Directed evolution for the development of conformation-specific affinity reagents using yeast display

Jane M. Weaver-Feldhaus1, Keith D. Miller1, Michael J. Feldhaus2 and Robert W. Siegel1,3

1Pacific Northwest National Laboratory, Richland, WA 99352 and 2Merrimack Pharmaceuticals, 101 Binney Street, Cambridge, MA 02142, USA

3 To whom correspondence should be addressed. Current address: Abbott Laboratories, Diagnostic Division, 100 Abbott Park Road, Abbott Park, IL 60064, USA E-mail: robert.siegel{at}abbott.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca2+ binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca2+-bound (Ca2+-CaM) or Ca2+-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (Kd = 0.8 nM) and specificity (>1000-fold) for Ca2+-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (Kd = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.

Keywords: calmodulin/directed evolution/flow cytometry/recombinant antibody/yeast display


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antibodies are essential tools in proteomics research used in numerous applications including protein localization studies using immuno-histochemical approaches, post-translational modification profiling with immuno-blots, immuno-precipitation of protein complexes, ELISA-based diagnostic assays and therapeutic applications (Bradbury et al., 2004Go). Over the past three decades, the research community has used antibodies derived from animals as undefined polyclonal sera of limited quantities or monoclonal murine antibodies of defined characteristics and of unlimited supply. The recent advent of phage/phagemid (McCafferty et al., 1990Go; Marks et al., 1991Go), ribosomal (Hanes et al., 1998Go) and yeast (Boder and Wittrup, 1997Go) in vitro display technologies allow new types of monoclonal affinity reagents, single-chain variable fragments (scFv) or Fab fragments to be readily isolated. Selected recombinant antibody fragments can also be produced as intact immunoglobulins (Igs) after genetic manipulation and subsequent production in mammalian cell lines. The in vitro nature of these display technologies also allow antibodies recognizing highly conserved antigens to be easily obtained by removing the selection process from self-tolerance mechanisms of the immune system. These in vitro display technologies are compatible with directed evolution approaches that allow investigators to engineer specifically desirable properties, such as affinity, specificity or stability into previously selected molecules.

Yeast display has been demonstrated to be a useful platform for both antibody discovery (Feldhaus et al., 2003Go; Weaver-Feldhaus et al., 2004Go) and modification (Boder et al., 2000Go; Graff et al., 2004Go). Recombinant antibodies are displayed on the yeast surface as a fusion protein to a cell wall component, Aga-2 (Boder and Wittrup, 1997Go), and library generation can be facilitated by the homologous recombination system inherent in yeast (Swers et al., 2004Go; Weaver-Feldhaus et al., 2004Go). Coupling fluorescence-activated cell sorting (FACS) with cell surface display permits monitoring of both antibody expression on the cell surface and the ability of that antibody to bind antigen. The unparalleled resolution offered by FACS during the selection process allows the visualization of discrete populations that differ in affinity and/or epitope specificity and the quantitative recovery of desired clones (Siegel et al., 2004Go). FACS analysis also facilitates the characterization of isolated clones directly on the yeast surface, eliminating the need for purified antibody. For those clones lacking a desired trait, yeast display has proven to be highly effective for various directed evolution applications (Kieke et al., 1999Go; Boder et al., 2000Go; Shusta et al., 2000Go; Colby et al., 2004aGo,bGo).

Calmodulin (CaM) is an ubiquitous Ca2+-dependent signaling molecule responsible for sensing cellular Ca2+ homeostasis [for a review, see James et al. (1995)Go]. As a vital cell signal component, CaM binds and activates numerous cellular enzymes, channels and receptors associated with intracellular signaling and metabolism (Cox, 1988Go). CaM contains four Ca2+-binding sites (Kd = 10–6–10–7 M) that each adopt a helix–loop–helix conformation, a motif termed EF-hand (Krestsinger and Knockolds, 1973Go). Each globular domain contains two Ca2+-binding sites that permit cooperative binding. Ca2+ binding induces a conformation change in CaM exposing two hydrophobic patches for target interaction that is crucial for transduction of the Ca2+ signal (Babu et al., 1988Go). The structure of the apo-CaM conformation has also recently been elucidated and shows that both the N- and C-terminal lobes form compact four-helix bundles that protect the hydrophobic patches (Schumacher et al., 2004Go). This conformation of CaM has been shown to serve as a regulator of neuronal growth as modulated by GAP 43 (Chan et al., 1986Go; Alexander et al., 1987Go).

As CaM is involved with a variety of signaling pathways in the cell, this protein offers an excellent opportunity to develop affinity reagents that would aid investigators in dissecting the multitude of protein complexes formed with CaM under specific environmental conditions. Unfortunately, the amino acid sequence of CaM is highly conserved among all eukaryotes and is identical within vertebrates (Jamieson et al., 1980Go), hampering traditional immunization processes. Studies have also demonstrated that antibody recognition can be dependent on the Ca2+-binding status of various EF-hand proteins (Winsky and Kurznicki, 1996Go; Goncalves et al., 1997Go). Given the absolute sequence conservation among vertebrates and conformational variability upon Ca2+ binding, there have been only a few reports of monoclonal antibodies recognizing mammalian CaM. Antibodies have been produced by immunizing mice with a mixture of mammalian CaM and phosphodiesterase (Hansen and Beavo, 1986Go), crosslinked brain extract (Wolf et al., 1995Go), invertebrate isoforms (Kobayashi et al., 1991Go) or thyroglobulin-linked fusion protein to C-terminal peptide of CaM (Sacks et al., 1991Go). In vitro immunization of mouse spleen cells has also been reported (Pardue et al., 1983Go). Some of the published antibodies specifically recognize Ca2+-CaM whereas only marginal specificity for apo-CaM has been observed. In addition, none of the published antibodies seem to have an especially high affinity (i.e. <10–9 M) for either conformation of CaM, preventing detailed proteomic investigations of each conformation state.

