conformational
antigen-binding surfaces including planar and ridged topologies [2]
The orientation of the loops is maintained by a combination
of their internal architecture,the underlying
immunoglo-bulin scaffold,and the hydrophobic interaction between the
antibody VHand VLdomains [3] In contrast,families of
antibody-like molecules characterized recently in camelids
and sharks rely on a single immunoglobulin VH-like domain
framework,which presents two or three CDR loops to form
the antigen-binding interface [4–7] For camelids,these VHH
single domain antibodies can bind an extensive range of
antigens,including large proteins,enzymes (either within or outside the active site clefts),haptens and dyes Biochemical and structural data now shows that VHH binding affinity resides in a variety of possible CDR conformations that can include all three CDRs,or an elongated CDR3 loop alone,
or a combination of CDR and framework side-chain and main-chain residues [8]
For sharks,the new antigen receptor (IgNAR) from Ginglymostoma cirratum (nurse sharks) and Orectolobus maculatus(wobbegong sharks) also utilizes a single VH-like domain,which we herein term VNAR[9,10] Structurally, the entire intact IgNAR antibody molecule is a disulphide-bonded dimer of two protein chains,each containing the single variable and five constant domains There is no associated light chain and immunoelectron microscopy confirms that the VNARdomains do not associate together across a VH/VL-like interface and thereby provide bivalent affinity to two separate antigen molecules [11] There is a striking evolutionary convergence at the molecular structure level between the shark VNAR and camelid VHH antigen-binding domains Similarities include,but are not limited
to (a) the presence of charged rather than hydrophobic residues in the conventional VLinterface of the immuno-globulin framework,which imparts a hydrophilic character
to the solvent-exposed areas; (b) larger CDR3 loops compared to those found in human and murine antibodies (murine, X ¼ 10; human, X ¼ 13; llama VHH, X ¼ 15; camel V H, X ¼ 17.5; V X ¼ 16) [10,12,13]; and
Correspondence to Stewart Nuttall,CSIRO Health Sciences and
Nutrition,343 Royal Parade,Parkville,Victoria 3052,Australia.
Fax: + 61 3 9662 7314,Tel.: + 61 3 9662 7100,
E-mail: Stewart.Nuttall@csiro.au
Abbreviations: IgNAR,new antigen receptor antibody from sharks;
VNAR,single variable domain of the IgNAR antibody; Tom70,
human 70 kDa component of the translocase of the outer
mito-chondrial membrane; CDR,complementarity determining region.
(Received 14 May 2003,revised 20 June 2003,accepted 1 July 2003)
Trang 2(c) the frequent presence of disulphide bridges,indicated
by paired cysteine residues,either within the CDR3 loop
(Type1 VNAR) or between the CDR1 and CDR3 loops
(Type2 VNAR) [5,11] Camelid VHHs typically possess
disulphide linkages either between the CDR1 and)3,or
CDR2 and)3 loops [13,14] Despite these similarities, the
camel and shark variable domains are clearly different,both
by sequence alignment (only 20% identity) and the
unusual focus of shark VNAR variability into only the
CDR1 and)3 regions (Type2 VNAR),or the CDR2 and)3
regions (Type1 VNAR) [7]
While the shark IgNARs have yet to be formally
demonstrated as in vivo molecules responsible for
immuno-surveillance,there is strong evidence for their functional
role in antigen binding First,an analysis of mutational
patterns of membrane-bound and secreted forms of nurse
shark IgNARs indicated that they are mutated in the latter
form,suggesting affinity maturation by somatic
hyper-mutation [15,16] Second,in a previous study,we showed
that the individual wobbegong VNARs could be expressed
as soluble single molecules in the E coli periplasm An
in vitro Type2 VNAR library was then designed with
randomized CDR3 loops,and displayed successfully on
the surface of fd bacteriophages and panned for specific
binding molecules [10] Third,and most recently we
isolated two naturally occurring VNAR domains targeting
the kgp protease from Porphyromonas gingivalis with
affinities within the nanomolar range [17] Finally,a third
type of VNAR (Type3) has very recently been identified in
neonatal shark primary lymphoid tissue that probably
functions as a protective low-specificity antibody early in
development,prior to maturation of the Type 1/2 IgNAR
antigen-driven response [7] The Type3 VNAR topology is
characterized by a constant sized CDR3 loop of limited
diversity,probably stabilized by a conserved tryptophan
residue within the CDR1 loop [7]
Here we describe the design and construction of an
expanded in vitro VNAR library with more extensive
synthetic CDR3 loop variations,and hypothesize that this
library should contain binding reagents to a wide range of
protein targets We have chosen as one target antigen the
cytosolic domain of the 70 kDa outer membrane
trans-locase (Tom70) from human mitochondria that is
impli-cated in mitochondrial import processes [18,19] Studies in
Saccharomyces cerevisiaeand the fungus, Neurospora crassa
have characterized Tom70 as a receptor peripheral to the
mitochondrial general insertion pore that preferentially
interacts with a subset of preproteins that typically contain
internal targeting signals and/or require the action of
cytosolic chaperones for their delivery to the mitochondrial
surface [20,21] The human homologue of Tom70 has only
