1 Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849 and 3 Division of Biological Sciences and 4 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA
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Abstract |
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Keywords: fibrinogen binding/landscape library/nanotechnology/phage display/substitute antibody
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Introduction |
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The major coat protein in viable filamentous phages tolerates different point mutations (Williams et al., 1995), deletion of N-terminal amino acids (V.A.Petrenko, unpublished work) and insertion of short peptides into the N-terminus (Ilyichev et al., 1989
; Felici et al., 1991
; Greenwood et al., 1991
; Kishchenko et al., 1991
; Iannolo et al., 1995
, 1997
; Petrenko et al., 1996
; Terry et al., 1997
). Foreign N-terminal peptides fused to every copy of pVIII can subtend as much as 2530% of the virion surface, dramatically changing the particles surface architecture and properties. Depending on the particular foreign peptide sequence, such phages can bind organic ligands, proteins, antibodies and cell receptors (Petrenko et al., 1996
; Iannolo et al., 1997
; Petrenko and Smith, 2000
; Romanov et al., 2001
), interact with proteases (Terry et al., 1997
), induce specific immune responses in animals (Minenkova et al., 1993
; di Marzo Veronese et al., 1994
; Perham et al., 1995
; De Berardinis et al., 2000
), resist stress factors such as chloroform or high temperature (Petrenko et al., 1996
) or migrate differently in an electrophoretic gel (V.A.Petrenko, unpublished work). Recombinant phages with foreign peptides fused to all pVIII subunits are called `landscape phages and collections of such phages are called `landscape libraries (Petrenko et al., 1996
; Petrenko and Smith, 2000
).
Here we describe a new type of landscape library, the `alpha landscape library, in which the randomized amino acids lie within the -helical portion of pVIII rather than at the N-terminus and are thus conformationally homogeneous (Bianchi et al., 1995
; Nord et al., 1995
). Our results show that phages tolerate multiple substitutions at these positions as long as they do not disturb general
-helical architecture of major coat protein. The new library can serve as a source of
-helical ligands and substitute antibodies and can be used in conjunction with another landscape library to construct a `mosaic library whose member particles simultaneously display different foreign peptides on their surface (V.A.Petrenko, unpublished work).
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Materials and methods |
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Solutions, preparations and reagents referred to in this paper are described in Table I.
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Escherichia coli and phage strains, general methods of phage display, titering infective particles as tetracycline transducing units (TU), spectrophotometric quantitation of phage particles, transfection, preparation of replicative form (RF) and viral single-stranded circular DNA, propagation and processing of phages, DNA sequencing and other standard microbiological methods have been described (Smith and Scott, 1993; Yu and Smith, 1996
; Barbas et al., 2001
) and are also detailed at the website (http://www.biosci.missouri.edu/SmithGP/index.html). DNA was extracted with phenolchloroform in Phage Lock Gel tubes (5 Prime
3 Prime, Inc.) and precipitated with ethanol as outlined by Sambrook et al. (Sambrook et al., 1989
).
Vector f85
The 9183 bp vector f85 (GenBank Accessions bankit439334) was constructed by standard methods and is illustrated in Figure 1. It has PstI, BamHI, NheI and MluI cloning sites in gene VIII and confers tetracycline resistance on the host cell.