Given the advantages that in vitro selection techniques, in general, and yeast display, in particular, have to offer for selecting antibodies with predefined qualities, we attempted to isolate human antibody fragments with high affinity and specificity for the different conformations of vertebrate CaM. A de novo selection of scFv clones recognizing this highly conserved antigen was performed using a human non-immune scFv library displayed on yeast. Directed evolution from variegated populations derived through error-prone polymerase chain reaction (PCR) of an individual clone obtained from this original selection was subsequently used to engineer improvements in both affinity and specificity. Two classes of CaM-specific antibodies, one recognizing Ca2+-CaM and the other apo-CaM, were obtained. The Ca2+-CaM scFv has an equilibrium dissociation constant (Kd) of 800 pM and >1000-fold specificity for this particular isoform relative to apo-CaM. A single-domain antibody (dAb) recognizing apo-CaM was engineered to have a Kd of 1 nM and is >300-fold more specific for this particular conformation relative to Ca2+-CaM. Some reactivity to other members of the EF-hand protein family was observed, but with greatly reduced affinities compared with CaM. Conversion of both of these CaM-binding clones into soluble scFv, dAb or IgG molecules resulted in the loss of binding activity which we cannot explain. Nonetheless, these results demonstrate that conformation-specific antibodies can be quickly and easily obtained by directed evolution of primary clones using the yeast display platform.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reagents

A Miltenyi MidiMacs system with LS columns in conjunction with streptavidin or anti-biotin Microbeads (http://www.miltenyibiotec.com) were used for all magnetic separations. Monoclonal antibodies anti-c-myc 9E10 (Covance, BAbco) and anti-HA 12CA5 (Roche, Indianapolis, IN) were used in conjunction with goat anti-mouse fluorochrome conjugated secondary antibodies (Molecular Probes, Eugene, OR). Streptavidin-conjugated phycoerythrin (SA-PE) was purchased from BD Pharmingen (San Jose, CA). Bovine calmodulin (P-1431), bovine brain S100 (S-6552) and chicken troponin C (T-1771) were obtained from Sigma (St Louis, MO). Initially, another source of calmodulin, kindly provided by Thomas Squier (PNNL) and purified as described (Strasburg et al., 1988Go), was used. Subsequent tests showed no differences between the two calmodulin sources. Purified antigens were biotinylated using a Pierce EZ-Link biotinylation kit (Pierce, Rockford, IL) with an average of two biotins per molecule as determined through the HABA assay (Pierce) following the manufacturer's protocols.

Plasmids, strains and media

The scFv library, plasmids and EBY100 yeast strain have been described previously (Boder and Wittrup, 1997Go, 1998Go; Feldhaus et al., 2003Go). Briefly, the scFv library was grown in selective liquid media (lacking tryptophan) containing glucose at 30°C. Surface expression was induced in selective media containing galactose at 25°C. Selected yeast clones were recovered as colonies or patches by growth on SD+CAA agar plates, which lack tryptophan, at 30°C. EBY100 Zeo is a derivative of EBY100 that carries the pTEF1 promoter Zeocin resistance gene (Sh ble gene) at the TRP loci that was deleted through homologous recombination of a PCR product amplified from the pPICZ{alpha} vector (Invitrogen, Carlsbad, CA). Yeast media were made as described (Feldhaus and Siegel, 2004Go).

Mutagenic library construction

The pPNL6 plasmid containing the parental Cam P4b VH3 gene was isolated from EBY100 yeast and electroporated into DH10b bacteria for subsequent plasmid purification using a Qiagen kit following conventional protocols (Ausubel et al., 2002Go). A 10 pg amount of plasmid DNA was used as template to amplify the dAb ORF using 0.5 µM PNL6 for (5'-TAGATACCCATACGACGTTC-3') and rev (5'-GTACGAGCTAAAAGTACAGTG-3') primers under mutagenic PCR conditions containing 1.5 mM MgCl2, 0.2 mM MnCl2, 0.2 mM each of dTTP/dGTP/dCTP, 0.1 mM dATP and 1 unit of Platinum Taq (Invitrogen, Carlsbad, CA) for 40 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C. PCR products were purified and co-transformed at a molar ratio of 7:1 with 3 µg of linearized pPNL6 vector digested with NheI/BamHI into EBY100-zeo yeast cells for homologous recombination following the lithium acetate protocol (Gietz and Woods, 2002Go). Transformed cells were selected using the tryptophan auxtrophic marker present on pPNL6 and passaged twice in 250 ml of SD+CAA medium to ensure homogeneity of transformed cells in the culture. The first library, named CaM P4b_M, had a diversity of 2.4 x 106 as determined by dilution plating on SD+CAA. The second mutagenic CaM P4b library, termed 2_CaM P4b_M (5 x 106 diversity), was constructed as above except that a pool of DNA from 10 clones isolated from the first mutagenic selection was used as template for the mutagenic PCR reaction. Sequence analysis of the library indicated that approximately five base changes per scFv were generated in the mutagenic library construction.

Yeast selections and flow cytometry

Selection from the non-immune yeast display library using the Miltenyi Macs system in conjunction with flow cytometric sorting has been described previously (Feldhaus et al., 2003Go; Siegel et al., 2004Go; Weaver-Feldhaus et al., 2004Go). The de novo selection of CaM clones was done in the following manner. The induced scFv library (1010 yeast) was incubated in 100 nM CaM for 30 min at room temperature and 10 min on ice. CaM was not specifically treated with calcium or EGTA. The yeast was washed three times with buffer (PBS + 0.25% BSA), before addition of streptavidin microbeads. Eluted yeast from the magnetic column (round 1 output) were re-grown to saturation in 250 ml of SD+CAA and 1010 cells were induced for another round of selection using the magnetic column as described above. The round 2 output was immediately stained for FACS using anti-myc/GaM-Alexa Fluor-633 to monitor scFv expression and SA-PE to label bound antigen. Typically, 104 yeast were sorted during this third round of selection. The sorted cells were grown for 48 h in 2 ml of SD + CAA and then induced for scFv expression. Then, 107 induced yeast, representing the round 3 output for CaM binders, were incubated with either 100 or 10 nM CaM pretreated with either 1 mM Ca2+ (generating Ca2+-CaM) or 1 mM EGTA (generating apo-CaM). The anti-myc antibody was also included during the incubation with antigen. All subsequent washes and incubations were done with PBS–0.25% BSA containing either 1 mM Ca2+ or 1 mM EGTA, respectively. Each sample was then incubated with secondary staining agents listed above for 30 min on ice before washing and FACS. Sorted yeast were plated on SD+CAA agar plates and incubated at 30°C for 2 days to allow colony formation. Individual colonies were then grown in liquid culture and induced for further characterization.