recently been identified [22],and attempts to generate high
specificity polyclonal or monoclonal antibodies have so far
been unsuccessful,perhaps due to extensive sequence
homology across species Here,we report the isolation
and characterization of a VNARthat binds with high affinity
to human Tom70 as an important demonstration that
VNARlibraries can provide novel binding reagents against
refractory and immunosilent targets Further,to
demon-strate the effect of removing the internal stabilizing
disul-phide bond in the manner of antibody V-domain
intrabodies [23],residues Cys22 and Cys82 were modified
by alkylation,or replaced with alanine and valine,respect-ively,and the resultant VNARs evaluated for retention of binding affinity
Material and methods
Equipment and reagents Restriction enzymes and Vent DNA polymerase were purchased from New England Biolabs; T4 DNA ligase was from Biotech (Australia) DNA fragment recovery and purification was by QIAquick Gel Extraction Kit,Qiagen and small-scale preparations of DNA from E coli were performed using the QIAprep Spin Miniprep Kit,Qiagen Monoclonal anti-(FLAG) Ig affinity resin was produced as previously described [17] BenchMarkTMPrestained Protein Ladder Cat # 10748–010 was from Gibco BRL Life Technologies Standard molecular biological techniques were performed as described [24] Goat anti-(mouse) IgG (Fc)-HRP was from Pierce
E coli strains The cell line used for library propagation and selection and protein expression was E coli TG1 (K12 supE D(lac-proAB) thi hsdD5 F¢{traD36 proAB+ lacIq lacZDM15]
E coli transformants were maintained and grown in
2· YT broth supplemented with 100 lgÆmL)1(w/v) ampi-cillin and/or 2% (w/v) glucose Solid media contained 2% (w/v) Bacto-agar Transformation of E coli was by stand-ard procedures [24] performed using electrocompetent cells Isolation of total RNA from Wobbegong sharks
Spotted Wobbegong sharks (Orectolobus maculatus) were housed and maintained at the Underwater World Aquar-ium,Mooloolaba,Queensland,Australia For isolation of peripheral blood lymphocytes,a blood sample (3 mL) was taken from the caudal vein of a young male (6.82 kg) Experiments were performed in accordance with CSIRO (Health Sciences and Nutrition) animal ethics requirements Total RNA was extracted using the AquaPure RNA Isolation Kit (BIO-RAD,Australia),stored at )80 C, and used in reverse transcription-polymerase chain reac-tions using the Titan one tube RT-PCR system (Roche, Germany) as described [10]
Library construction and panning DNA library cassettes encoding the Wobbegong VNARwith randomization of the CDR3 loop were constructed as described [10],using CDR3 randomization oligonucleotide primers to generate synthetic CDR3s of 15 residues (#6981);
16 residues (#7211); 17 residues (#6980) and 18 residues (#7210) in length (Table 1) In addition,natural Wobbe-gong VNAR sequences were amplified direct from cDNA using only the variable domain terminal primers (Table 1) [10,17] Cassette fragments were cut with the restriction endonucleases NotI and SfiI and ligated into similarly cut phagemid display vector,pFAB5c.His [25] Library liga-tions were purified,pooled and transformed into E coli TG1,yielding a total library size of approximately 4.0· 108
Trang 3independent clones,which included a subset ( 7 · 106) of
clones derived from natural CDR3 sequences Phagemid
particles carrying the VNAR–gene3protein fusion were
propagated and isolated by standard procedures [26] For
biopanning of the phagemid library,recombinant human
Tom70 (5 lgÆmL)1in NaCl/Pi) was coated onto Maxisorb
Immunotubes and incubated at 4C overnight
Immuno-tubes were rinsed (NaCl/Pi),blocked with NaCl/Pi/Blotto
(2%,w/v; Diploma skim milk powder,Bonlac Foods Ltd.,
Melbourne,Australia) for 1 h,and incubated with freshly
prepared phagemid particles in NaCl/Pi/Blotto (2%,w/v)
for 30 min at room temperature with gentle agitation
followed by 90 min without agiation After incubation,
immunotubes were washed [NaCl/Pi/Tween20 (0.1%,v/v);
7,8,10 and 10 washes for panning rounds 1–4],followed by
an identical set of washes with NaCl/Pi Phagemid particles
were eluted using 0.1M HCl,pH 2.2/1 mgÆmL)1 BSA,
neutralized by the addition of 2MTris base [26],and either
immediately reinfected into E coli TG1 or stored at 4C
Nucleic acid isolation and cloning
Following final selection,phagemid particles were infected
into E coli TG1 and propagated as plasmids,followed by
DNA extraction The VNAR cassette was extracted as a
NotI/SfiI fragment and subcloned into the similarly
restric-ted cloning/expression vector pGC [27] DNA clones were
sequenced on both strands using a BigDye terminator cycle
sequencing kit (Applied Biosystems) and a Perkin Elmer
Sequenator The nucleotide sequence of clone 12F-11 is
deposited in the GenBank database under accession number
AY069988
Recombinant Tom70 protein
DNA encoding the receptor domain of Tom70 (residues
111–608) was amplified by PCR,cloned into the vector
pET3a as described by Young et al [21] For expression,
cells were grown to D600¼ 0.6,induced by the addition of
isopropyl thio-b-D-galactoside (IPTG,1 mM final),and
recombinant protein purified using Ni-NTA
chromatogra-phy (Qiagen) according to the manufacturer’s instructions [21]
Soluble expression of VNARconstructs from expression vector pGC
