Phage Display of Peptides and Proteins: A Laboratory Manual

Webster-- principles and applications of phage display, B. Hoess-- vectors for phage display, N. Kay-- construction of random peptide libraries in bacteriophage M13, A. Kay-- construction and screening of antibody display libraries, J. Johnson-- phagemid-displayed peptide libraries, D. Winter-- phage libraries displaying random peptides derived from a target sequence, D. Jordaan-- display and selection of proteins on genetic packages, R.

Crameri-- multicombinatorial libraries and combinatorial infection - practical considerations for vector design, R. Sudoyer-- use of filamentous virus particles as immunogens, R. Perham-- screening phage displayed random peptide libraries, A.

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Check copyright status Cite this Title Phage display of peptides and proteins: Other Authors Kay, Brian K. Bacteriophages -- Laboratory manuals. Microbial biotechnology -- Laboratory manuals. Viral proteins -- Laboratory manuals. Affinity chromatography -- Laboratory manuals. Peptides -- Biotechnology -- Laboratory manuals.

Phage Display of Peptides and Proteins: A Laboratory Manual

Proteins -- Biotechnology -- Laboratory manuals. Microbiology Laboratory techniques Summary Both novices and experts will benefit from this insightful step-by-step discussion of phage display protocols. Phage Display of Peptides and Proteins: A Laboratory Manual reviews the literature and outlines the strategies for maximizing the successful application of phage display technology to one's research. It contains the most up-to-date protocols for preparing peptide affinity reagents, monclonal antibodies, and evolved proteins.

Contents Machine derived contents note: Table of contents for Phage display of peptides and proteins: Bibliographic record and links to related information available from the Library of Congress catalog Information from electronic data provided by the publisher. May be incomplete or contain other coding. Biology of the Filamentous Bacteriophage. Principles and Applications of Phage Display. Vectros for Phage Display. Construction of Random Peptide Libraries in Bacteriophage.

Construction and Screening of Antibody Display Libraries. Phage Libraries Displaying Random Peptides. Derived Form a Target Sequence. Display and Selection of Proteins on Genetic Packages. Multicombinational Libraries and Combinational Infection: Practical Considerations for Vector Design. Use of Filamentous Virus Particles as Immunogens. Proteins involved in DNA replication remain in the cytoplasm. When the phage specific single-stranded DNA binding Chapter I Biology of the Filamentous Bacteriophage protein, pV, reaches the proper concentration, it sequesters the newly synthesized phage single-stranded DNA into a complex.

Assembly occurs at the bacterial envelope, at a site where the inner and outer membranes are in close contact. During the assembly process, the molecules of the pV single-stranded binding protein are displaced and the capsid proteins assemble around the DNA as it is extruded through the envelope. The transfer of the capsid proteins from their integral cytoplasmic membrane location to the assembling phage particle is directed by three phage-specific noncapsid assembly proteins and the bacterial protein thioredoxin.

Assembly continues until the end of the DNA is reached and the phage is released into the media. About phage particles are produced during the first generation following infection, after which the host produces approximately phage per generation. This continues for as many generations as has been measured so the curing rate is very low Model and Russel, Plaques are quite turbid and of varying size and contain approximately infective particles.

As a consequence of their structure and life cycle, the Ff phage have served as valuable tools for biological research. Since replication of DNA or assembly of the phage is not constrained by the size of the DNA, the Ff phage are excellent cloning vehicles see Smith, , for review.

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The result of an insertion of foreign DNA into a nonessential region merely results in a longer phage particle. Thus, large quantities of single-stranded DNA containing foreign DNA inserts ranging from a few to thousands of nucleotides can be easily obtained. These can be used for sequence analysis as well as other manipulations, such as the creation of substrates to study DNA mismatch repair Suet al. By combining the best features of plasmids and the phage, new cloning vectors called phagemids have been constructed see Mead and Kemper, , for review.

