Department of Biotechnology, Royal Institute of Technology (KTH), S-100 44 Stockholm, Sweden
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Abstract |
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Keywords: affibody/affinity maturation/phage display/staphylococcal protein A/Taq DNA polymerase
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Introduction |
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A previously described strategy to circumvent the need for large naive libraries to isolate high-affinity antibody fragments is the harvesting of immunoglobulin-encoding sequences from donors immunized with the target of interest (Clackson et al., 1991; Hoogenboom et al., 1998
). However, such biased libraries are of limited use for the isolation of antibodies to a wide range of targets. Alternatively, binders of higher affinities can be obtained through affinity maturation of lead binders (`first generation' binders) isolated from naive libraries. This approach includes selection of variants from hierarchical libraries constructed on the basis of sequences of already identified binders. For antibodies, different methods for construction of such libraries for the isolation of high-affinity variants have been described involving, for example, heavy and/or light chain shuffling (Marks et al., 1992
; Schier et al., 1996a
), CDR re-randomization (Yang et al., 1995
; Schier et al., 1996b
), step-wise sexual PCR (Crameri et al., 1996
) or by using a bacterial mutator strain (Low et al., 1996
). Similar principles, although directed to other relevant portions of the structure, have also been applied to the increase of affinities of non-immunoglobulin proteins, including scaffold proteins (Martin et al., 1996
) or peptides (Wrighton et al., 1996
) obtained from primary selections from combinatorial libraries, or naturally occurring binding proteins (Lowman and Wells, 1993
; Ballinger et al., 1998
).
We have previously described the phage display-facilitated selection of novel binding proteins (affibodies) from combinatorial libraries of the three-helix bundle, 58 residue Z domain derived from staphylococcal protein A (SPA), in which 13 amino acid positions distributed over the first two helices were targeted for randomization (Nord et al., 1995, 1997
) (Figure 1
). Using standard technology, the library sizes obtained were approximately 4.5x107 members, which only corresponds to a minute fraction of the number of clones (approximately 1019) required for a full sampling of the variability. The affinities observed for binders to the different targets investigated so far have all been in the micromolar (KD) range, albeit with different binding kinetics (Nord et al., 1997
). The moderate affinities obtained for selected affibodies could possibly be a consequence of the sparse library sizes or reflect a conceptual limitation of the approach involving the randomization of a discontinuous `paratope' distributed over two separate
-helices.
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Materials and methods |
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Escherichia coli strain RRIM15 (Rüther et al., 1981
) was used as host during library construction and phagemid work. Strain RV308 (Maurer et al., 1980
) was used as host for production of soluble affibodies and strain BL21(DE3) (Novagen, Inc., Madison, WI) for the production of the Aff2Taq DNA polymerase fusion protein. Phagemid vector pKN1 was used for library constructions as described earlier (Nord et al., 1995
) and the vector pAff2c (Nilsson et al., 1996
), encoding a multiaffinity fusion partner containing an in vivo biotinylated domain, a hexahistidyl sequence and a serum albumin binding protein (ABP), was employed for intracellular production (T7 system) of Taq DNA polymerase.
Construction of the hierarchical library
The library was constructed based on the sequence of a previously described Taq DNA polymerase binding protein (ZTaq4:8), selected from a library of the Z domain derived from staphylococcal protein A (Nord et al., 1997). For library constructions, a solid phase gene assembly strategy was employed as described earlier (Nord et al., 1995
) where all the oligonucleotides used were as described for Z-lib 1 except for the oligo GUEL8 (5'-CAAAGAACTGGGTTGGGCGACCTGGGAGATCTTCAACTTACCTA-3'), encoding the sequence of helix one, which was kept unchanged in the hierarchical library. Briefly, 350 ng NheI/Esp3I-digested PCR product, encoding helices one and two [NN(G/T) randomization at six positions] was ligated into 6 µg MluI/NheI-restricted pKN1 phagemid. The ligation mixture was digested with SacI and thereafter purified by extraction with phenol/chloroform/isoamylalcohol (25:24:1), washed twice with chloroform, ethanol precipitated and finally dissolved in 30 µl sterile water. Samples of 2.5 µl ligation mix were electroporated into 100 µl electrocompetent RRI
M15 cells. Cells were grown in 1 ml SOC medium Tryptone Soy Broth (TSB) medium supplemented with 2% glucose, 10 mM MgCl2, 10 mM MgSO4, 10 mM NaCl and 2.5 mM KCl for 1 h and plated on TYE (15 g/l agar, 8 g/l NaCl, 10 g/l tryptone and 5 g/l yeast extract) medium supplemented with 100 µg/ml ampicillin and 2% glucose (TYEAmpGlu) and grown over night at 37°C. Colonies were pooled in TSB medium supplemented with 100 µg/ml ampicillin and 2% glucose (TSBAmpGlu) and frozen at 80°C in aliquots with addition of glycerol to a final concentration of 30%. The frequency of religated phagemid vector was determined by PCR screening using phagemid-specific primers RIT27 and NOKA-2. The diversity of the library was analysed by solid-phase DNA sequencing (Hultman et al., 1991
) using the same set of oligonucleotides but with a biotinylated version of NOKA-2.