Recombinant proteins were expressed in the bacterial periplasm as described [10] Briefly, E coli TG1 starter cultures were grown overnight in 2YT medium containing
100 lgÆmL)1 ampicillin and 2.0% glucose (w/v),diluted 1/100 into fresh 2YT containing 100 lgÆmL)1ampicillin and 0.1% glucose (w/v) and then grown at 37C and shaken at
200 r.p.m until D550¼ 0.2–0.4 Cultures were then induced with IPTG (1 mMfinal concentration),grown for a further
16 h at 28C and harvested by centrifugation (Beckman JA-14/6K,5500 g 10 min,4C) Periplasmic fractions were isolated by the method of Minsky [28] and either used as crude fractions or recombinant protein purified by affinity chromatography using an anti-(FLAG) Ig/Sepharose col-umn (10· 1 cm) The affinity column was equilibrated in NaCl/Pi,pH 7.4 and bound protein eluted with Immuno-PureTMgentle elution buffer (Pierce) Eluted proteins were dialysed against two changes of NaCl/Picontaining 0.02% sodium azide,concentrated by ultrafiltration over a 3-kDa cutoff membrane (YM3,Diaflo),and analysed by FPLC on
a precalibrated Superdex 200 column (Pharmacia) equili-brated in NaCl/Pibuffer pH 7.4 Recombinant proteins were analysed,by SDS/PAGE through 15% Tris/glycine gels Enzyme linked immunosorbent assays
Protein antigens (0.5 lg per well) in NaCl/Piwere coated onto Maxisorb Immuno-plates (Nunc) and incubated at
4C overnight Plates were rinsed,blocked with 5% (w/v) Blotto in NaCl/Pifor 1 h,and incubated with periplasmic fractions or recombinant protein for 1 h at room tempera-ture Plates were rinsed with NaCl/Pi,washed three times with 0.05% Tween20 in NaCl/Pi,and anti-(FLAG) Ig (diluted 1/1000 in 5% Blotto in NaCl/Pi) added Plates were incubated and washed as above,and the horseradish peroxidase conjugated secondary anti-(mouse Fc) Ig added
Table 1 Oligonucleotide primers used in generation of V NAR libraries Sense primer (fi); antisense primer (‹).
5¢ Amplification 8406 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCACAAGGGTAGACCAAACACC N-terminus ¼ TRVDQTP … 5¢ Amplification 8407 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCGCAAGGGTGGACCAAACACC N-terminus ¼ ARVDQTP … 5¢ Amplification 8408 (fi) GTCTCGCGGCCCAGCCGGCCATGGCCGCATGGGTAGACCAAACACC N-terminus ¼ AWVDQTP … 3¢ Amplification 8404 (‹) CACGTTATCTGCGGCCGCTTTCACGGTTAATGCGGTGCC C-terminus ¼ … GTALTVK
3¢ Amplification 8405 (‹) CACGTTATCTGCGGCCGCTTTCACGGTTAATACGGTGCCAGCTCC C-terminus ¼ … GTVLTVK
CDR3 Library
construction
6981 (‹) GGTTAATACGGTGCCAGCTCCCYYMNNMNNMNNMNNMNNRYHRYH
RYHRYHMNNMNNMNNMNNMNNMNNTGCTCCACACTTATACGTGCCACTG
15 residue randomised loop CDR3 Library
construction
7211 (‹) TTTCACGGTTAATACGGTGCCAGCTCCTTTCTCMNNMNNMNNMNNRYHR
YHRYHRYHRYHMNNMNNMNNMNNMNNGNATGCTCCACACTTATACGT GCC
16 residue randomised loop
CDR3 Library
construction
6980 (‹) GGTTAATACGGTGCCAGCTCCCYYMNNMNNMNNMNNMNNMNNMNNRYHRY
HRYHRYHMNNMNNMNNMNNMNNMNNTGCTTGACACTTATACGTGCC ACTG
17 residue randomised loop
CDR3 Library
construction
7210 (‹) TTTCACGGTTAATACGGTGCCAGCTCCTTTCTCMNNMNNMNNMNNMNN
MNNRYHRYHRYHRYHRYHMNNMNNMNNMNNMNNGNATGCTTGA CACTTATACGTGCC
18 residue randomised loop
Trang 4[1/1000 in NaCl/Pi/5% Blotto] Plates were washed again
and developed using 2,2-azino di-(ethyl) benzthiazoline
sulphonic acid (Boehringer Mannheim) and read at A405
Biosensor binding analysis
A BIAcoreTM 1000 biosensor (BIAcore AB,Uppsala,
Sweden) was used to measure the interaction between
VNARprotein 12F-11 and Tom70 FPLC-purified Tom70
at a concentration of 20 lgÆmL)1in 10 mMsodium acetate
buffer,pH 4.5 was immobilized onto a CM5 sensor chip via
amine groups using the amine coupling kit (BIAcore AB)
[29] The immobilization was performed at 25C and
5 lLÆmin)1flow rate Injection of 28 lL of Tom70 coupled
990 RU to the surface Binding experiments were performed
in Hepes buffered saline (HBS; 10 mMHepes,0.15MNaCl,
3.4 mMEDTA,0.005% surfactant P20,pH 7.4) at 25C
and a constant flow rate of 5 lLÆmin)1 with a series of
12F-11 concentrations (2.2–17.8 nM) Binding experiments
were performed immediately,as prolonged washing with
the HBS buffer resulted in a decrease in activity of the
immobilized Tom70 Regeneration of the Tom70 surface
was achieved by running the dissociation reaction to
completion before the next injection of analyte
For binding experiments in the reverse orientation,
recombinant protein 12F-11 was immobilized by the
standard amine coupling method 12F-11 protein at a
concentration of 20 lgÆmL)1 in 10 mM sodium acetate
pH 6.0 was injected for 8 min (40 lL) over an activated
surface to couple 650 RU of protein onto the sensor surface
The 12F-11 surface was regenerated with a 10-lL aliquot of
50 mMHCl with negligible loss of binding activity Binding
experiments were performed in HBS buffer at 25C and a
flow rate of 5 lLÆmin)1with a range of Tom70
concentra-tions (37.0–296 nM) The binding data was evaluated with
BIAEVALUATION3.0.2 [30]
Structural modeling
VNAR domains 12F-11 (this study) and 7R-1 [10] were
modelled using the program MODELLER [31] The PDB
database was searched with the 12F-11 VNARsequence and
17 structures with the best Z-scores selected as templates
The PDB accession numbers for the template molecules
were as follows with the chain Ids indicated: 1B88:B,
1D9K:E,1H5B:B,1KB5:A,1KJ2:A (T-cell receptor alpha
domains); 1BJM:A,2RHE (Bence–Jones proteins);
domains); 1IAI:M,1F6L:L,iIFF:L,1JNL:L and 35C8:L
(VLdomains) The resulting model structures were refined