They contain the replication origin and packaging signal PS of the filamentous phage together with the plasmid origin of replication and gene expression systems of the chosen plasmid. Phagemids can maintain themselves as plasmids directing the expression of the desired proteins in the bacteria. Infection with a filamentous helper phage activates the phage origin of replication and the resulting phagemid single-stranded DNA is encapsulated into phage-like particles using helper phage proteins. In recent years, vectors have been developed that allow the display of foreign peptides on the surface of the filamentous phage particle Cesareni, By insertion of specific oligonucleotides or entire coding regions into genes encoding specific phage capsid proteins, chimeric proteins can be produced which are able to be assembled into phage particles Hoess, ; Smith and Scott, ; Winter et al.

This results in the display of the foreign protein on the surface of the phage particle. It is this use of the Ff phage that is the subject of this book. To aid in the understanding of this powerful technique, this chapter will describe aspects of the 4 Robert E. The initiating methionine is included in proteins which do not contain an amino terminal signal sequence biology of the Ff phage. It will concentrate on the present knowledge of the structure and location of the phage proteins in both the infected bacteria and the mature virion. An attempt will be made to relate this "static" picture of these proteins to the interactions which might occur between them during the dynamic processes of phage assembly and infection.

Both of these processes occur at the bacterial membranes, making detailed biochemical analysis difficult. Consequently, we have only a crude picture of the mechanism of phage assembly and disassembly based on molecular biological and genetic analysis. Still, such knowledge should aid in designing better display vectors which, in turn, may further our understanding of the biology of these interesting bacteriophage. The excellent review by Model and Russel contains a complete compendium of references on the subject up to Therefore, references cited in this chapter will primarily be to papers published subsequent to that review.

It encodes 11 genes, the products of which are listed in Table 1. The genes are grouped on the DNA according to their functions in the life cycle of the bacteriophage Fig. One group encodes proteins required for DNA replication, the second group encodes the proteins which make up the capsid and the third group encodes three proteins involved in the membrane associated assembly of the phage. The single-stranded DNA contains 11 genes. It has nucleotides which are numbered from the unique HindlI site located in gene II represented by 0. The figure is adapted from Model and Russel Dimers of pV bind single-strand DNA in a highly cooperative manner.

The structure appears to be wound in a left-handed helix with the pV dimers binding two strands of single-stranded DNA running in opposite directions alongthe helix. The PS is at one end of this rod-like structure, allowing it to be available for the initiation of phage assembly Bauer and Smith, Webster II and therefore has the same sequence as the carboxyl-terminal third of pII.

It is essential for the proper replication of the phage DNA and appears to function, in part, as an inhibitor of pII function Fulford and Model, All reside in the cytoplasmic membrane Fig. It is synthesized as a amino acid precursor containing a residue amino-terminal signal sequence which is removed after the protein is inserted into the membrane in a manner independent of the bacterial Sec system Kuhn and Troschel, The mature amino acid pVIII resides in the cytoplasmic membrane via its hydrophobic region residues 2 0 - 4 0 with its amino terminus in the periplasm Wickner, and its positively charged carboxyl-terminal 10 amino acids in the cytoplasm Ohkawa and Webster, Recent evidence suggests that the membrane associated pVIII adopts an alpha helical conformation with the carboxyl-terminal 30 residues traversing the membrane and extending into the cytoplasm in an orientation perpendicular to the surface of the membrane McDonnell et al.

The remaining four minor capsid proteins, which make up the ends of the phage particle, are also located in the membrane prior to assembly into mature bacteriophage FIGURE 3 A schematic representation of the known topologies of the membrane-associated phage capsid and assembly proteins. The interactions between the pI, pIV, and pXI assembly proteins shown are based on the evidence cited in the text. The numbers on the cytoplasmic side of the inner membrane refer to the residue at the end of the membrane-spanning region for that protein.