Preparation of phage stocks
TSBAmpGlu (100 ml) was inoculated with approximately 0.52x109 cells from the constructed library or from between selection rounds and incubated with shaking at 37°C to give an A600 of 0.5. About 5x1010 M13K07 helper phage particles (New England Biolabs, Beverly, MA) were added to 10 ml culture and incubated 30 min without shaking at 37°C. The superinfected cells were spun down and used to inoculate 100 ml TSB medium supplemented with 100 µg/ml ampicillin, 25 µg/ml kanamycin and 100 µM isopropyl-ß-D-thiogalactoside (IPTG). The culture was grown at 30°C for approximately 15 h before it was pelleted by centrifugation and subjected to rounds of PEG/NaCl precipitation. The phages were redissolved in 1 ml phosphate buffered saline (PBS; 50 mM phosphate, 100 mM NaCl, pH 7.2) and filtered through a 0.45 µm filter. This procedure routinely resulted in a phage titre of 10111012 phages/ml.
Selections
Selections were performed against an in vivo biotinylated Aff2Taq DNA polymerase (Aff2Taq) fusion protein, produced and purified as described earlier (Nilsson et al., 1997), either in solution or immobilized onto streptavidin coated paramagnetic beads (Dynabeads® M280-SA, Dynal AS, Oslo, Norway).
Solid phase selections
For each panning round, a 5 mg portion of beads was washed twice in washing/binding buffer (W/B; 1 M NaCl, 10 mM TrisHCl, pH 7.5, 1 mM EDTA), followed by incubation with 40 µg Aff2Taq protein in 310 µl storage solution (100 mM KCl, 100 µM EDTA, 20 mM TrisHCl, pH 8.0, 0.5% Tween 20 and 50% glycerol) overnight at 4°C, resulting in a target protein concentration of approximately 4 µg Aff2Taq/mg beads. Approximately 100 µl phage stock was added to 5 mg beads with immobilized Aff2Taq, previously washed four times in PBS with 0.1% Tween 20 (PBST), in a pretreated (PBST with 0.1 % gelatin) Eppendorf tube together with 5 µl 2% gelatin (final concentration 0.1%). The panning mixture was incubated with rotation overnight at 4°C and subsequently washed 10 times with 1 ml PBST. The beads were transferred to a new pretreated tube followed by 5 washes in 1 ml PBST. Bound phages were eluted with 500 µl 0.1 M glycinHCl, pH 2.2 for 20 min and the eluate was neutralized with 50 µl 1 M TrisHCl and 450 µl PBS. Eluted phages were used to infect 10 ml log phase RRIM15 cells for 20 min which were subsequently plated on TYEAmpGlu agar plates. This panning procedure was repeated during four rounds of selection.
Soluble selections
Eppendorf tubes, phage stocks and beads were pretreated with PBST containing 0.1% gelatin for 1 h before use in selections. Aff2Taq protein was added to 100 µl phage stock (PBS and 0.1% gelatin) to a final concentration of 10 nM or 1 nM and incubated on a rotator for 2.5 h at room temperature. After washing, 0.5 mg beads in PBS (washed twice in PBST) were added to the phage mix to a total volume of 200 µl and incubated for 15 min as before. The beads were immediately washed 10 times with 1 ml portions of PBST and transferred to a new tube before bound phages were eluted during 10 min and neutralized and used for reinfection as described above.