CaM affinity maturation selections

A 100-fold over-representation of the mutagenic library diversity was induced at 20°C overnight in SG/R-CAA medium at a density of 0.5 OD600/ml. Yeast cells displaying mutagenized CaM P4b antibodies on the surface were resuspended in wash buffer (1x PBS supplemented with 1 mM EGTA and 0.25% BSA) and incubated with various concentrations of biotinylated apo-CaM (all below the Kd of the parental clone; see Results section) that had been pretreated with 1 mM EGTA. Cells were also co-incubated with anti-HA mAb recognizing an N-terminal epitope present in the dAb clones to normalize for variations in antibody expression levels. Reactions were performed at 25°C for 30 min in excess antigen for the first mutagenic selection and 10 min at 25°C with limiting antigen for the second selection. Antigen dissociation was quenched by placing reactions on ice and unbound antigen was removed by washing. The cells were then incubated for 20 min on ice with appropriate secondary staining reagents to detect bound antigen and dAb expression. Cells displaying the highest levels of bound apo-CaM were selectively isolated by FACS. Typically, the brightest 0.1–0.2% of dAb-expressing, antigen-binding cells were sorted. Two rounds of sorting and re-growth were performed per selection to isolate highly enriched populations of improved mutants.

Analysis of isolated scFv clones

Affinities of the selected clones were determined directly on the yeast cell as described previously (Boder et al., 2000Go; VanAntwerp and Wittrup, 2000Go). Briefly, 106 induced cells were used per reaction and titrated with various concentrations of CaM in molar excess. Apo-CaM was titered from 1.5 to 100 nM for clones from the first CaM P4b_M library selection and from 0.125 to 15 nM for clones from the 2_CaM P4b_M library selection. Ca2+-CaM was titered from 8 to 1000 nM for clones from both mutagenic library selections. Reactions with the monovalent antigen were allowed to reach equilibrium before quenching on ice. Unbound antigen was removed and the amount of bound antigen was determined using flow cytometry after incubation with SA-PE on ice to prevent dissociation. The antibody-normalized antigen mean fluorescence intensity was plotted against antigen concentration and a non-linear least-squares fit was used to determine the equilibrium dissociation constant (Kd). Individual Kd determinations were performed at least three times. Troponin C and S100 binding affinities were determined in triplicate by equilibrium measurements for each of the proteins titrated from 1 to 900 nM in the presence and absence of Ca2+. The pPNL6 plasmids containing the selected clones were isolated as described above. The antibody ORFs were sequenced using P2 for (5'-TCTGCAGGCTAGTGGTGGTG-3') and rev (5'-CCGCCGAGCTATTACAAGTC-3') oligos. Vector NTI (Informax) was used to align sequences from the various clones and highlight mutations from the parental gene.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Selection of CaM-specific scFv

The Miltenyi Macs magnetic isolation system was used to enrich CaM binders through two rounds of selection from a human non-immune scFv library displayed on yeast. The yeast eluted from the second magnetic column were immediately incubated with fluorescently labeled secondary reagents and analyzed by flow cytometry. scFv expression was observed using anti-myc mAb/GaM-Alexa Fluor-633 recognizing the C-terminal epitope tag of the displayed scFv and bound biotinylated antigen was detected using streptavidin-PE. The first three rounds of selection used 100 nM biotinylated CaM that was not pretreated with either EGTA or Ca2+ resulting in a stochastic process that presumably allows both conformations of the antigen to be present. Ca2+-dependent conformational changes in CaM were utilized for the fourth round of selection to isolate scFv clones with different CaM binding characteristics.

FACS analysis of the round 2 magnetic column output revealed the presence of many different CaM-binding populations (Figure 1A). Two different sort gates (P1a and P1b) were used to enrich binders in the third round of selection. The P1a gate (Figure 1A) isolated c-myc negative yeast capable of binding 100 nM CaM. While the presence of the C-terminal c-myc epitope is routinely used to ensure the selection of full-length clones and avoid potential non-specific interactions due to exposed hydrophobic residues present in truncated clones, the unexpected high percentage of binders present caused us to examine this population more closely. Subsequent analysis demonstrated that these clones did not bind unrelated antigens (data not shown), indicating that this population was binding CaM in a specific fashion. The P1a population was expanded by growth and subsequently induced for a fourth round of selection using 10 nM Ca2+-CaM (Figure 1B). Two gates (P8 and P7) were used when sorting the amplified P1a population with the P8 gate isolating c-myc negative cells and the P7 gate isolating a distinct population protruding from the main group of cells. Individual colonies from the P7 and P8 sort gates were analyzed for antigen-binding characteristics (Table I).



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 1. Ca2+-dependent calmodulin binding selection. (A) CaM third round of selection. Two populations were sorted: P1a (c-myc negative) and P1b (c-myc positive). (B) The fourth round of selection using cells amplified from the R3 P1a sort gate in (A). The P7 and P8 sort gates are shown. (C and D) Percentage of yeast originating from R3 P1b population binding 100 and 10 nM Ca2+-CaM, respectively. The P2 and P3 sort gates used for the fourth round of selection with this antigen treatment are shown. (E) Percentage of yeast originating from the R3 P1b population binding 100 nM apo-CaM. The P4, P5 and P6 sort gates used for the fourth round of selection with this antigen treatment are shown. All panels show scFv expression (anti-myc mAb/GaM-633) on the x-axis and antigen binding (biotinylated CaM/SA-PE) on the y-axis.

 

View this table:
[in this window]
[in a new window]
 
Table I. Summary of clones isolated from de novo CaM selection

 
The second sort gate (P1b) used during the third round of selection isolated c-myc positive yeast capable of binding 100 nM CaM (Figure 1A). The P1b population was expanded by growth and subsequently induced for a fourth round of selection. Three separate aliquots of the induced P1b output were stained and sorted using different antigen treatments to elucidate the presence of high-affinity and conformation-specific clones (Figure 1C–E). Cells within the gate shown in Figure 1C were not sorted, but were included in order to compare the results obtained after incubation with either 10 or 100 nM Ca2+-CaM (Figure 1C and D, respectively). Analysis of these results indicated the presence of a number of CaM-binding scFv clones that had different relative affinities for Ca2+-CaM and demonstrates how low-affinity clones can be easily excluded from subsequent analysis during the selection process. Those clones retaining the highest level of fluorescence, and therefore a high level of CaM binding in the presence of 10 nM Ca2+-CaM, were sorted and individual clones isolated for analysis (P2 gate, Figure 1D). Those clones having a low affinity for Ca2+-CaM were also sorted for subsequent analysis (P3 gate, Figure 1D). Three additional populations were sorted after incubating P1b cells with 100 nM apo-CaM (Figure 1E). Both P4 and P5 sort gates contained clones with relatively high affinity for apo-CaM but differed in the degree of antibody expression while the P6 sort gate isolated those clones having poor affinity for apo-CaM.