and energy minimized using molecular dynamics restrained
to the template structure,except where gaps occurred in the
alignments and for CDRs 1 and 3 In these cases extensive
loop modelling was undertaken and the final model
selection based on the modeller objective score
Construction of the 12F-11DCys mutant and 12F-11
reduction/alkylation
For reduction and alkylation of 12F-11,recombinant
protein (1.3–1.5 mg) was denatured/reduced using 6M
guanidine HCl and 50 m dithiothreitol (pH 8.0) for 1 h
at 45C under nitrogen Cysteine residues were then alkylated by the addition of 100 mM(final) iodo-acetamide (pH 8.0)/1 h (room temperature) followed by quenching with additional dithiothreitol Samples were dialysed against four changes of NaCl/Pi,concentrated,and analysed by FPLC,SDS/PAGE and ELISA,as above The disulphide minus variant of 12F-11 incorporating mutations Cys22Ala and Cys82Val was constructed by overlapping PCR using oligonucleotide primers N8517 (Forward: 5¢-ACAAGGG TAGACCAAACACCAAGAACAGCAACAAAAGAG ACGGGCGAATCACTGACCATCAACgccGTCCTGA GAGAT-3¢) and N8518 (Reverse: 5¢-TTTCACGGTTAA TGCGGTGCCAGCTCCCCAACTGTAATAAATACC AGACAAATTATATGCTCCaacCCTATACGTGCCA CTG-3¢); followed by secondary PCR using VNARterminal oligonucleotide primers [17] to complete the framework and incorporate NotI and SfiI restriction endonuclease sites for cloning Bacterial expression was as described above Affinity maturation by error-prone PCR
The 12F-11 VNARcassette was mutagenized by error prone PCR using Taq DNA polymerase [32] Pools of mutated
VNARcassettes were isolated,cut with SfiI/NotI,cloned into the phagemid vector pFAB.5c,and transformed into E coli TG1 as above The resulting library ( 1 · 106independent clones) showed on average 1–2 residue changes/100 amino acids Two rounds of panning under high stringency conditions were performed on immobilized Tom70 as above,except that the final five washes for each selection round incorporated a further 2 min incubation to promote dissociation The selected VNARcassettes were then rescued, subcloned and analysed
Results
Construction of an expanded Wobbegong VNARlibrary
We previously designed and constructed a Wobbegong IgNAR variable domain (VNAR) library with long synthetic CDR3 loops of either 15 or 17 residues in length inserted into
a mixed scaffold repertoire of 26 naturally occurring VNAR domains This small library ( 3 · 107independent clones) was displayed on the surface of fd bacteriophage and successfully panned against protein antigens [10] In order to increase the diversity of possible antigen-binding fragments, this library was expanded in three ways: (a) the extended library comprised increased complexity with CDR3 lengths ranging form 15–18 residues to reflect the predominant natural diversity in Wobbegong and Nurse shark VNARs (Fig 1A) Additionally,a different randomization pattern was used,biased toward the incorporation of cysteine at CDR3 loop residue positions 1 and 7–11,to enhance the possibility of inter- and intra CDR disulphide cross-links These strategies are summarized in Fig 1B and Table 1,and details of the library construction are given in greater detail in the Materials and methods (b) The extended library was based on CDR3 loops grafted into a large scaffold repertoire
of natural VNARdomains generated by direct RT-PCR from total RNA extracted from Wobbegong shark peripheral blood lymphocytes Thus,many differing CDR1 sequences and minor framework variations were represented in the
Trang 5extended library (c) A subset of the extended library now
also included naturally occurring CDR3 loops derived
directly from the immune repertoire of several sharks These
natural sequences form part of the matured shark immune
response,generated in response to exposure to antigen in the
natural environment,and have extensive size heterogeneity
(as indicated in Fig 1A) and different randomizations from
those used for the synthetic CDR3s [17] Taken together, these changes resulted in an expanded VNARlibrary consist-ing of over 4· 108independent clones,representing signi-ficantly enhanced diversity compared to our original VNAR repertoire This library was displayed as a fusion with the gene3protein of fd bacteriophage in the vector pFAB5c.HIS (Fig 1C) allowing for standard phage display and selection
Biopanning against immobilized mitochondrial import receptor Tom70
Tom70 is an integral membrane protein consisting of a large globular receptor domain exposed to the cytosol and a short N-terminal transmembrane anchor A truncated Tom70 protein construct consisting of residues 111–608 of human Tom70 was expressed in E coli,purified as a soluble extracellular 60 kDa protein,and immobilized on immuno-tubes as a target protein [21]
The VNARlibrary was transformed into E coli TG1 and phagemid particles rescued and panned against the immo-bilized Tom70 antigen Four rounds of biopanning were performed with an increase in the stringency of washing at each step,and between selection rounds three and four a significant ( 1000-fold) increase in the titre of eluted bacteriophage was observed Colony PCR on transfected bacteriophage showed that 100% of colonies were positive for VNARsequences,and this combined with the increase in the titre,indicated positive selection Thus,VNARcassettes were rescued from phagemids,cloned into the periplasmic expression vector pGC,and transformed into E coli TG1 Periplasmic fractions from recombinant clones were tested for binding to Tom70 and negative control antigens by ELISA (not shown) Several clones showed significant binding above background,and all of these proved to be identical sequences One of these,designated clone 12F-11, was chosen for further analysis