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We used E.coli K91BlueKan cells (Yu and Smith, 1996) for propagation of phages. For sequencing, phage was propagated in 2 ml of culture and separated by PEG precipitation (Haas and Smith, 1993
). For ELISA, phage was propagated in 20 ml of culture and purified by double PEG precipitation. For CD analysis, phage was grown in 200 ml of culture and purified by double PEG precipitation followed by ultracentrifugation in a gradient of CsCl as described (Smith and Scott, 1993
)
Construction of the f8 library
Double-stranded f85 RF DNA (600 µg) was cleaved with BamHI (1340 units) and MluI (670 units) in 10 ml of NEB buffer 3 (both enzymes and buffer from New England Biolabs), diluted with 400 µl of 250 mM EDTA, treated with phenolchloroform, precipitated with ethanol and dissolved in 12 ml of TE buffer. A short stuffer fragment that lies between BamHI and MluI sites was removed by ultrafiltration of 2 ml portions of DNA solution through six Centricon 100 kDa units (Amicon) followed by washing of each unit with 6x2 ml of TE buffer. DNA in a total volume of 5 ml was treated with phenol and chloroform and precipitated with ethanol; the yield was 365 µg. Meanwhile, the degenerate insert was prepared by annealing two partially complementary synthetic oligonucleotides, 1 and 2 (Table II), filling in the gaps at each end with DNA polymerase and digesting the product with BamHI and MluI as follows. A mixture of 768 pmol of 32-mer 1 and 624 pmol of 73-mer 2 in 80 µl of 3.75x Klenow buffer, divided into three equal portions, was incubated in PCR microtubes for 5 min at 70°C, 10 min at 37°C and 10 min at 20°C. The tubes containing 1 µl of 5 mM dNTPs, 50 units of Klenow fragment and water to 100 µl were incubated at 30°C for 45 min. Reactions were stopped with 4 µl of 250 mM EDTA; mixtures were pooled, adjusted to 800 µl with TE; DNA was treated in two portions with phenol and chloroform and precipitated with ethanol. The resulting 85-mer degenerate duplex was digested in 660 µl of NEBuffer 3 with 1400 units of BamHI and 700 units MluI, reactions being controlled by electrophoresis in a 12% polyacrylamide gel along with pGEM DNA markers (Promega). The insert was excised from a preparative 12% polyacrylamide gel (Yu and Smith, 1996
), electroeluted in an ISCO device on to Whatman DE81 paper, eluted from the paper with 3x25 µl of 1 M NaCl and precipitated with ethanol. The BamHI/MluI-cut RF DNA (13.8 µg) was ligated with a 2.7-fold molar excess of gel-purified degenerate insert in 1.5 ml Ligase buffer with 380 units of T4 ligase (Boehringer) at 16°C for 16 h. Reaction was stopped with 60 µl of 250 mM EDTA; DNA product was isolated by phenolchloroform extraction and ethanol precipitation. It was treated with NheI to destroy traces of uncut vector, isolated by phenolchloroform extraction and ethanol precipitation and dissolved in 40 µl of water. Four 100 µl portions of frozen electrocompetent cells of E.coli MC1061 (Yu and Smith, 1996
) (competence 9.8x108 TU per µg of f85 RF DNA) were electroporated with 10 µl of ligation mixture as described (Yu and Smith, 1996
). Transformed cells were inoculated into four 1 l cultures of NZY medium containing 20 µg/ml tetracycline. After spreading 200 µl portions of appropriate dilutions on Tc plates to determine the number of independent transformed clones (4.4x108), the bulk cultures were shaken vigorously overnight at 37°C. Meanwhile, TcKan plates were seeded with K91BlueKan cells (Yu and Smith, 1996
) and 100 transformed MC1061 colonies were grided on to the seeded plate. In order for a colony to grow at a grid-point, the corresponding MC1061 colony (resistant to tetracycline by virtue of the phage it carries, but sensitive to kanamycin) must have released infectious phage particles that could transduce the kanamycin-resistant K91BlueKan cells to tetracycline resistance. This occurred at 40% of the grid-points, indicating that 40% of the original transformed clones release infectious phages; 55% of these clones were shown by sequence analysis to harbor random peptides.
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A 50 ml culture of NZY was inoculated with 1 ml of an overnight culture of K91BlueKan cells, shaken at 37°C until the optical density at 600 nm reached 1.0, infected with ~2x1010 TU of f88mer or f8 library phages, shaken gently for 15 min at 37°C, transferred to a 3 l flask containing 500 ml of NZY supplemented with 0.2 µg/ml tetracycline and shaken vigorously for 35 min at 37°C. Additional tetracycline was added to a final concentration of 20 µg/ml, a portion was withdrawn and dilutions of that portion were spread on Tc plates; the total yield of transductants was 2.1x1010 and 5x1010 TU for f8
and f88mer libraries, respectively. Meanwhile, the 500 ml cultures were shaken vigorously overnight at 37°C. Two 1 ml portions were withdrawn and cleared of cells by two 5 min centrifugations in a microfuge. The resulting first-round supernatants contained ~5x1010 TU/ml for f8
and f88mer libraries. They served as inputs for a second round of propagation carried out in the same way, yielding second-round supernatants containing ~5x1010 TU/ml. These served in turn as inputs for a third round of propagation, yielding third-round supernatants. Individual clones in the third-round supernatants were propagated and their viral DNAs sequenced in the relevant regions to determine sequences of their displayed guest peptides.