After removal of the signal sequence, the mature residue pIII spans the cytoplasmic membrane one time with only the final five carboxyl-terminal residues in the cytoplasm Davis et al. This places the amino-terminal residues in the periplasm. Evidence suggests that the first amino-terminal residues are responsible for phage adsorption and DNA penetration during infection when pIII is part of the mature phage particle see The Infection Process.

This region contains 8 cysteines which are involved in intramolecular disulfide bonds and buried in the interior of the folded portion of pIII Kremser and Rasched, Both pVII and pIX are presumed to span the membrane with their amino-terminal portion exposed to the periplasm. This topology is based only on the observation that these molecules retain their amino-terminal formyl group after membrane insertion Simons et al.

In the case of pIX, such an orientation would put the positively charged carboxyl-terminal region in the cytoplasm as expected. The sequence of the amino acid pVI predicts one hydrophobic membrane-spanning region residues It has been shown that pVI is an integral membrane protein Endemann and Model, but the topology of this protein in the membrane is unknown. Assembly Proteins Gene products pI, pIV, and pXI are required for phage assembly but are not part of the phage particle, pI is synthesized without an amino-terminal signal sequence but its insertion into the membrane appears to be dependent on the Sec system of the host Rapoza and Webster, It spans the membrane once with its aminoterminal residues in the cytoplasm and its carboxyl-terminal 75 amino acids exposed to the periplasm Guy-Caffey et al.

Production of moderate amounts of pI from plasmid-encoded gene I results in cessation of bacterial growth and loss of membrane potential Horabin and Webster, This suggests that pI monomers may interact in the membrane to allow the transmembrane regions to form a channel through the cytoplasmic membrane. Consistent with this hypothesis is the observation that chimeric proteins which contain the pI transmembrane region in the middle of the chimeric molecule also are able to span the membrane and cause cessation of cell growth when present in moderate amounts Horabin and Webster, ; Guy-Caffey and Webster, The resulting residue pXI is homologous to the carboxyl-terminal portion of pI.

Like pI, pXI spans the membrane once with its carboxyl-terminal 75 amino acids in the periplasm Fig. Production of moderate amounts of pXI from a plasmid does Robert E. Webster not stop cell growth, suggesting that pXI cannot interact to form a channel in a manner similar to pI Rapoza and Webster, However, like pI, pXI has been shown to be essential for phage assembly.

The product of gene IV is synthesized with a residue amino-terminal signal sequence. It is secreted across the cytoplasmic membrane in a manner dependent on the Sec system of the host Brissette and Russel, ; Russel and Kazmierczak, ; Rapoza and Webster, The amino-terminal half of pIV is exposed to the periplasm while the carboxylterminal half appears responsible for forming the portion of the pIV oligomer embedded in the outer membrane. The carboxyl-terminal region of pIV shows significant sequence homology to a number of bacterial proteins which are involved in the export of certain bacterial factors into the medium Russel, a.

All these observations suggest that the pIV oligomer may form a large exit port in the outer membrane through which the assembled phage particle can pass. However, this port must be gated in some way since bacteria containing pIV oligomers in the outer membrane remain impervious to compounds which normally do not pass through the outer membrane Russel, b.

However, if the assembly proteins were from the same phage, some heterologous phage could be assembled Russel, Periplasmic interactions between these assembly proteins could form a channel connecting the inner and outer membranes to allow extrusion of the assembling phage particle Fig.

It is proposed that the outer membraneassociated pIV oligomer acts as the gate in this channel to maintain the integrity of the membrane in the absence of phage assembly Russel, b. The existence of such a channel is consistent with the earlier observation that assembly of the bacteriophage occurs at sites where the inner and outer membrane appear to be in close contact Lopez and Webster, There is evidence suggesting possible interactions between the capsid and assembly proteins within the inner membrane. As outlined above, direct interaction of the carboxyl-terminal regions of pI and pXI with the pIV oligomers would suggest an inner membrane oligomer of pI and pXI, as depicted in Fig.