The primary and deduced amino acid sequences of clone 12F-11 are presented in Fig 2A,including in-frame dual octapeptide FLAG epitope tags and two alanine linker regions The protein represents a 103 residue VNARdomain, with a predicted molecular mass of 14 054 Da (including the affinity tags) An alignment of protein 12F-11 with four other VNAR proteins showed a high level of sequence conservation except in the CDR regions (Fig 2B) How-ever,and most surprisingly,the alignment also revealed that the Thr39 residue,present in the other VNARs,was absent in the 12F-11 protein (Fig 2B,arrowed) Equally unusual was the CDR3 structure that at 10 residues (NYN LSGIYYSWC) is significantly shorter than the 15–18 residues encoded within the library design This truncation
in the CDR3 size probably occurred either during the initial PCR-based library construction,or under selection pressure within the early rounds of panning The presence of such CDR deletions in large libraries is not uncommon [33],and indeed one VNARprotein we reported previously was an obvious deletion mutant [10]
Recombinant protein 12F-11 is a monomeric, correctly folded protein
Loss of a nonCDR residue is potentially destructive to immunoglobulin domain structures Thus,to determine
Fig 1 Design of the V NAR library (A) Cumulative frequency
histo-gram of IgNAR CDR3 loop lengths,from Wobbegong sharks (26
sequences) and Nurse sharks (35 sequences) (B) Schematic diagram
of synthetic CDR3s used in V NAR library construction,showing the
randomization patterns used for the varying length CDRs X
repre-sents use of the nucleotide randomization strategy (NNK) that encodes
any residue or an amber stop codon Surrounding framework regions
are also shown (C) V NAR cassettes were ligated into phagemid vector
pFAB.5c at the SfiI and NotI restriction endonuclease sites The
phagemid vector incorporates a lacZ promoter,and in-frame PelB
leader,Ala 3 linker,and DGene3 protein domains prior to a translation
termination codon.
Trang 6whether the absence of residue Thr39 had an adverse effect
upon protein 12F-11 expression,folding,and stability,we
undertook a thorough protein chemical analysis
Recom-binant 12F-11 protein was expressed in E coli and then
isolated from the bacterial periplasm by affinity
chromato-graphy using an anti-(FLAG) Ig affinity resin Expression
levels obtained were routinely between 2 and 3 mgÆL)1
of protein from shake-flask cultures Analysis of
affinity-isolated protein by FPLC through a precalibrated Superdex
200 gel filtration column showed a single peak eluting from
the column at approximately 36 min,corresponding to a
protein of 14–15 kDa molecular mass and consistent with
the size of a monomeric VNAR domain (Fig 3A) There
was no evidence of protein aggregation,nor were higher
order multimers such as protein dimers or tetramers
observed Indeed,protein 12F-11 appeared to have at least
equal stability to other VNARproteins we have analysed as
this FPLC profile was maintained consistently,even after
prolonged storage at 4C or multiple freeze-thaw cycles,
with no evidence of protein degradation To confirm that
the isolated protein was being correctly processed in the
E coliperiplasm,N-terminal amino acid sequence analysis
on material eluted from the FLAG column showed that
only one protein species was present (1TRVDQTP-)
corresponding to the predicted N-terminus (Fig 2A) Far
ultraviolet circular dichroism (CD) spectra of aqueous
solutions of protein 12F-11 showed a profile with a negative band with kmaxat 217–219 nm (Fig 3B) This spectrum is characteristic of a protein with a b-sheet structure with unstructured loops contributed by the CDRs and FLAG affinity tags,and is not a disordered structure [34] Indeed, the 12F-11 specturm is very similar to CD spectra obtained for other VNARproteins,for example VNAR12A-9 that has different CDR loops and a slightly different b-sheet framework,and which is shown for comparison (Fig 3B) [17] Together,these results suggest that despite the absence
of Thr39,protein 12F-11 folds into compact,b-sheet immunoglobulin in the E coli periplasm Indeed,in preliminary structural studies,protein 12F-11 crystallises
in the monoclinic P2 space group (results not shown),
Fig 2 Nucleotide and deduced amino acid sequences of the V NAR
12F-11 variable domain (A) Nucleotide and deduced amino acid sequences
of clone 12F-11 The conserved termini dictated by the oligonucleotide
primer sequences used in library construction are underlined,and the
alanine linker and dual octapeptide FLAG tags are italicised The
positions of the CDR1 and )3 regions are indicated in bold type.
(B) Alignment of protein 12F-11 with four other V NAR domain amino
acid sequences (GenBank AY069988; AF336094; AF336087;
AF336088; AF336089) Amino acids are designated with the
single-letter code,and identical residues (dark shading) and conservative
replacements (light shading; I/V/L/M,D/E,K/R,A/G,T/S,Q/N,F/Y)
are indicated The framework residue at position 39,absent in 12F-11,is
indicated by the arrow,and the CDR1 and )3 regions are highlighted.