Affinity selection of fibrinogen-binding phages
Six 35 mm Petri dishes were coated with 400 µl of 10 µg/ml bovine fibrinogen (Sigma) in TBS for 16 h at 4°C. These were used for three rounds of affinity selection from the f8 library (2x1010 virions) and the f88mer library (2x1011 virions) as described (Petrenko and Smith, 2000
). Phages from 40 individual clones were propagated in 20 ml and partially purified by double PEG precipitation (first precipitation from 20 ml of supernatant and second from 1 ml of TBS solution to obtain a titer of ~2x1012 in 200 µl of 1/20 TE) for sequence analysis and binding studies.
Biotinylated fibrinogen
Bovine fibrinogen (38 mg dissolved in 9 ml of water; Sigma Chemical) was dialyzed against PBS, mixed with 1 ml of 1 M NaHCO3 and 644 µl of freshly prepared 1.8 mM sulfo- succinimidyl 6-(biotinamido)hexanoate (Biotin-LC-NHS; Pierce Chemical) and reacted at 20°C for 2 h. To quench the residual reagent, 2.5 ml of 1 M TrisHCl, pH 8.9 was added and the mixture incubated for an additional 1 h at 20°C. After dialysis against TBS, the solution was concentrated to 3.2 ml using a Centriprep-30 Concentrator (Amicon), diluted with an equal volume of glycerol and stored at 20°C; the concentration was 3 mg/ml protein, assuming that an A280 of 1 corresponds to 0.61 mg/ml.
ELISA of fibrinogen binders
Phages, 5x1011 virions/ml in 40 µl TBS, were absorbed in wells of 96-well polystyrene ELISA dishes for 16 h at 4°C. The wells were washed, blocked with BLOTTO solution, washed, filled with various amounts of biotinylated bovine fibrinogen in 40 µl of TBS, incubated for 30 min at 20°C, washed and developed with alkaline phosphatase-conjugated streptavidin and p-nitrophenyl phosphate as described (Yu and Smith, 1996). In competition ELISA, biotinylated fibrinogen at a fixed concentration of 76 nM was preincubated for 45 min with various amounts of inhibitor phage before being added to the wells.
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Results and discussion |
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Construction of the alpha library
The major coat protein of filamentous phages consists of four functional domains AD:
N-Terminal mobile surface domain A (Ala1 to Asp5) appears to be in a non-helical, possibly disordered conformation (Kishchenko et al., 1994). Domain B is an amphipathic, slowly curving
-helix, extending from Pro6 to about Tyr24 (Glucksman et al., 1992
). Domain C, a highly hydrophobic helix extending from Ala25 to Ala35, is entirely buried in the interior of the protein coat. The remainder of the protein, D, from Thr36 to Ser50, constitutes an amphipathic helix forming the inside wall of the protein coat. Four basic residues near the C-terminus interact with the viral DNA.
Foreign peptides fused to N-terminal domain A can adopt various conformations depending on their sequence (Ilyichev et al., 1989; Felici et al., 1991
; Greenwood et al., 1991
; Kishchenko et al., 1994
; Iannolo et al., 1995
; Petrenko et al., 1996
; Petrenko and Smith, 2000
). In this work we found that unlike promiscuous N-terminal peptides, foreign peptides loaded into domain B submit to
-helical architecture of wild-type protein.
According to an -helical model of segment B, amino acids K8, D12, S13, Q15, A16, S17, T19, E20, Y21, G23 and Y24 belong to a polar area and are exposed on the surface of the phage, while another amino acids belong to a non-polar region and interact with phage body (Makowski, 1993
; Marvin et al., 1994
; Williams et al., 1995
). We designed a 108 clone library f8
(see the structure above) of phages randomized in the polar area of the B segment (amino acids 12, 13, 1517 and 19).