Protein expression and purification
After four rounds of selections, 10 randomly picked clones from each protocol were subjected to solid-phase DNA sequencing (Hultman et al., 1991; Nord et al., 1997
). Plasmid DNA from selected clones were prepared using a standard alkali lysis protocol (Sambrook et al., 1989
). Purified plasmid (phagemid) constructs were transformed into Escherichia coli strain RV308 for production of soluble affibodyABD fusion proteins as follows. Overnight cultures in TSBAmp supplemented with 5 g/l yeast extract at 37°C were used to inoculate (diluted 1:200) 100 ml TSBAmp in baffled shake flasks and allowed to grow until A600nm reached 1 before protein production was induced with IPTG to a final concentration of 1 mM. Cultures were further grown at 25°C for 24 h before subjected to an osmotic shock treatment to release periplasmic proteins (Nygren et al., 1988
). Periplasmic fractions were loaded onto human serum albumin (HSA)Sepharose columns for affinity chromatography (Nygren et al., 1988
). Proteins were analysed by SDSPAGE on 20% polyacrylamide gels stained with Coomassie brilliant blue R-250 using the Pharmacia PhastTM system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and the protein concentrations were determined spectrophotometrically at A280nm.
Binding studies using BIAcore
Selected affibodies were affinity ranked using surface plasmon resonance (SPR) employing a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden). AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) or polyclonal hIgG (used as control) (Pharmacia & Upjohn AB, Stockholm, Sweden) was immobilized in different flow cells by amine coupling according to the manufacturer's recommendations onto the carboxylated dextran layer on surfaces of a CM-5 chip (research grade) resulting in approximately 2100 and 5900 resonance units (RU), respectively. A third flow cell surface was activated and deactivated for use as a blank during injections. Twelve newly selected Taq DNA polymerase binding affibody variants and the original ZTaq4:8 affibody were injected over the surfaces at a concentration of 50 nM, diluted in HEPES buffered saline (HBS; 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.5% surfactant P-20) in duplicates in random order at a constant flow rate of 30 µl/min for 7 min. After each injection, the surfaces were regenerated with 0.05% SDS. The five affibodies showing the highest equilibrium responses were selected for further binding analyses.
KD values of selected affibodies were determined using a CM-5 chip surface containing 1500 RU of immobilized AmpliTaq DNA polymerase. Samples of Taq DNA polymerase binding affibodies were injected (duplicates in random order) at different concentrations (ZTaq4:8: 0.75 nM to 25 µM; second generation variants: 0.75 nM to 7.5 µM) at a flow rate of 20 µl/min. Injections were made during 5 min and the surfaces were regenerated using 0.05% SDS. Binding curves were based on the equilibrium responses obtained and KD values were calculated using the BIAevaluation 2.1 software (Biacore AB). Kinetic rate constants were calculated using BIAevaluation 2.1 software assuming a one-to-one binding model.
Dimerization of the ZTaqS1-1 affibody
The gene encoding the ZTaqS1-1 variant was amplified in eight 50 µl PCR reactions using oligonucleotides NOKA-6 (5'-CCCCGTCGACCGTAGACAACAAATTCAACAAAG-3') and NOKA-7 (5'-CCCCCTCGAGCTTTTCGGCGCCTGAGCATC-3') introducing recognition sites for the restriction enzymes SalI and XhoI, respectively. Fragments were pooled, ethanol precipitated, restricted and agarose gel purified before ligation into the vector pKN1-ZTaqS1-1, restricted with XhoI and dephosphorylated. Transformants were analysed by solid phase DNA sequencing and plasmid DNA of the resulting construct pKN1-di-ZTaqS1-1 was prepared using a Jetstar miniprep column (Genomed, Inc., NC). Di-ZTaqS1-1ABD fusion protein was produced and purified as described above. A comparative binding analysis between ZTaqS1-1ABD and di-ZTaqS1-1ABD fusion proteins was performed using a CM-5 sensor chip containing 2200 RU of immobilized AmpliTaq DNA polymerase and 4600 RU hIgG in different flow cells. A blank surface (200 RU) was prepared through activation/deactivation for use as an additional control. For calculations of binding kinetic parameters, samples of ZTaqS1-1 and di-ZTaqS1-1ABD fusion proteins were injected at random order during 5 min at a flow rate of 50 µl/min at concentrations ranging between 0.32 nM and 25 µM (ZTaqS1-1) or 64 pM and 5 µM (di-ZTaqS1-1).