Fig 3 Size exclusion chromatographyand CD analy sis of protein 12F-11 (A) Elution profile of affinity purified 12F-11 protein on a calibrated Superdex 200 gel-filtration column equilibrated in NaCl/P i ,
pH 7.4 and run at a flow rate of 0.5 mLÆmin)1 Protein 12F-11 elutes at approximately 36 min consistent with a monomeric domain (12F-11 calculated M r of 14 054 Da,including linker and dual FLAG octa-peptide tags) Approximate elution times for a series of protein standards are indicated by arrows,and the absorbance at A 214
(unbroken line) and A 280 (dashed line) is given in arbitrary units The
A 214 absorbance peak at approximately 46 min represents sodium azide The inset shows the same sample analysed by SDS/PAGE through a 15% (w/v) polyacrylamide Tris/glycine gel and stained with Coomassie Brilliant Blue (B) Circular dichroic spectrum of affinity purified V NAR 12F-11 in 0.05 M sodium phosphate buffer,pH 7.4 (unbroken line) For comparison,the spectrum for the naturally occurring V NAR domain 12A-9 [17] is also shown (dotted line).
Trang 7providing further evidence for folding into an ordered
domain structure
To explain more fully why protein 12F-11 folds into a
functional protein while missing a framework residue,we
modelled the variable domain structure and compared it to
a model of a conventional VNAR (Fig 4) Our modelling
studies indicate that Thr39 is located at the end of the
C strand,and that the adjacent C-C¢ loop is therefore
amenable to structural changes imposed by the residue
deletion without disruption to the framework Otherwise,
there is good agreement between the two VNAR models,
with the obvious exception of the CDR loop diversity
Specificity and binding activity of recombinant
protein 12F-11
The specificity of protein 12F-11 for Tom70 was
demon-strated by ELISA (Fig 5A) Recombinant protein reacted
specifically with Tom70 but not several other antigens tested
and was concentration dependent to 0.6 pmol of protein
(Fig 5B) The binding kinetics of the 12F)11/Tom70
interaction were also measured by BIAcore biosensor
analysis with Tom70 protein immobilized via amine coup-ling to the sensor surface As immobilized Tom70 on the sensor surface was found to be unstable,with a 70% loss of binding activity in 24 h,binding experiments were per-formed immediately upon immobilization and Fig 6A shows the interaction of varying concentrations (17.8 nM, 8.9 nM,4.5 nM,2.2 nM) of peak-purified 12F-11 monomer with the immobilized Tom70 Analysis of the binding data with the 1 : 1 Langmuir binding model showed a good fit (Fig 6A) for the monovalent analyte (12F-11) binding to a Tom70 epitope consistent with a 1 : 1 binding interaction There was no binding of the 12F-11 monomer to a blank surface (activated and then blocked with ethanolamine) indicating that there is no nonspecific interaction with the sensor surface (Fig 6A,inset) Kinetic analysis of 12F-11 binding to immobilized Tom70 revealed a rapid association rate constant (ka¼ 1.68 ± 0.27 · 106M )1Æs)1) and a dis-sociation rate constant (kD) of 3.49 ± 0.36–3Æs)1to yield a
KDof 2.2 ± 0.31)9M )1
VNARprotein 12F-11 monomer was also immobilized via amine coupling onto the sensor chip to measure the binding interaction in the reverse orientation The 12F-11 surface was stable and could be regenerated with 50 m HCl
Fig 4 Models of V NAR domains V NAR s 12F-11 (purple framework
with magenta CDRs) and 7R-1 (dark green framework with light
green CDRs) were modelled on existing immunoglobulin superfamily
variable domain structures The position of residue Thr39,missing in
12F-11,is indicated.
Fig 5 Analysis of protein 12F-11 by ELISA (A) Protein 12F-11 was purified from the periplasmic fraction of E coli TG1 by affinity chromatography through an anti-FLAG M2 antibody column and
150 pmol tested for binding to Tom70,lysozyme,kgp (lysine specific gingipain protease from Porphyromonas gingivalis),and a-amylase Results represent the average of triplicate wells (B) As for (A) except serial twofold dilutions of protein 12F-11 were tested for binding to Tom70 and lysozyme Results represent the average of duplicate wells.