Composition of f8 library and its evolution
It was interesting to compare the evolution of the phage library in vitro and the natural evolution of filamentous phages. Phages f1, fd and M13 presumably have arisen through independent mutations of a common ancestor (Hill and Petersen, 1982). Their evolution resulted in accumulation of dozens of mutations in DNA, which, however, did not change substantially the primary structures of the capsid proteins. In mature forms of the major coat protein there is only one difference between the wild-type phages: D12 in phage f1 and fd is replaced for N12 in phage M13, as shown above, which probably is not essential for phage reproduction. In phage libraries, however, some peptide inserts can confer a selective growth advantage or disadvantage for the host phage, as shown by Iannolo et al. (Iannolo et al., 1997
). In our `evolution experiments we used populations of phages from f8
library, mutated in positions 12, 13, 1517 and 19 and, for comparison, f88 library of clones having random multiple mutations in the N-terminus (deletion of EGD sequence with insertion of random 8-mers) (Petrenko et al., 1996
). 108 phage clones from the f8
library were propagated three times in liquid media with an excess of bacterial cells. The proportion of the wild-type phages in the phage population did not increase substantially during propagation: 2/120 (1.7%) before and 7/160 (4.3%) after three rounds of propagation. Data on the diversity of amino acids in 64 randomly chosen clones from the initial library and 114 clones from the propagated library are summarized in Table III
. There are some clear biases in the distribution of some amino acids in the libraries. For example, cysteine, proline and tryptophan are prohibited in all positions, while the occurrence of negatively charged and polar amino acids is considerably higher than expected. However, the composition of the library is not dramatically changed through the propagation of the clones, indicating that clones from the alpha library have about equal advantages in the growing process. It is interesting to note that natural phage M13 successfully uses the less advantaged asparagine instead of the commonly dominant aspartic acid present in the strains fd and f1.
Evolution of the f8 library contrasted dramatically with that of the landscape library f88, composed of the clones having insertions of foreign peptides in N-terminal part of the major coat protein (Petrenko et al., 1996
; Petrenko and Smith, 2000
). To characterize the primary library f88 we determined the structure of the foreign peptides in 140 clones among which 13 clones (11%) were identified as wild-type phages (vector). Occurrences of different amino acids in foreign peptides in 73 non-wild-type phages are shown in Table IV
. The distribution of amino acids in the inserts does not differ dramatically from random, with some exceptions: cysteine is absent and proline and threonine are favored in all positions.
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aELSNTTTSdpak....
aASTVSTSEdpak...
aENSNPLTTdpak...
aAQESSMVPdpak...
aDRSLIDGTdpak...
aGTEDAALPdpak...
aDSGAPRYEdpak...
aGADGDdpak...
aVPdpak...
aegedpak... vector
where non-wild-type amino acids are shown as capitals and dominant serine and threonine are bold.
After four rounds of amplification, 40 clones were sequences and 35 (88%) were found to be vector. The structure of the other five clones are as follows:
aAxNTSATTdpak...
aDTATSRSTdpak...
aDSTAAGLTdpak
aEPGQDdpak...
aEdpak...
These results clearly indicate powerful censoring during growth of phages from the f88 library in favor of the wild-type phages and probably weak or no censoring of the f8 library.
-Helical conformation of random peptides in f8
library
The absence of proline and cysteine and the frequent appearance of glutamic acid and glutamine in the random peptides of the f8 library agree with their
-helical conformation (Chou and Fasman, 1974
). More detailed analysis using Protean 4.0 Expert Sequence Analysis Software from DNASTAR showed that, according to the ChouFasman and GarnierRobson algorithms, in all 18 randomly chosen clones the exposed part of the major coat protein between 9 and 21 amino acids preferably appears in an
-helical conformation. In contrast, not one of 10 randomly chosen fusion peptides from the f88 library, used as a control, was present in an
-helical conformation. An intense positive band at 194 nm along with two negative bands at about 223 and 208 nm in the CD spectra of randomly chosen clones from the f8
library (Figure 2
) also demonstrate that they are exposed in an
-helical conformation (Johnson, 1990
; Williams and Deber, 1996
). As a control we used phages fused to the peptide identical with a loop segment in concanavalin A (Hardman et al., 1982
; Petrenko et al., 1996
). The lower magnitude of positive and negative bands in its CD spectrum, as expected, corresponds to lower
-helicity of the fusion protein in phage capsid in comparison with clones from the alpha library (Toumadje and Johnson, 1993
).
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It can be expected that peptides of the alpha library, which are constrained in the -helical conformation, are not promiscuous as peptides from another phage libraries, which are flexible enough to bind various ligands and receptors in their native conformations (Smith and Petrenko, 1997
). However, the rigid conformation of the displayed peptides can provide the entropic advantage of binding with the matching molecule, thus increasing the affinity of binding. It can give also precise information on the structure of the complex, which can be used for the design of organic peptidomimetics (Bianchi et al., 1995
).