Mutational studies are consistant with interactions between the transmembrane helices of pVIII itself Deber et al. It should be pointed out that the evidence for any membrane associated interactions between these phage proteins is indirect, and thus any conclusion is preliminary.

At one end of the particle, there are about 5 molecules each of pVII and plX. The other end contains approximately 5 molecules each of pill and pVI. X-ray diffraction and other physical studies have given a fairly complete description of the pVIII cylinder portion of the virion Opella et al. Except for the amino-terminal five amino acids, pVIII appears to be present in an uninterrupted alpha helical conformation. The pVIII monomers are arranged in an overlapping manner so that the carboxyl-terminal residues form the inside wall of the particle.

This region contains four positively charged lysine residues which are arranged to form the positive face of an amphiphilic helix. The top is an electron micrograph of a negatively stained particle with the pVII-plX end at the right. The bottom is a schematic representation of the phage particle showing the location of the capsid proteins and the orientation of the DNA.

The figure is adapted from Webster and Lopez Webster of the DNA. Mutational studies suggest that these lysine residues do have some electrostatic interaction with the DNA Hunter et al. The additional mutant pVIII molecules in the longer particle presumably are required to maintain the appropriate ratio of positive charges of the capsid to negative charges of the DNA backbone. The anfino-terminal portion of pVIII, with its acidic residues, is exposed on the outside of the particle.

The residues connecting the amino and carboxyl regions of pVIII interact with each other to form the inner core of the protein cylinder Williams et al. Much of this middle portion of pVIII is part of the membrane spanning region of the protein before it is assembled into the particle Fig. One end of the particle contains approximately five molecules each of the small hydrophobic pVII and plX proteins.

The arrangement of these proteins in the capsid and their interaction with pVIII is not known. The observation that antibodies to plX but not pVII are able to interact with one end of the phage particle would suggest that only plX is exposed and pVII is buried Endemann and Model, Immunoprecipitation experiments on detergent-disrupted phage suggest an interaction between pVII and pVIII major capsid protein in the intact particle.

Approximately 5 molecules each of pill and pVI make up the other end of the particle and account for about nm of the phage length Specthrie et al. A major portion of the amino-terminal portion of pill can be removed from the phage particle by protease treatment Gray et al. Under certain conditions, this portion of plII can be visualized by electron microscopy as a knob-like structure at one end of the phage Gray et al.

It is this amino-terminal portion which is required for the infection process. The carboxylterminal residues of pill, together with pVI, presumably interact with pVIII to form one of the ends of the particle Crissman and Smith, ; Kremser and Rasched, Proteins pVI and pill appear to specifically recognize each other as they remain associated following disruption of the phage particle with certain detergents Gailus and Rasched, ; Endemann and Model, It is proposed that the hydrophobic amino terminus of pVI is buried within the particle Makowski, Presumably, the rather hydrophilic carboxyl-terminal region of this protein is near the surface, although antibody against the carboxyl-terminal 13 amino residues of pVI did not react with phage Endemann and Model, However, the observation that phage particles containing foreign proteins fused to the carboxyl end of pVI can be produced would argue that the carboxyl terminus of pVI is very near the surface of the phage particle Jespers et al.

The first involves the interaction of the pill end of the phage particle with the tip of the F conjugative pilus Fig. Retraction of the pilus, presumably by depolymerization of the pilin subunits into the inner membrane, brings the tip of the phage to the membrane surface. It is not known whether this event is a result of the normal polymerization-depolymerization cycles inherent to the pili or whether attachment of the phage can trigger pilus retraction Frost, The second step involves the integration of the pVIII major capsid proteins and perhaps the other capsid proteins into the inner membrane together with the translocation of the DNA into the cytoplasm.

This translocation event requires the products of the bacterial tolQRA genes Russel et al. Mutations in any one of these genes block the uptake of the phage DNA into the cytoplasm. However, the phage can still adsorb to the tip of the F pilus and conjugation is normal in these mutant bacteria Sun and Webster, These bacteriocins are able to bind to their outer membrane receptor in the tol mutant bacteria but such binding does not lead to cell death.