Two clones from each of the seven different R4 sorted populations were analyzed for sequence and affinity for both Ca2+- and apo-CaM (Table I). The two clones from the P2 gate (P2a and P2b) are identical with the clones in the P6 gate, which correspond to the Ca2+-sensitive populations (Figure 1D and E, respectively). These clones have at least a 1250-fold preference for Ca2+-CaM, with a Kd of 800 pM. As expected, clones from the P3 sort gate have a much lower affinity for Ca2+-CaM (>1 µM) than the P2 clones and have only slightly improved affinity for apo-CaM (>500 nM). Clones from the P4 gate have a moderate affinity (48 nM) and preference (5-fold) for apo-CaM, but unexpectedly lack a VL gene, resulting in a truncated clone. Clones isolated from gates P5 and P7 are identical with one another and bind apo-CaM ~8-fold better than Ca2+-CaM (29 vs 249 nM, respectively). Like clones from the P4 sort, the P5/P7 clones were also determined to be truncated even though they were initially thought to be c-myc positive. In contrast to the P4 clones that simply do not contain a VL gene, the P5/P7 clone goes out of frame (OOF) in the linker between the VH and VL genes. Analysis of the obvious c-myc negative clones from the P8 population reveals an expected frameshift in the VL gene preventing full-length expression. Unlike the previous truncated clones, P8a prefers Ca2+-CaM (81 nM vs >1 µM).

We were puzzled by the number of single-domain antibodies (dAbs) isolated during the CaM selection that did not contain a functional C-terminal epitope, yet appeared to be c-myc positive during the selection. Subsequent work demonstrated that the particular fluorophore present on the secondary staining reagent used during the selection interacted with the dAb clones from the P4 and P5/P7 gates in the absence of the c-myc C-terminal tag. The secondary reagent interaction appears to be limited to the Alexa Fluor-633 dye, as these cells did not react with similar goat anti-mouse antibodies conjugated to the Alexa Fluor-488 fluorophore (data not shown). The interaction with the Alexa Fluor-633 fluorophore may be dependent on the number of tyrosine residues present in the VH CDR3. The P4 clones (showing the highest degree of Alexa Fluor-633 labeling) contain the JH6 joining region encoding a stretch of four continuous tyrosines at the end of the CDR3, whereas the P5/P7 clones encode four tyrosines distributed throughout CDR3 and the Alexa Fluor-633 negative clones from P8 contain only three tyrosine residues. The Alexa Fluor-633 secondary reagent binding also appears to be competitively inhibited by CaM. Preincubation of the cells with saturating amounts of CaM eliminates interaction with the Alexa Fluor-633 goat anti-mouse conjugate (data not shown), indicating that both bind to the same region of the dAb.

Conformation-specific directed evolution

CaM clone P4 was chosen for affinity maturation based on both the starting affinity (48 nM) and initial preference for apo-CaM (5-fold). The CaM P4 VH3 gene was randomly mutagenized using error-prone PCR. The inherent homologous recombination system in yeast was used to construct the library. This library, designated CaM P4b_M, had a diversity of 2.4 x 106 as determined by dilution plating on selective media. In order to obtain dAbs with improved conformational specificity, the first round of selection contained 5 nM apo-CaM (one-tenth the parental Kd) and clones exhibiting improved binding characteristics were identified and isolated by FACS (Figure 2). In the initial round of selection, the top 0.2% antigen-positive cells were sorted (Figure 2B). The round 1 output was grown, induced for dAb expression and re-analyzed for antigen binding. Almost the entire antigen-binding population falls within the original sort gate whose boundary was set above the degree of antigen-binding observed with the parental CaM P4 clone, indicating that most of the clones present after the first round of sorting had improved affinities (Figure 2C). Another aliquot of cells was incubated with 1 nM apo-CaM and the top 0.1% antigen-positive cells were sorted.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Directed evolution of apo-CaM binders. (A) The mutagenic CaMP4b_M library was induced for antibody expression and incubated with SA-PE in the absence of antigen. (B) Round 1 sort of CaMP4b_M library incubated with 5 nM apo-CaM. (C) Analysis of R1 sort output re-induced for antibody expression. dAb expression was detected using anti-HA mAb/GaM-488 (N-terminal tag) and is indicated with an arrow on the x-axis. Antigen binding was detected using biotinylated-CaM/SAPE and is indicated with an arrow on the y-axis. Sort gate is indicated with a box.

 
Ten individual colonies from the second round of the selection were randomly selected for further characterization. Sequence analysis demonstrated that eight of the 10 clones were unique. All eight clones contained at least one amino acid change relative to the VH3 sequence of the parental clone, with each having amino acid changes within the first 11 positions of framework 1(Figure 3A). Only five clones had additional amino acid changes in the complementarity-determining regions (CDRs) and two of those were conservative tyrosine to histidine changes (clones 2-2 and 2-5). Those clones that contained the most significant amino acid changes in the CDR regions were further analyzed (Figure 3B). The affinities of CaM P4b_M clone 2-1 (5 vs 82 nM), clone 2-8 (7 vs 130 nM) and clone 2-10 (1.4 vs 101 nM) were determined for apo- and Ca2+-CaM, respectively. From this simple analysis (10 clones from one round of mutagenesis), one clone (CaM P4b_M 2-10) was identified as having single nM affinity and nearly 60-fold specificity for apo-CaM, translating into a 28-fold affinity improvement and a 12-fold specificity improvement relative to the parental clone (Figure 4B).



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3. Analysis of evolved CaM clones. (A) Amino acid alignment of parental CaM P4 VH3 dAb and unique clones selected from CaMP4b_M and 2_CaMP4b_M libraries. Parental sequence is on top. CDR regions are denoted and the line separates clones from each mutagenic library. (B) Affinity analysis of CaMP4b_M clones. (C) Affinity analysis of 2_CaMP4b_M clones. Reported values are averages from at least two independent experiments.

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Evolved CaM clone comparison. (A) Bivariate plots showing amount of antigen binding for each clone incubated with 1 nM apo-CaM and dAb expression. The particular clone used is labeled on top of each plot with corresponding Kd value listed inside. dAb expression was detected using anti-HA mAb/GaM-488 and is indicated with an arrow on the x-axis. Antigen binding was detected using biotinylated apo-CaM/SAPE and is indicated with an arrow on the y-axis. (B) Comparison of improvements in apo-CaM affinity and specificity. The affinities for apo- and Ca2+-CaM for parental clone CaM P4 and clone 2-10 from CaMP4b_M library selection and clone -2 from 2_CaMP4b_M library selection are shown. Affinity and specificity improvements are relative to the parental clone. All values are means of at least four independent experiments with standard deviations listed.