We compared the repertoires of the binding phages from two libraries, alpha library f8 and landscape library f88 (Petrenko et al., 1996
; Petrenko and Smith, 2000
), using bovine fibrinogen as a model acceptor of the phage clones. Fibrinogen is a 45 nm long rod-like 340 kDa glycoprotein composed of six polypeptide chains joined by disulfide bonds (Doolittle et al., 1998
). To select fibrinogen-binding phages, fibrinogen was absorbed on plastic dishes and treated with landscape libraries. After three rounds of selection (Petrenko and Smith, 2000
), fibrinogen-binding clones were propagated and sequenced (Table V
). The fibrinogen-binding phage displayed five families of guest peptides with dominant motifs AYLADRAD, FDLQLLAE, EAGPRXXP, (D/E)G(Y,F)LRP(E/D)Z and DSSVRFTG, where the positions marked X are occupied with an unusually high proportion (50%) of S and T and positions marked Z have P, S or T. The majority of clones selected from the alpha library displayed peptides AYLADRAD or FDLQLLAE, while clones selected from the landscape library f88 had diverse structures of guest peptides.
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Earlier we demonstrated that landscape phages, harboring foreign peptides fused to all copies of major coat protein, can demonstrate emergent properties intrinsic to the whole phage architecture, such as the ability to bind small organic compounds and protein antigen, resistance to stress conditions, mobility in an electric field, etc. (Petrenko et al., 1996; Petrenko and Smith, 2000
). The architecture of landscape phages depends on the structure of the guest peptide and the mode of its fusion to the major coat protein. For example, Terry et al. (Terry et al., 1997
) showed that peptide bonds located approximately three residues or more from the N-terminus of the major coat protein are resistant to proteases, presumably because of their proximity to the viral surface and conformational constraints. On the other hand, amino acids 112 of the major coat protein serve as antigenic epitopes and interact with antibodies (Kneissel et al., 1999
). In phages from the f88 library the foreign octamers are introduced into the N-terminus of the major coat protein between amino acids A-1 and D-5. The C-terminal part of the insert is probably accessible for binding but can be conformationally constrained. Furthermore, in some clones whole peptides can be constrained, being squeezed between neighboring subunits, as was shown by Kishchenko et al. (Kishchenko et al., 1994
). In contrast to the f88 library, whose clones are promiscuous enough to bind different receptors and ligands (Petrenko et al., 1996
; Petrenko and Smith, 2000
), the f8
library contains a repertoire of clones with a strongly constrained conformation of guest peptides, which allows them to bind only receptors and ligands having a flexible conformation, matching the shape of
-helically constrained peptides, such as fibrinogen, used in this work as a model antigen.
The two libraries differ dramatically in their evolution. Most of the clones in the f88 library with foreign inserts in the N-terminal part of the major coat protein replicate in bacterial cells considerably slower than wild-type phages and are lost during several rounds of amplification. Different mechanisms of the biases intrinsic to the libraries, such as protein synthesis, inner membrane insertion, signal peptide cleavage, assembly, etc., can be considered (Rodi and Makowski, 1999). In contrast, the diversity of the f8
library is not changed much after three rounds of amplification, leading to the conclusion that clones in this library, having multiple mutations in the central displayed part of the major coat protein, apparently have similar replication rates.
Conclusion
The alpha library is a new type of phage display library, in which biological selection helps to generate a great variety of conformationally biased -helical ligands. Strong biological censoring during phage growth probably prevents the appearance of amino acids which disturb the
-helical conformation of the phage major coat protein. Phage-borne
-helical peptides have a very rigid structure and cannot be used with sure success for the selection of ligands for any receptors and antibodies. However, if selected they provide precise information about the structure of the ligand, including its conformation, and give a clue for the design of lead compounds for this receptor. Furthermore, in combination with other landscape libraries, the alpha library is a partner for the generation of mosaic phage libraries with a very high diversity of antigen-binding sites, reminding antibodies and limited only by the volume of infected bacteria, 1012 clones being a realistic number. The alpha library is a source of new fiber materials for nanotechnology with emergent physical and chemical properties. There is a need for such materials, for example, in the construction of diagnostics, chemical and biological detectors, vaccines, hemostatics, hemosorbents, affinity sorbents, gene- and drug-delivery vehicles, matrices for artificial tissues or transplants, etc. (Smith and Petrenko, 1997
; Koivunen et al., 1999
).
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Notes |
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Acknowledgments |
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References |
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Received April 12, 2002;