Since both the phage and the colicins can bind to their respective receptors but do not infect or kill bacteria containing mutations in the tolQRA genes, these mutant bacteria have been designated "tolerant" tol to filamentous phage or the group A colicins. The TolQRA proteins are located in the inner membrane.

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TolQ spans the membrane three times with the bulk of the protein in the cytoplasm Kampfenkel and Braun, ; Vianney et al. Both TolR and A span the membrane one time via a region near their amino terminus, leaving major portions of each protein exposed in the periplasm Levengood et al. The structure and length of the periplasmic portion of TolA suggest that a region near the carboxyl-terminal end may interact with the outer membrane.

There is some evidence that the transmembrane regions of these three Tol proteins interact with each other Lazzaroni et al. All these observations suggest that the TolQRA proteins form some type of complex which can communicate between the inner and outer membranes. One of the normal functions of these Tol proteins may be to maintain the integrity of the outer membrane as bacteria containing mutants in these tol genes freely leak periplasmic proteins into the media see Webster, Recognition of the receptor pilus tip and the appropriate Tol proteins appears to be a function of the amino-terminal region of pill.

Removal of the amino-terminal 36 kDa of pill by subtilisin renders the phage particle noninfectious Gray et al.

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Infection of the bacteria by the phage is inhibited by the addition of this proteolytic fragment, presumably because the fragment competes with the phage for the pilus tip or the TolQRA proteins. This is consistent with the observation that production of pill or its amino-terminal half confers resistance to infection by the Ff phages Boeke et al. Webster that confers resistance to both superinfection by the Ff phage and the effect of the group A colicins in Ff-infected bacteria.

The pilus recognition site is present in residues while residues are involved in the penetration of the DNA into the bacterium. Presumably it is this latter region which specifically interacts with the TolQRA inner membrane proteins. The colicins also have a specific amino-terminal domain which appears to recognize the TolQRA proteins Webster, This region of colicins E 1 and A has been shown to interact with the carboxylterminal portion of TolA Benedetti et al.

This would explain why the overproduction of a fragment containing the first 96 residues of gene III is able to confer tolerance resistance to the effect of some group A colicins Boeke et al. Comparison of the amino-terminal domains of pill with the E and A colicins reveal that all contain regions of glycine-rich repeats. Whether these regions are involved in any interactions with TolA or any of the other Tol proteins is unclear. It is not known whether the absorption and penetration domains must be contiguous for pill to function in the infection process.

Also unclear is the relation of these two domains to the carboxyl-terminal third domain of pill which interacts with the phage particle. Until a clear picture of the function of these regions of pill emerges, it is best to continue to fuse foreign proteins at or near the amino terminus of plII if one wishes to create an infectious particle. However, it is possible to create infectious hybrid phage containing a mixture of wild-type and chimeric pill for example, see Lowman et al.

All that is needed is to have the foreign peptide fused to the carboxyl-terminal region of pill so it will assemble with wild-type pill into particles. Recently, attempts have been made to design a type of "two-hybrid system" in which active phage will be produced based on the interaction between two different peptides Duenas and Borrebaeck, ; Gramatikoff et al. In this case, one of the interacting peptides is fused to the carboxyl terminus of the amino-terminal adsorption and penetration domain of pill and the other interacting species is attached to the amino-terminal portion of the carboxyl-terminal part of plII responsible for phage assembly see Chapter 3 for description of these display techniques.

Some experiments suggest that it may be cleaved during the infection process but this has not been confirmed. There is no evidence that intact pill integrates into the membrane in a state competent to be incorporated into a newly assembled phage particle. This is in contrast to the pVIII major capsid protein of the infecting bacteriophage, which has been shown to join the pool of newly synthesized pVIII and be assembled into newly formed particles see Model and Russel, This is based on the observation that infection of nonsuppressing bacteria with gene VII or IX amber mutant phage results in the production of one to three infectious polyphage per cell Webster and Lopez, Polyphage are multilength phage particles containing many unit length molecules of the single-stranded phage DNA.