 
A second round of error-prone PCR was employed using the eight improved clones characterized from the first mutagenic library as template. The pool of PCR products was co-transformed with linearized display vector for homologous recombination in yeast. We expected that recombination events between individual PCR products would combine separate beneficial mutations and perhaps remove neutral or deleterious mutations (Swers et al., 2004Go). This second CaM mutagenic library, designated 2_CaM P4b_M, was 5 x 106 diverse. More stringent selection criteria were employed for screening the second mutagenic library in that the first round of sorting used only 200 pM apo-CaM. The isolation of clones having increased association rates was also facilitated by limiting incubation with the antigen to 10 min prior to quenching the reaction. FACS was used to collect the top 0.1% of antigen-binding clones. The output was amplified though growth, re-induced for dAb expression and subjected to a second round of selection using 100 pM of apo-CaM for a 10 min incubation period. The top 0.1% antigen binders were sorted and the output was plated on selective media to obtain individual colonies for further analysis.

Sequence analysis of five individual 2_CaM P4b_M clones revealed all to be different from one another, and also from any clone analyzed from the first CaM P4b_M library (Figure 3A). All the 2_CaM P4b_M clones contain the same glutamate to glycine change at position 6 in framework 1. All five clones also contain a serine to arginine change within CDR1. Both of these mutations were observed in clone 2-1 from the original CaM P4b_M library. In addition, all the 2_CaM P4b_M clones have a number of differences in CDR3 that were not observed in clones from the first library. All the 2_CaM P4b_M clones have similar affinities for the apo-CaM ranging from 0.74 to 1.6 nM; however their affinities for Ca2+-CaM range from 128 to 351 nM (Figure 3C). Of these clones, 2_CaM P4b_M 2-2 appeared to have the highest specificity for apo-CaM (319-fold).

A comparison of the starting parental clone and selected clones isolated from each mutagenic library is shown in Figure 4. As would be expected, increased levels of antigen binding are observed with each improvement in affinity for apo-CaM (Figure 4A); however, the difference in antigen-binding fluorescence between the affinity matured clones seems disproportional to affinities. Close examination of the scFv expression (Figure 4A, x-axis) shows that CaM P4b_M clone 2-10 reproducibly expresses ~2-fold less dAb on the cell surface, limiting the amount of antigen, and therefore fluorescence, which can be captured. A combination of additional mutations, removal of deleterious mutations and setting sort gates on the highest scFv expressing clones restored the surface expression in the clone from the second mutagenic library to wild type levels (Figure 4C). Overall, 2_CaM P4b_M clone 2-2 had a 43-fold affinity and 64-fold specificity improvement for apo-CaM relative to the parental CaM P4_WT clone (Figure 4B). In comparing individual clones from each mutagenic library, a relatively minor improvement in the apo-CaM affinity was observed; nonetheless, a >5-fold improvement in specificity was obtained, due in part to decreased affinity for Ca2+-CaM (101 vs 351 nM, respectively).

Cross-reactivity of CaM clones for other EF-hand calcium-binding proteins, S100 and troponin C

To characterize the CaM-specific binders in greater detail, we determined the cross-reactivity and affinities of a panel of our binders for the EF-hand Ca2+-binding proteins CaM, troponin C and S100 heterodimer in the presence and absence of Ca2+ using yeast display and equilibrium-based Kd measurements by flow cytometry. All the CaM-selected clones bind both Ca2+ and apo conformations of S100 and troponin C to varying degrees (Table II). For example, the CaM P2a clone has sub-nanomolar affinity and a >1000-fold specificity for Ca2+-CaM. However, this clone binds either form of troponin C with moderate affinity (Kd = 160 nM) and has high affinity and preference for Ca2+-S100 (Kd = 9 vs 143 nM, respectively). Clone P3a, which binds CaM poorly regardless of conformation, has good affinity for both conformations of troponin C (Kd = 40 and 16 nM, respectively), but has a clear preference for apo-S100 protein (Kd = >1 µM vs 55 nM, respectively). Finally, 2_CaM P4b_M clone 2-2, engineered for greater specificity to apo-CaM (>300-fold), has moderate to poor affinity for either conformation of the other EF-hand family members. Although it seems that the immunoreactivity of the scFv/dAbs for the three Ca2+ binding EF-hand proteins is inconsistent and not predictable, it is important to note that both of the high-affinity CaM clones (P2a and 2_CaMP4bM_2-2) bind their preferred CaM conformation with higher affinity and specificity than any conformation of the other structurally similar EF-hand proteins tested (Table II, last column).


View this table:
[in this window]
[in a new window]
 
Table II. Affinity and specificity of CaM clones for EF-hand proteins

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Yeast display was used to isolate a suite of recombinant scFv and dAb antibodies recognizing specific antigen conformations. The in vitro nature of these selections allowed binders recognizing the highly conserved vertebrate CaM to be quickly and easily obtained. Antibodies recognizing CaM were first selected from a previously described yeast non-immune scFv library (Feldhaus et al., 2003Go). Initial rounds of selection were absent of any intentional bias towards one conformation of CaM over another. Subsequent analysis and sorting of enriched CaM-binding populations revealed the presence of clones that specifically recognized Ca2+-CaM (e.g. CaM P2 and P8) and other clones that preferred apo-CaM (e.g. CaM P4 and P5) (Figure 1 and Table I). Of note is the fact that CaM P2 scFv, having a Kd of 800 pM with >1000-fold specificity for the Ca2+-CaM conformation, was obtained without any subsequent manipulation. In contrast, directed evolution was used to improve both the affinity and specificity for apo-CaM of a parental dAb clone (CaM P4) with moderate affinity (48 nM) and slight preference (5x) for this particular conformation (Table I and Figures 2 and 3). Clone 2_CaM P4b_M 2-2 was obtained that has a Kd of 1 nM and >300-fold specificity for the apo-CaM, which translates into a 40-fold improvement in affinity and >60-fold improvement in specificity relative to the parental clone (Figure 4).