Trang 8without loss of binding activity The binding data for the
interaction of Tom70 to immobilized 12F-11 however,did
not fit the theoretical Langmuir model for 1 : 1 binding
(Fig 6B) but displayed biphasic binding characteristics
indicative of multivalent binding This result is consistent
with the observation that Tom70 elutes as an equilibrium
mixture between dimer (Mr 136 kDa) and monomer on
size exclusion chromatography
Removal of the 12F-11 VNARframework disulphide
linkage
Next,protein 12F-11 was used to test the utility of VNAR
domains as possible intrabodies for expression and use in
in vivotargeting applications Specifically,we asked whether
binding affinity was retained in the absence of the conserved
immunoglobulin intradomain disulphide bond In an initial
series of experiments,recombinant 12F-11 protein was denatured and reduced using guanidine HCl and dithio-threitol,followed by alkylation of the cysteine residues and refolding However,in a result seen for many VH/VL antibodies,the modified protein almost exclusively precipi-tated in the soluble fraction Only a small proportion consistently remained in the soluble fraction,and this protein showed a similar FPLC profile to the unmodified protein (Fig 7A) This soluble protein retained binding affinity for Tom70 by ELISA (Fig 1C),and we hypothesize that this fraction represents protein that was not fully alkylated and was thus able to refold,with probable reoxidation of the disulphide bond In contrast,the insoluble fraction most likely represents irreversibly aggre-gated alkylated material
In order to more systematically test this hypothesis,we elected to eliminate the possibility of disulphide bond formation genetically by replacement of residues Cys22 and Cys82 with alanine and valine,respectively,to give a cysteine minus mutant (12F-11DCys) Use of alanine and valine was initially determined in a set of competitive selection experiments [35],and replacement with this pair of residues maintains a hydrophobic character suitable for amino acids buried deep within the protein interior and closely approximates the relative size of the cysteine side chains When 12F-11DCys was expressed into the E coli periplasm,expression levels comparable to those obtained for the wild-type were obtained,and both wild-type and mutant proteins were indistinguishable by gel filtration chromatography (Fig 7B) However,when binding to Tom70 was assessed by ELISA under stringent washing conditions,12F-11DCys did not appear to target Tom70 compared to 12F-11 and the refolded 12F-11 (reduced/alkylated soluble fraction) proteins (Fig 7C) This surprising result was further tested by kinetic ana-lysis by biosensor,which showed an approximately 200-fold decrease in binding affinity (KD¼ 2 nM to
KD¼ 500 nM) (Fig 7D) Interestingly,this loss in bind-ing affinity was completely attributed to a slower association phase,with the dissociation curve indistinguishable from the parent protein We suggest that removal of the stabilizing rigidity provided by the disulphide bond results in a slight perturbation of the orientation of the immunoglobulin b-sheets relative to each other However,upon binding,the interaction with antigen functions to lock the b-sheet and CDR loop conformations,resulting in the original dissoci-ation kinetics
Affinity maturation of 12F-11
In order to affinity mature VNAR 12F-11,a library of mutant proteins was generated by error-prone PCR The resultant bacteriophage-displayed library ( 106 independ-ent clones) was panned against Tom70 under conditions designed to select for variants with enhanced off-rate kinetics After two rounds of selection a >4000-fold increase in titre was observed indicating strong selection
Of the resultant clones,a large proportion (64%) were
12F-11 wild-type,while those with mutations almost exclusively showed conservative framework variations well-removed from the VNARbinding site Only one variant,designated 15Z-2,showed changes mapping to either the CDR or
Fig 6 Analysis of protein 12F-11 by BIAcore (A) Binding of
mono-meric V NAR protein 12F-11 to immobilized Tom70 protein (990 RU)
was measured at a constant flow rate of 5 lLÆmin)1with an injection
volume of 35 lL Dissociation was continued with HBS buffer until
the response returned to the initial value before injecting the next
sample The inset shows the binding profile of protein 12F-11
(20 lgÆmL)1) to immobilized Tom70 and a blank surface (NHS/EDC
activated and blocked with ethanolamine) The circles show the fit to
the data obtained on analysis with the 1 : 1 Langmuir binding model
for evaluation of the kinetic rate constants (B) Sensorgram showing
the binding of Tom70 (74 n M ) to immobilized V NAR protein 12F-11
monomer (650 RU) at a constant flow rate of 5 lLÆmin)1 The circles
show the fit to the data on analysis with the 1 : 1 Langmuir binding
model when only the first 110 s of the dissociation phase is analysed.
Trang 9CDR-framework junctions The two mutations in this clone
were Lys33fi Arg,which is in the CDR1 loop (Fig 1A),
and Thr43fi Ile,which is a conservative framework
variant Protein 15Z-2 expressed at levels similar to the
parental type and exhibited similar behaviour upon FPLC
analysis (Fig 8A) When tested by ELISA,15Z-2 showed a
slightly higher binding response to Tom70,but not negative
control antigens (Fig 8B) However,this difference was not
apparent in kinetic measurements by biosensor,where
12F-11 and 15Z-2 showed indistinguishable binding
kinet-ics,including no differences in dissociation rates Moreover,
upon extended storage,protein 15Z-2 showed a slight
tendency to precipitation,suggesting that its coselection
with wild-type,and slightly higher ELISA responses,may be
attributable to an increased tendency toward aggregation
Discussion
The aim of this study was to generate an in vitro library
of VNAR domains,containing both synthetic and natural
CDR3 loops,and then to isolate specific binding molecules
using as an initial target antigen the mitochondrial outer
membrane receptor Tom70 The resulting protein,12F-11,
shows a high degree of affinity and specificity for Tom70,
and with a KDof 2 nMcompares very well with affinities
reported for camelid V H domains (that vary in the range
2–300 nM [36]) and for scFv and disulphide stabilized Fv fragments [37] The high affinity of 12F-11 is directly attributable to a relatively rapid association rate (ka 1.7 · 106 M )1Æs)1),while the dissociation rate (kDa
3.5 · 10)3Æs)1) is more typical of other VNARand camelid
VHH single domain antibodies [36] For example,previously
we described a naturally occurring VNARtargeting the kgp protease from Porphyromonas gingivalis,and there the KD was 1.31· 10)7M,primarily due to the lower association rate (ka¼ 4.3 · 104
M )1Æs)1) [17]
Structurally,protein 12F-11 is a slightly unusual member
of the VNAR family Firstly,the absence of a framework residue which is commonly present (Thr39),could be hypothesized to deform the underlying b-framework However,the data clearly demonstrates that this is not the case,and molecular modelling maps this residue to a region distant to the antigen-binding site,and on the periphery of the immunoglobulin-like core scaffold and adjacent to the C-C¢ loop (see Fig 4) Further,there may well be even greater latitude for mutations within this region,as in unrelated experiments we have also discovered a naturally occurring VNARlacking five residues in this loop position (S Nuttall,unpublished data) Secondly,the mean size of the VNARCDR3 loop is 16 residues,yet protein 12F-11 achieves low nanomolar affinity binding with a CDR3 only
10 residues in length While this apparently contradicts the
Fig 7 Effect of removal of the 12F-11 disulphide bond (A) Elution profiles of affinity purified 12F-11 protein (dotted line),and the soluble fraction remaining after reduction and alkylation (unbroken line),on a calibrated Superose 12 gel-filtration column equilibrated in NaCl/P i ,pH 7.4 and run
at a flow rate of 0.5 mLÆmin)1 The inset shows relative amounts of material recovered from the soluble (S) and insoluble (P) fractions after reduction and alkylation compared to untreated protein (U) (B) Elution profiles of affinity purified 12F-11 (unbroken line) and 12F-11DCys (dotted line) proteins analysed as in (A) (C) ELISA comparing binding of 12F-11,12F-11 reduced and alkylated soluble fraction,and 12F-11DCys proteins to Tom70 and negative control antigens Results represent the average of duplicate wells (D) Binding of monomeric proteins 12F-11 and 12F-11DCys (40 lgÆmL)1) to immobilized Tom70 protein The inset shows the binding profile of protein 12F-11DCys to immobilized Tom70 and
to a nonspecific antigen and a blank surface (NHS/EDC activated and blocked with ethanolamine).