These assembly sites may be the result of specific interactions between pI, plV, and pXI as previously described Fig. However, at some stage in the assembly process the capsid proteins must become part of the assembly site since they are all present as integral membrane proteins until assembled into the phage particle. Interaction of the pV-phage DNA complex with proteins in the assembly site presumably is the initiating event for assembly of the particle. This is followed by a progressive process in which the pV dimers are displaced from, and the appropriate capsid protein added to the DNA as it is extruded through the bacterial envelope into the media.

Throughout this whole process, the membrane must retain its integrity, as the bacteria continues to grow and divide. The assembly process is conveniently divided into three parts, initiation, elongation and termination, reflecting the events required for packaging the different ends and the long cylinder of the phage reviewed in Russel, Presently, there is no direct biochemical evidence for any of these specific PS-protein interactions.

It is also possible that these three proteins might interact to form the complete tip of the phage in the membrane which then recognizes the PS in a pI-directed reaction. This is based on the observation that assembly sites are able to form in bacteria infected with phage containing amber mutants in genes VII or IX Lopez and Webster, Webster 5 A schematic representation of the elongation process in phage assembly. Trx represents molecules of thioredoxin required for assembly. The arrows in the periplasm suggest that capsid proteins are able to enter the assembly site from the adjacent membrane area.

The proteins are represented by the same shapes shown in Fig. This figuce is only meant to depict interactions that occur during assembly and does not represent any specific structure for the proteins or DNA involved in the process. In the case of mutant-infected bacteria, they are defined as the increase in the number of membrane contact regions over the number found in uninfected bacteria Lopez and Webster, The assembly sites seen in the gene VII or IX mutant-infected bacteria may be defective as bacteria quickly cease to grow and divide after infection with these mutant phage see Model and Russel, These reactions are probably catalyzed by the cytoplasmic portion of pI and may require the hydrolysis of ATP Russel, The host protein, thioredoxin, also is required and genetic evidence suggests that it interacts with the cytoplasmic domains of pI Russel and Model, , ; Lim et al.

Assembly does not require the redox activity of thioredoxin but only that thioredoxin be present in its reduced conformation. It is suggested that thioredoxin confers processivity on the elongation process see Model and Russel, Removal of the pV dimers by pI would result in two antiparallel strands of naked DNA being present near the membrane surface of the assembly site Fig. The 12 amino acids of both pI and pXI adjacent to the cytoplasmic face of the membrane can form a positively charged amphiphilic helix similar to that present in the carboxyl 10 residues of the pVIII protein.

Whether the proposed interaction between the periplasmic portions of the pI, pIV, and pXI assembly proteins is maintained during elongation is unknown. Once the assembling phage particle is being extruded through the pIV oligomer in the outer membrane, the presence of a continuous channel between the pI proteins and the pIV oligomer might not be needed. This is consistent with the observation that phage can be assembled with pVIII carrying an amino-terminal extension containing up to six additional amino acids Kishchenko et al.

Since these large foreign proteins are present in the amino terminus of pVIII, they would be in the periplasm. Any tight channel between the gene I proteins and pIV might hinder these large periplasmic inserts from assembling into phage. Webster continuing to encapsulate another DNA. In this way polyphage containing multiple copies of the genome are produced. Polyphage produced in the absence of pVI are very unstable compared to polyphage produced in the absence of pill. This observation suggests that the addition of pVI stabilizes the phage structure and provides a structure for the binding of plII.