Ideally, the CaM conformation-specific antibodies should not cross-react with any other member of the Ca2+-binding EF-hand protein family. There is a high degree of sequence (64% similarity and nearly 50% identity) and structural homology between CaM and TnC. There is less sequence homology between CaM and S100 (28% similarity and 17% identity); however, it is interesting to note the increasing number of common protein targets (e.g. tau and p53) that both have been shown to bind (Schafer and Heizmann, 1996Go), presumably mediated by the conserved EF-hand architecture. We used a flow cytometry-based assay to determine the affinity each CaM clone had for both the Ca2+-bound and apo conformations of other members in the EF-hand family (Table II). Further characterization of these clones show that other EF-hand family members are recognized to varying extents, although this reactivity is inconsistent and not predictable (Table II). For instance, the Ca2+-CaM specific clone P2a has the highest degree of reactivity towards the Ca2+-bound form of S100, which at least conforms to this clone's preference for the Ca2+-bound state. On the other hand, the only EF-hand protein that the apo-CaM specific clone 2_CaM P4b_M 2-2 reacts with to any significant degree is the Ca2+-bound conformation of troponin C. It would appear as if both CaM-binding clones might recognize at least a portion of the EF-hand motif that is conserved among all of these antigens and indicates a conformational rather than linear epitope is involved. Nevertheless, the selected clones described in this work still prefer their respective CaM isoform with 11- and 75-fold greater specificity for P2 and 2_CaM P4b_M 2-2, respectively (Table II).

A direct comparison of all the CaM antibodies that have been produced is difficult given inconsistencies in their characterization. Immunization with a C-terminal peptide from bovine CaM produced a monoclonal antibody with reasonable affinity (5 x 108 l/mol as determined by a displacement assay) that did not react to any significant degree with other EF-hand members with or without Ca2+ (Sacks et al., 1991Go). This is not surprising given the antigen used for immunization; however, this antibody recognized both conformations of CaM and was only obtained after immunization in a specific strain of mice. Interestingly, the hybridoma fusions did not survive routine limiting dilution and required multiple cells per well for viability (Sacks et al., 1991Go). Other groups have produced Ca2+-specific CaM antibodies with either moderate (Hansen and Beavo, 1986Go) or undetermined affinities (Wolf et al., 1995Go). Neither reported exhaustive characterization for cross-reactivity with other EF-hand proteins in both the presence and absence of Ca2+; although Hansen and Beavo reported that binding to CaM was inhibited in the presence of calcineurin and Wolf et al. saw some reactivity with S100a with some of their antibodies. Another publication describes what appear to be Ca2+-CaM and apo-CaM antibodies present in the sera of hepatitis patients (Ikeda et al., 1987Go). The affinities for each conformation and degree of cross-reactivity with other EF-hand proteins were not determined and the source of these antibodies limits practical use.

It is difficult to predict which changes impact affinity, specificity and stability of an antibody. A simple, random and typically asexual error-prone PCR approach was used to generate mutations for all of the mutagenic libraries used in this study. Taking advantage of the homologous recombination system in yeast, we were able to combine the benefits of Stemmer's sexual DNA shuffling (Stemmer, 1994Go) with an elegant and simple means of library creation. Wittrup and co-workers have recently demonstrated that multiple crossover events can occur when co-transforming a pool of scFv PCR products with linearized vector, potentially combining mutations from individual clones (Swers et al., 2004Go). Homologous recombination also avoids clonal interference that has been observed using a strictly asexual PCR-only mutagenesis scheme (Rowe et al., 2003Go). As the efficiency of any directed evolution selection is a function of its throughput, sensitivity, precision and dynamic range, it seems that FACS is particularly well suited for this endeavor (Daugherty et al., 2000Go; VanAntwerp and Wittrup, 2000Go). Yeast display is also capable of simultaneously improving multiple parameters such as affinity and expression (this work and Graff et al., 2004Go) during the selection process. Consideration of all these points may explain how clones with such dramatic affinity and specificity improvements could have been so easily identified from such a small sampling (typically 5–10) of clones after each round of selection.

Analysis of mutations isolated from each round of directed evolution indicates that a number of changes in both of the framework regions and the antigen-binding CDR loops are responsible for the observed improvements. Of the eight unique clones analyzed from the first mutagenic CaM library, only five contained changes within the CDR loops (Figure 3A). All of the clones contained changes within framework 1 with a majority containing a change at position 6. This position, in particular, has been previously observed to impact biophysical properties such as affinity and stability of antibodies (Jung et al., 2001Go). All of these mutations had the opportunity to recombine during the construction of the second CaM mutagenic library and analysis of selected clones shows that the glutamate to glycine change at position 6 of framework 1 region and the serine to arginine change in VH CDR1 became fixed. A number of residues were also changed in VH CDR3 that were not previously observed (Figure 3A). It is intriguing to speculate that these CDR3 changes are largely responsible for the increase in specificity for the apo-CaM conformation as there is very little difference in affinities for apo-CaM between the first and second mutagenic libraries (1.7 vs 1.1 nM, respectively), but a >3-fold difference in Ca2+-CaM affinities (100 vs 350 nM, respectively). However, without detailed structural studies, we are not able to determine which mutations are in direct contact with the antigen and which mutations change the architectural presentation of the CDR loops without making direct contact and/or make some other indirect contribution to binding.

An unexpected finding from this work was that many of the clones, four out of six, and regardless of conformation specificity, isolated from the original de novo selection were single-domain antibodies (dAbs). Many selections have been performed using this yeast non-immune scFv library (Feldhaus et al., 2003Go; Siegel et al., 2004Go); however, we have not isolated any dAb clones until working with CaM in this study. A phage scFv library (Sheets et al., 1998Go) converted to the yeast display format and screened with CaM in a similar manner also produced both dAb and intact scFv antibodies similar to those identified in this work (data not shown). This suggests that the results presented here are not just a reflection of the particular library used for selection. The inherent properties of this antigen that cause the selection of these single-domain clones remain unknown. Yeast displaying single-domain antibodies arise from two sources: (1) clones that have both VH and VL genes but contain a frame-shift introducing a stop codon between the two domains and (2) clones that do not contain any VL coding sequence. Both types are artifacts of library construction. However, single domain antibodies are not without precedent. Functional VH-only {gamma}-immunoglobulins occur naturally in Camelidae (Hamers-Casterman et al., 1993Go). Artificial, non-Camelidae, VH-only libraries have also been constructed (Reiter et al., 1999Go; Holt et al., 2003Go). A VL-only intracellular dAb that inhibits huntingtin aggregation has also been described recently (Colby et al., 2004aGo). Normally, most human VH-only domains have a tendency to aggregate without changing key hydrophobic residues left exposed by the absence of the VL domain. Plückthun and co-workers have shown that the particular VH gene family (VH3) and length of CDR3 (≥17 residues) dramatically improve single-domain expression and stability (Ewert et al., 2003Go). Interestingly, the parental CaM P4 is a VH3 dAb containing 18 residues in CDR3. The surface-displayed version of this construct also encodes an additional 23 residues of the G4S linker, which, taken together with the gene family and CDR3 length, may explain its ability to be expressed on the surface of yeast.