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Results |
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From a phagemid-displayed combinatorial library of the 58 residue, three-helix bundle staphylococcal protein A domain analogue Z, the selection of two affibody variants (ZTaq4:1 and ZTaq4:8) showing micromolar affinities (KD) to Taq DNA polymerase have been previously described (Nord et al., 1997). An alignment of their amino acid sequences showed that out of the 13 positions subjected to randomization using NN(G/T) degenerate codons, two positions in helix one [positions 10 (G) and 17 (F)] and a single position in connection to helix 2 [position 24 (G)] were identical in the two selected affibodies (Figure 1
). Despite the relatively extensive amino acid substitutions compared with the wild-type Z domain, studies using circular dichroism spectroscopy had shown that the secondary structure contents in the two affibodies closely resembled that of the parental wild-type Z domain, suggesting similar overall
-helical content (Nord et al., 1997
; Tashiro et al., 1997
).
To investigate if related affibody variants could be isolated with higher binding affinities to Taq DNA polymerase, an affinity maturation strategy involving the construction of a secondary library followed by re-selection against the Taq DNA polymerase target was performed. A hierarchical phagemid library based on the affibody ZTaq4:8 was constructed such that helix one (containing two identities compared with the ZTaq4:1 affibody) was kept as initially selected from the naive library whereas the six positions previously variegated in helix two were re-randomized using NN(G/T) degenerate codons including all 20 amino acids (Figure 2). This strategy should potentially allow for the selection of novel helix two variants contributing to Taq DNA polymerase binding, from a more extensively sampled diversity than present in the naive library.
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Biosensor binding analyses
To determine binding affinities using biosensor measurements, 12 variants were chosen for further analyses and produced as soluble affibodyABD fusion proteins from their respective phagemid constructs and HSA-affinity purified employing the 5 kDa serum albumin binding fusion partner. Expression levels were in the range of 630 mg/l shake-flask culture and an SDSPAGE analysis showed that the purified proteins were of expected size (approximately 13 kDa, data not shown). To obtain an initial ranking of binding affinities, the 12 affibodies were separately injected at an approximate concentration of 50 nM over a Taq DNA polymerase coated sensor chip surface, using the first generation ZTaq4:8 affibody as reference. An overlay plot of recorded sensorgrams shows that all except one of the injected second generation affibodies bind to the target with higher affinities than the reference variant, as indicated by their higher equilibrium (plateau) responses (Figure 3). From a visual inspection of the post-injection parts of the sensorgrams, it could be seen that the interactions between the target and the newly selected variants were characterized by slower off-rate kinetics (Figure 3
).
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To investigate if the apparent affinity for the Taq DNA polymerase target could be further increased by linking two affibody moieties in a dimeric construct, the affibody variant with the highest affinity for Taq DNA polymerase (clone ZTaqS1-1) was produced as a genetically fused (head to tail) dimeric fusion protein, di-ZTaqS1-1ABD and analysed for binding to sensor chip immobilized target protein. A comparison between response curves obtained for the dimeric variant and its monovalent counterpart clearly shows that the binding of the dimeric version is characterized by a higher equilibrium response and slower off-rate kinetics (Figure 5). The apparent affinity (KD) of the dimeric di-ZTaqS1-1 affibody was calculated to approximately 8 nM, corresponding to a threefold increase in affinity compared with the monomeric counterpart. However, in affinity determinations of interactions where avidity effects are present, the obtained numbers are dependent on the density of immobilized target protein and will therefore vary depending on experimental conditions. Nevertheless, the obtained increase in apparent affinity for the dimeric construct shows upon the potential of further engineering of affibody moieties to obtain, for example, multimeric constructs of high binding avidities.
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Discussion |
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The affibody with the highest observed affinity (clone ZTaqS1-1) was efficiently enriched using a low concentration of soluble target during selections. In this variant, four of the six randomized positions are occupied by amino acids previously observed at the corresponding positions in either of the two Taq DNA polymerase binding affibodies ZTaq4:1 and ZTaq4:8 previously isolated from the naive Z-library (Nord et al., 1997). This suggests that the two initially selected Taq DNA polymerase binding affibodies as well as the newly selected and affinity matured variants bind to the same site on the target. Thus, during selections, Taq DNA polymerase binding determinants present in the unaltered helix one could have contributed to a directed selection of variants, resulting in a selection advantage over variants binding to other parts of the target mediated by helix two only. This notion is supported by results from affinity recovery of recombinant Taq DNA polymerase from crude Escherichia coli lysates in which both the ZTaq4:8 and the ZTaqS1-1 affibodies have been used as affinity ligands. Using both ligands, a Taq DNA polymerase degradation product is co-purified together with the full-length protein, indicating that both ligands recognize an `epitope' located within this fragment (Nord,K., Gunneriusson,E., Uhlin,M. and Nygren,P.-Å., manuscript in preparation).