Trang 10theory that VNARs encompass most of their binding affinity
within a long and diverse CDR3 loop,it is significant that
some naturally occurring VNARs,presumably selected by
the shark antigen-driven immune response,have even
shorter CDR3 lengths [10] Indeed,not all antibody V-like
domains display extended CDR3 loops,by analogy to a
recent structural analysis of camelid VHH domains [38] a
significant proportion of the VNARrepertoire may comprise
CDR3 loops overlayed onto and across the scaffold surface
This conclusion means that,in the context of antigen
binding surfaces,shark VNAR and camelid VHH libraries
will contain structural homologues similar to antibody VH
and VLlibraries,as well as providing discrete and distinctly
different structural repertoires
In an attempt to further improve the 12F-11 binding
affinity,we generated a library of affinity-matured variants
However,selection failed to isolate any mutants with
improved binding kinetics and instead there was strong
selection for the wild-type protein This probably reflects a
precise interaction between the CDR1 and relatively short
CDR3 loop with only minor variations being tolerated
Thus,within the relatively limited context of a library of a
million independent clones covering the entire V
cassette,it is unlikely that the rare beneficial mutations will
be present In contrast,in experiments aimed at affinity maturing other VNAR domains,we have isolated several variants with approaching order-of-magnitude enhanced affinities from similar sized error-prone PCR libraries However,in these cases the mutations targeted an extended and flexible CDR3 loop,which provided greater latitude for the introduction of positive mutations (S Nuttall,unpub-lished observation)
The experiments testing the impact of removal of the intradomain disulphide bond provide further evidence that
VNAR 12F-11 is a tightly folded protein domain relatively intolerant to manipulation Reduction and alkylation most probably had a fatal impact upon protein stability and folding resulting in aggregated protein This is not uncom-mon for immunoglobulin domains,and only relatively few antibody VHor VLdomains remain functional after such modification [39–41] This is unsurprising given that during alkylation,cysteine is replaced by S-carboxymethylate cysteine,resulting in a significant increase in molecular mass that must be accommodated within the otherwise tightly folded antibody core More elegant is genetic replacement of the two half-cystines with an alanine-valine pair that is virtually mass neutral and represents an optimum replacement strategy for antibodies determined
in a competitive selection system [35] Our finding that such
a substitution,with consequent ablation of the intramole-cular disulphide bond,decreases the kinetics of association but not dissociation can be explained as due to a slight perturbation of the orientation of the immunoglobulin b-sheets relative to each other thereby reducing the associ-ation rate In contrast,antigen binding presumably locks the b-sheet and CDR loop conformation,resulting in the original dissociation kinetics This interpretation is consis-tent with the current dogma that antigen binding is critically dependant upon the precise interaction of CDR1,CDR3, and underlying framework residues Specifically,we noted that the three CDR3 tyrosine residues in 12F-11 (Tyr residues are over-represented in CDR loop structures [42]), may combine with Tyr35 which lies just C-terminal to the CDR1 loop,to form the predominant antigen binding site Additionally,the noncanonical disulphide bridge often found in VNAR domains,that links the CDR1 and )3 loops together providing additional conformational stabi-lity,is absent in this case However,any more detailed analysis of the 12F-11 paratope and assessment of the varying contributions of CDR and framework regions clearly requires definitive structural data; this analysis is currently in progress
The isolation of 12F-11 from the extended VNARlibrary demonstrates that synthetic CDR3 libraries selected in vitro can generate proteins with antigen-binding affinities at least equal to those of natural systems (i.e immunization of animals followed by isolation of the variable gene reper-toire) This is especially important where conventional (i.e murine) antibodies are difficult to generate The human Tom70 receptor is an important example of such refractory targets,and the generation of a high-affinity reagent specific for the cytosolic domain is a potentially valuable tool in the study of preprotein targeting However,in a series of in vitro import inhibition experiments,designed to test the ability of 12F-11 protein to block the import of precursor proteins
Fig 8 Affinitymaturation of 12F-11 (A) Elution profiles of affinity
purified 12F-11 (dotted line) and 15Z-2 (unbroken line) proteins on a
calibrated Superose 12 gel-filtration column equilibrated in NaCl/P i ,
pH 7.4 and run at a flow rate of 0.5 mLÆmin)1 (B) ELISA comparing
binding of 12F-11 and 15Z-2 protein to Tom70 and negative control
antigens Results represent the average of duplicate wells.