It is expected that antigen binding by the dAb clones can be mediated through CDR-specific contacts and/or exposed hydrophobic residues normally buried in the VH–VL interface. We have evidence suggesting that the hydrophobic interface is involved, at least in part, in the binding of CaM. The ability to bind CaM is lost upon converting clones P2 and P4 clones to intact IgG molecules, indicating that the hydrophobic interface buried between the paired heavy and light chains contributes to antigen binding (data not shown). The lack of binding of the CaM IgG molecules is not a reflection of conversion of scFv to the IgG format, as other scFv that we have converted to IgG antibodies retain the ability to bind their cognate antigens. Although this is not entirely unexpected for the single domain P4 clone, the P2 scFv clone expresses both the VH and VL domains and was thought to bind CaM only in a CDR-specific fashion (Table I). These data once again highlight the fact that any isolated affinity ligand will not necessarily function on conversion to formats different from that used during the original selection process.

Although the hydrophobic face is probably involved in binding, it is unlikely to be solely responsible for the affinity or specificities that we observed, for several reasons. First, none of the single-domain P4, P5 and P8 clones preferentially bind the same CaM conformation. In fact, even the VH3-derived dAb clones (P4 and P8) containing the same interface hydrophobic residues do not share the same CaM conformation specificity (Table I), suggesting that other residues (most likely CDRs) are involved in conformation discrimination. Second, various structural studies (Babu et al., 1988Go; Ikura et al., 1990Go) have indicated that it is the Ca2+-bound and not the apo form of CaM that exposes hydrophobic regions. If hydrophobic interactions were solely responsible, then it seems that all of the dAbs isolated from our selection would prefer the Ca2+-CaM conformation. Third, labeling of the naïve unselected library with CaM is not observed and multiple rounds of selection (>104-fold enrichment) were required in order to observe binders from the library. We estimate that truncated clones account for ~10% of our library (Feldhaus et al., 2003Go) and would be readily observed to bind CaM prior to any selection if non-specific hydrophobic interactions were solely responsible for binding. Fourth, except for the structurally related EF-hand proteins, the CaM scFv/dAb clones do not bind unrelated antigens such as epidermal growth factor, vascular endothelial growth factor and a host of other secondary reagents (data not shown). This indicates that these clones specifically bind CaM and, to a much lesser extent, other EF-hand proteins. Hence it would appear that these affinity reagents interact with CaM in both a CDR-dependent and specific hydrophobic interface-dependent manner.

We will continue to investigate why none of the CaM selected scFv/dAb clones produced to date in Escherichia coli are active. The dAb clones are not active after re-folding solublized inclusion bodies, which can be readily explained given the complexities of those procedures. More disturbing is the lack of activity observed from soluble CaM P2 scFv. It would appear as if these clones are either stabilized on the yeast surface as a translational fusion and that this stability and activity is lost when expressed as a soluble fragment or that the Aga-2 fusion contributes to the binding observed with these scFv/dab clones. Alternatively, CaM binding could be mediated by the scFv/dAb on the yeast cell surface in conjunction with another unknown cellular protein. However, ample evidence exists to conclude that binding must include expression of these specific antibodies on the cell surface and is not simply due to the yeast cell itself. Future efforts will focus on producing active fractions of these antibody fragments for immuno-precipitation reactions to determine if different complexes can be identified with each conformation of CaM.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank the members of the ADD Hybridoma Research group for critical review of the manuscript and excellent suggestions. Portions of this research were supported by the NIH National Center for Research Resources (RR18522) and Laboratory Directed Research and Development and Genomes to Life programs of the DOE. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the US Department of Energy under contract DE-AC06-76RLO-1830.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Alexander,K.A., Cimler,B.M., Meier,K.E. and Storm,D.R. (1987) J. Biol. Chem., 262, 6108–6113.[Abstract/Free Full Text]

Babu,Y.S., Bugg,C.E. and Cook,W.J. (1988) J. Mol. Biol., 204, 191–204.[CrossRef][ISI][Medline]

Boder,E.T. and Wittrup,K.D. (1997) Nat. Biotechnol., 15, 553–7.[CrossRef][ISI][Medline]

Boder,E.T. and Wittrup,K.D. (1998) Biotechnol. Prog., 14, 55–62.[CrossRef][ISI][Medline]

Boder,E.T., Midelfort,K.S. and Wittrup,K.D. (2000) Proc. Natl Acad. Sci. USA, 97, 10701–10705.[Abstract/Free Full Text]

Bradbury,A.R., Velappan,N., Verzillo,V., Ovecka,M., Marzari,R., Sblattero,D., Chasteen,L., Siegel,R. and Pavlik,P. (2004) Methods Mol. Biol., 248, 519–546.[Medline]

Chan,S.Y., Murakami,K. and Routtenberg,A. (1986) J. Neurosci., 6, 3618–3627.[Abstract]

Colby,D.W., Chu,Y., Cassady,J.P., Duennwald,M., Zazulak,H., Webster,J.M., Messer,A., Lindquist,S., Ingram,V.M. and Wittrup,K.D. (2004a) Proc. Natl Acad. Sci. USA, 101, 17616–17621.[Abstract/Free Full Text]

Colby,D.W., Garg,P., Holden,T., Chao,G., Webster,J.M., Messer,A., Ingram,V.M. and Wittrup,K.D. (2004b) J. Mol. Biol., 342, 901–912.[CrossRef][ISI][Medline]

Cox,J.A. (1988) Biochem. J., 249, 621–629.[ISI][Medline]

Daugherty,P.S., Iverson,B.L. and Georgiou,G. (2000) J. Immunol. Methods, 243, 211–227.[CrossRef][ISI][Medline]

Ewert,S., Huber,T., Honegger,A. and Plückthun,A. (2003) J. Mol. Biol., 325, 531–553.[CrossRef][ISI][Medline]