In this study, three different selection protocols were used, based on either having the target in solution (two different concentrations) or immobilized onto paramagnetic particles. Interestingly, the ZTaqS1-1 variant, shown to have one of the highest affinities of the variants tested, was present among clones isolated using both strategies. However, a more selective enrichment (8/10) of this variant was seen using the target in solution, relative to when an immobilized target was used (1/10). This suggests that the soluble selection strategy was favourable for efficient selection of stronger binders from a background of lower affinity variants. Of the variants analysed, the five with the highest affinities were all isolated using this strategy. The obtained increase in affinity of those affibody variants was found to primarily be a result from slower off-rate kinetics. These results are in accordance with results from affinity maturation of antibody fragments using similar selection principles (Schier et al., 1996a).
Although affibodies with affinities in the micromolar range (KD) can be readily selected from medium sized naive libraries of the Z-domain (Nord et al., 1997; Hansson et al., 1999
) in some applications ligands of higher affinities could prove important. In this work, the affinity of a Taq DNA polymerase-specific affibody previously selected from a naive library of the Z-domain derived from staphylococcal protein A was increased 15-fold after a single round of affinity maturation directed to only one of the
-helices taking part in the interaction. This strategy resulted in a panel of related second generation variants with KD values in the range of 3050 nM. Interestingly, the affinity between the ancestral wild-type Z domain and its binding partner Fc of IgG has earlier been determined by several groups and KD values between 10 and 40 nM have been reported (Nilsson et al., 1994
; Jendeberg et al., 1995
; Braisted and Wells, 1996
; Jansson et al., 1998
). The fact that the dimeric version of the ZTaqS1-1 showed cooperative binding effects also suggests that higher multimeric forms of selected affibodies could be constructed to make this novel class of ligands attractive reagents in different applications. Taken together, the results demonstrate that it is possible to isolate variants of the IgG binding Z domain, directed to an entirely different target, without loss of binding strength.
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Acknowledgments |
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Notes |
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References |
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Ballinger,M.D., Jones,J.T., Lofgren,J.A., Fairbrother,W.J., Akita,R.W., Sliwkowski,M.X. and Wells,J.A. (1998) J. Biol. Chem., 273, 1167511684.
Braisted,A.C. and Wells,J.A. (1996) Proc. Natl Acad. Sci. USA., 93, 56885692.
Clackson,T., Hoogenboom,H.R., Griffiths,A.D. and Winter,G. (1991) Nature, 352, 624628.[ISI][Medline]
Crameri,A., Cwirla,S. and Stemmer,W.P. (1996) Nature Med., 2, 100102.[ISI][Medline]
Dunn,I.S. (1996) Curr. Opin. Biotechnol., 7, 547553.[ISI][Medline]
Georgiou,G., Stathopoulos,C., Daugherty,P.S., Nayak,A.R., Iverson,B.L. and Curtiss,R.,III (1997) Nature Biotechnol., 15, 2934.[ISI][Medline]
Griffiths,A.D. et al. (1993) EMBO J., 12, 725734.[Abstract]
Griffiths,A.D. et al. (1994) EMBO J., 13, 32453260.[Abstract]
Hanes,J. and Plückthun,A. (1997) Proc. Natl Acad. Sci. USA, 94, 49374942.
Hansson,M., Ringdahl,J., Robert,A., Power,U., Goetsch,L., Nguyen,T.N., Uhlén,M., Ståhl,S. and Nygren,P.Å. (1999) Immunotechnol., 4, 237252.[ISI][Medline]
He,M. and Taussig,M.J. (1997) Nucleic Acids Res., 25, 51325134.