This does not preclude the possibility of an interaction of the carboxyl region of plII with pVI in the membrane, as Endemann and Model observed that pill could be immunoprecipitated from membranes of infected bacteria using anti-pVI antisera. The final step in assembly is the release of the completed phage particle from the bacterial envelope. Presumably, this requires the extrusion of the completed phage through the outer membrane. It is not known whether an assembly site can be used for the synthesis of more than one phage particle. It is possible that the addition of the pVI and pill during termination results in a breakdown of at least the inner membrane portion of the assembly site.

In this case, a new site, capable of initiating assembly, would have to be formed. If this were the case, then initiation might be expected to be one of the rate-limiting steps in phage assembly. It is the membrane-associated assembly that has made the filamentous phage such a valuable tool in biological research. Since the capsids are assembled around the DNA as it is extruded through the membrane-associated assembly site, there are no constraints, other than a possible shear constraint, on the length of DNA packaged.

This property led to its use as a cloning vehicle. Similarly, the membrane-associated assembly properties of the capsid proteins allow the packaging of chimeric proteins into the phage particle. Most of the interactions involved in the assembly process appear to occur near the portion of the capsid proteins which initially span the membrane. Thus foreign proteins fused to the periplasmic portions of the pVIII or plII capsid proteins have little, if any, influence on the assembly of the capsid proteins around the DNA.

Theoretically, any protein has a good chance of being packaged into a phage particle when fused to the periplasmic portion of pVIII, pill, or perhaps the pVI capsid protein, providing that at least two criteria can be satisfied. The foreign protein must be translocated efficiently across the inner membrane into the periplasm so that enough of the protein is available for assembly into particles.

Once in the periplasm, it must be able to enter the assembly site and not interfere with the processes that occur at this site during assembly. The apparent flexibility of the assembly process has led to an impressive array of applications for the use of phage display, as discussed in the next chapter. Further knowledge about the assembly mechanism of the filamentous phage should lead to the design of better display vectors. Domain structure of bacteriophage fd adsorption protein. Filamentous phage morphogenetic signal sequence and orientation of DNA in the virion and gene V protein complex.

Nucleotide sequence and genome organization of filamentous bacteriophages fl and fd. Protein import into Escherichia coli: Colicins A and E1 interact with a component of their translocation system. Effects of bacteriophage fl gene III protein on the host cell membrane. Secretion and membrane integration of a filamentous phageencoded morphogenetic protein. Peptide display on filamentous phage capsids. A powerful new tool to study proteinligand interaction. Gene 3 protein of filamentous phages: Evidence for a carboxylterminal domain with a role in morphogenesis. Fine structure of a membrane anchor domain.

Val - Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M 13 coat protein. Clonal selection and amplification of phage displayed antibodies by linking antigen recognition and phage replication. Bio Technology 12, Location of filamentous phage minor coat proteins in phage and in infected bacteria. Conjugative pili and pilus-specific phages. In "Bacterial Conjugation" D. Analysis of the sequence and gene products of the transfer region of the F factor. Gene X of bacteriophage fl is required for phage DNA synthesis: Mutagenesis of in-frame overlapping genes.

The adsorption protein of bacteriophage fd and its neighbour minor coat protein build a structural entity. The role of the adsorption complex in the termination of filamentous phage assembly. Three-dimensional structure of a cloning vector. X-ray diffraction studies of filamentous bacteriophage M 13 at 7A resolution.

Direct interaction rescue, a novel filamentous phage technique to study protein-protein interactions. IKe and fd gene 5 proteins from left-handed helices with single-stranded DNA. Adsorption complex of filamentous fd virus. Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA. Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens. The membrane domain of a bacteriophage assembly protein: Membrane insertion and growth inhibition. Membrane localization and topology of a viral assembly protein.

Nucleotide sequence of bacteriophage fl DNA. Phage display of peptides and protein domains. An amino acid sequence which directs membrane insertion causes loss of membrane potential. Interactions between DNA and coat protein in the structure and assembly of filamentous bacteriophage fd. Nature London , Modifying filamentous phage capsid: Limits in the size of the major capsid protein. Conjugation among enteric bacteria: Mating systems dependent on expression of pili.