Feldhaus,M. and Siegel,R. (2004) Methods Mol. Biol., 263, 311–332.[Medline]

Feldhaus,M.J. et al. (2003) Nat. Biotechnol., 21, 163–170.[CrossRef][ISI][Medline]

Gietz,R.D. and Woods,R.A. (2002) Methods Enzymol., 350, 87–96.[CrossRef][ISI][Medline]

Goncalves,C.A., Gottfried,C., Kommers,T. and Rodnight,R. (1997) Anal. Biochem., 253, 127–130.[CrossRef][ISI][Medline]

Graff,C.P., Chester,K., Begent,R. and Wittrup,K.D. (2004) Protein Eng. Des. Sel., 17, 293–304.[Abstract/Free Full Text]

Hamers-Casterman,C., Atarhouch,T., Muyldermans,S., Robinson,G., Hamers,C., Songa,E.B., Bendahman,N. and Hamers,R. (1993) Nature, 363, 446–448.[CrossRef][ISI][Medline]

Hanes,J., Jermutus,L., Weber-Bornhauser,S., Bosshard,H.R. and Plückthun,A. (1998) Proc. Natl Acad. Sci. USA, 95, 14130–14135.[Abstract/Free Full Text]

Hansen,R.S. and Beavo,J.A. (1986) J. Biol. Chem., 261, 14636–14645.[Abstract/Free Full Text]

Holt,L.J., Herring,C., Jespers,L.S., Woolven,B.P. and Tomlinson,I.M. (2003) Trends Biotechnol., 21, 484–490.[CrossRef][ISI][Medline]

Ikeda,Y., Toda,G., Hashimoto,N., Maruyama,T. and Oka,H. (1987) Biochem. Biophys. Res. Commun., 144, 191–197.[CrossRef][ISI][Medline]

Ikura,M., Kay,L.E. and Bax,A. (1990) Biochemistry, 29, 4659–4667.[CrossRef][ISI][Medline]

James,P., Vorherr,T. and Carafoli,E. (1995) Trends Biochem. Sci., 20, 38–42.[CrossRef][ISI][Medline]

Jamieson,G.A.,Jr, Bronson,D.D., Schachat,F.H. and Vanaman,T.C. (1980) Ann. N. Y. Acad. Sci., 356, 1–13.[Medline]

Jung,S., Spinelli,S., Schimmele,B., Honegger,A., Pugliese,L., Cambillau,C. and Plückthun,A. (2001) J. Mol. Biol., 309, 701–716.[CrossRef][ISI][Medline]

Kieke,M.C., Shusta,E.V., Boder,E.T., Teyton,L., Wittrup,K.D. and Kranz,D.M. (1999) Proc. Natl Acad. Sci. USA, 96, 5651–5656.[Abstract/Free Full Text]

Kobayashi,K., Yoshida,M., Shinoda,Y., Yazawa,M. and Yagi,K. (1991) J. Biochem. (Tokyo), 109, 551–558.[Abstract]

Krestsinger,R.H. and Knockolds,C.E. (1973) J. Biol. Chem., 248, 3313–3326.[Abstract/Free Full Text]

Marks,J.D., Hoogenboom,H.R., Bonnert,T.P., McCafferty,J., Griffiths,A.D. and Winter,G. (1991) J. Mol. Biol., 222, 581–597.[CrossRef][ISI][Medline]

McCafferty,J., Griffiths,A.D., Winter,G. and Chiswell,D.J. (1990) Nature, 348, 552–554.[CrossRef][ISI][Medline]

Pardue,R.L., Brady,R.C., Perry,G.W. and Dedman,J.R. (1983) J. Cell Biol., 96, 1149–1154.[Abstract]

Reiter,Y., Schuck,P., Boyd,L.F. and Plaksin,D. (1999) J. Mol. Biol., 290, 685–698.[CrossRef][ISI][Medline]

Rowe,L.A., Geddie,M.L., Alexander,O.B. and Matsumura,I. (2003) J. Mol. Biol., 332, 851–860.[CrossRef][ISI][Medline]

Sacks,D.B., Porter,S.E., Ladenson,J.H. and McDonald,J.M. (1991) Anal. Biochem., 194, 369–377.[CrossRef][ISI][Medline]

Schafer,B.W. and Heizmann,C.W. (1996) Trends Biochem. Sci., 21, 134–140.[CrossRef][ISI][Medline]

Schumacher,M.A., Crum,M. and Miller,M.C. (2004) Structure (Camb.), 12, 849–860.[Medline]

Sheets,M.D. et al. (1998) Proc. Natl Acad. Sci. USA, 95, 6157–6162.[Abstract/Free Full Text]

Shusta,E.V., Holler,P.D., Kieke,M.C., Kranz,D.M. and Wittrup,K.D. (2000) Nat. Biotechnol., 18, 754–759.[CrossRef][ISI][Medline]

Siegel,R.W., Coleman,J.R., Miller,K.D. and Feldhaus,M.J. (2004) J. Immunol. Methods, 286, 141–153.[CrossRef][ISI][Medline]

Stemmer,W.P. (1994) Nature, 370, 389–391.[CrossRef][ISI][Medline]

Strasburg,G.M., Hogan,M., Birmachu,W., Thomas,D.D. and Louis,C.F. (1988) J. Biol. Chem., 263, 542–548.[Abstract/Free Full Text]

Swers,J.S., Kellogg,B.A. and Wittrup,K.D. (2004) Nucleic Acids Res., 32, e36.[Abstract/Free Full Text]

VanAntwerp,J.J. and Wittrup,K.D. (2000) Biotechnol. Prog., 16, 31–37.[CrossRef][ISI][Medline]

Weaver-Feldhaus,J.M., Lou,J., Coleman,J.R., Siegel,R.W., Marks,J.D. and Feldhaus,M.J. (2004) FEBS Lett., 564, 24–34.[CrossRef][ISI][Medline]

Winsky,L. and Kurznicki,J. (1996) J. Neurochem., 66, 764–771.[ISI][Medline]

Wolf,T., Fleminger,G. and Solomon,B. (1995) J. Mol. Recognit., 8, 67–71.[CrossRef][Medline]

Received May 12, 2005; revised August 11, 2005; accepted August 16, 2005.

Edited by Ian Tomlinson





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
18/11/527    most recent
gzi060v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Weaver-Feldhaus, J. M.
Articles by Siegel, R. W.
PubMed
PubMed Citation
Articles by Weaver-Feldhaus, J. M.
Articles by Siegel, R. W.