Hoogenboom,H.R., de Bruine,A.P., Hufton,S.E., Hoet,R.M., Arends,J.W. and Roovers,R.C. (1998) Immunotechnology, 4, 120.[ISI][Medline]
Hultman,T., Bergh,S., Moks,T. and Uhlén,M. (1991) Biotechniques, 10, 8493.[ISI][Medline]
Jansson,B., Uhlén,M. and Nygren,P.Å. (1998) FEMS Immunol. Med. Microbiol., 20, 6978.[ISI][Medline]
Jendeberg,L., Persson,B., Andersson,R., Karlsson,R., Uhlén,M. and Nilsson,B. (1995) J. Mol. Recognit., 8, 270278.[ISI][Medline]
Low,N.M., Holliger,P.H. and Winter,G. (1996) J. Mol. Biol., 260, 359368.[ISI][Medline]
Lowman,H.B. and Wells,J.A. (1993) J. Mol. Biol., 234, 564578.[ISI][Medline]
Marks,J.D., Griffiths,A.D., Malmqvist,M., Clackson,T.P., Bye,J.M. and Winter,G. (1992) Biotechnology, 10, 779783.[ISI][Medline]
Martin,F., Toniatti,C., Salvati,A.L., Ciliberto,G., Cortese,R. and Sollazzo,M. (1996) J. Mol. Biol., 255, 8697.[ISI][Medline]
Maurer,R., Meyer,B. and Ptashne,M. (1980) J. Mol. Biol., 139, 147161.[ISI][Medline]
McConnell,S.J. and Hoess,R.H. (1995) J. Mol. Biol., 250, 460470.[ISI][Medline]
Nilsson,J., Nilsson,P., Williams,Y., Pettersson,L., Uhlén,M. and Nygren,P.Å. (1994) Eur. J. Biochem., 224, 103108.[Abstract]
Nilsson,J., Larsson,M., Ståhl,S., Nygren,P.Å. and Uhlén,M. (1996) J. Mol. Recognit., 9, 585594.[ISI][Medline]
Nilsson,J., Bosnes,M., Larsen,F., Nygren,P.Å., Uhlén,M. and Lundeberg,J. (1997) Biotechniques, 22, 744751.[ISI][Medline]
Nord,K., Nilsson,J., Nilsson,B., Uhlén,M. and Nygren,P.Å. (1995) Protein Engng, 8, 601608.[Abstract]
Nord,K., Gunneriusson,E., Ringdahl,J., Ståhl,S., Uhlén,M. and Nygren,P.Å. (1997) Nature Biotechnol., 15, 772777.[ISI][Medline]
Nygren,P.Å., Eliasson,M., Abrahmsén,L., Uhlén,M. and Palmcrantz,E. (1988) J. Mol. Recognit., 1, 6974.[Medline]
Perelson,A.S. and Oster,G.F. (1979) J. Theor. Biol., 81, 645670.[ISI][Medline]
Rüther,U., Koenen,M., Otto,K. and Muller-Hill,B. (1981) Nucleic Acids Res., 9, 40874098.[Abstract]
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
Schatz,P.J. (1993) Biotechnology, 11, 11381143.[ISI][Medline]
Schier,R., Bye,J., Apell,G., McCall,A., Adams,G.P., Malmqvist,M., Weiner,L.M. and Marks,J.D. (1996a) J. Mol. Biol., 255, 2843.[ISI][Medline]
Schier,R., McCall,A., Adams,G.P., Marshall,K.W., Merritt,H., Yim,M., Crawford,R.S., Weiner,L.M., Marks,C. and Marks,J.D. (1996b) J. Mol. Biol., 263, 551567.[ISI][Medline]
Smith,P.G. and Petrenko,V.A. (1997) Chem. Rev., 97, 391410.[ISI][Medline]
Tashiro,M., Tejero,R., Zimmerman,D.E., Celda,B., Nilsson,B. and Montelione, G.T. (1997) J. Mol. Biol., 272, 573590.[ISI][Medline]
Tramontano,A., Bianchi,E., Venturini,S., Martin,F., Pessi,A. and Sollazzo,M. (1994) J. Mol. Recognit., 7, 924.[Medline]
Vaughan,T.J., Williams,A.J., Pritchard,K., Osbourn,J.K., Pope,A.R., Earnshaw, J.C., McCafferty,J., Hodits,R.A., Wilton,J. and Johnson,K.S. (1996) Nature Biotechnol., 14, 309314.[ISI][Medline]
Wrighton,N.C., Farrell,F.X., Chang,R., Kashyap,A.K., Barbone,F.P., Mulcahy, L.S., Johnson,D.L., Barrett,R.W., Jolliffe,L.K. and Dower,W.J. (1996) Science, 273, 458464.[Abstract]
Yang,W.P., Green,K., Pinz-Sweeney,S., Briones,A.T., Burton,D.R. and Barbas, C.F.,III (1995) J. Mol. Biol., 254, 392403.[ISI][Medline]
Received January 7, 1999; revised June 7, 1999; accepted June 18, 1999.