In "Microbial Cell-cell Interactions" M. A hybrid toxin from bacteriophage fl attachment protein and colicin E3 has altered cell receptor activity. Bio Technology 13, Inactivation of TolQ by a missense mutation in the proposed first transmembrane segment. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Filamentous phage plV forms a multimer that mediates phage export across the bacterial cell envelope.

Structure of a foreign peptide displayed on the surface of bacteriophage M The adsorption protein of filamentous phage fd: Assignment of its disulfide bridges and identification of the domain incorporated in the coat. Distinct steps in the insertion pathway of bacteriophage coat proteins. Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex. E, Jr, and Webster, R.

A membrane protein involved in colicin uptake contains an extended helical region. Thioredoxin is the bacterial protein encoded by tip that is required for filamentous bacteriophage fl assembly. Assembly site of bacteriophage fl corresponds to adhesion zones between the inner and outer membranes of the host cell. Selecting high-affinity binding proteins by monovalent phage display. Terminating a macromolecular helix. Structural model for the minor proteins of bacteriophage M Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages.

Chimeric single-stranded DNA phage-plasmid cloning vectors. In "The Bacteriophages" R. Membrane topology of the Escherichia coli Tol R protein required for cell envelope integrity. The orientation of the major coat protein of bacteriophage fl in the cytoplasmic membrane of Escherichia coli. Biology of the Filamentous Bacteriophage 19 Olah, G. Structures of fd gene 5 protein-nucleic acid complexes: A combined solution scattering and electron microscopy study.

Protein structure by solid-state NMR spectroscopy. Raman spectroscopy of the filamentous virus Ff fd, fl, M Structural interpretation for coat protein aromatics. The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane.

The products of gene I and the overlapping in-frame gene XI are required for filamentous phage assembly J. Protein-protein interactions during filamentous phage assembly. A paradigm for bacterial virulence factor export. Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phage.

Analysis of the structure and subcellular location of filamentous phage plV. A bacterial gene,fip, required for filamentous bacteriophage fl assembly. Thioredoxin is required for filamentous phage assembly. Genetic analysis of the filamentous bacteriophage packaging signal and of the proteins that interact with it. Low-frequency infection of F-bacteria by transducing particles of filamentous bacteriophages. Structure of the gene V protein of bacteriophage fl determined by multiwavelength x-ray diffraction on the selenomethionyl protein. Filamentous phages as cloning vectors.

Libraries of peptides and proteins displayed on filamentous phage. In "Methods in Enzymology" R. Construction of a microphage variant of filamentous bacteriophage. Dissection of functional domains in phage fd adsorption protein-discrimination between attachment and penetration sites. Mispair specificity of methyl-directed DNA mismatch correction in vitro. Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: Comparison with phage fd. Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. The tol gene products and the import of macromolecules into Escherichia coli. Structure and assembly of the class 1 filamentous bacteriophage.

In "Virus Structure and Assembly" S. Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli. Structure and function of the F factor and mechanism of conjugation. In "Escherichia coli and Salmonella typhimurium" E C. American Society for Microbiology, Washington, D. Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M Role of smallresidues in protein oligomerization. Making antibodies by phage display technique. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems.

The origins of phage display date to the mids when George Smith, on sabbatical in Bob Webster's laboratory at Duke University, first expressed a foreign segment of a protein on the surface of bacteriophage M13 virus particles. Using a polyclonal antibody specific for the endonuclease, Smith demonstrated that phage containing the EcoRI-gIII fusion could be enriched more than fold from a mixture containing wild-type nonbinding phage with an immobilized polyclonal antibody.

From these first experiments emerged two important concepts. First, using recombinant DNA technology, it should be possible to build large libraries i. Second, the methodology provides a direct physical link between phenotype and genotype. That is, every displayed molecule has an addressable tag via the DNA encoding that molecule. Hoess ease and rapidity of DNA sequence analysis, selected molecules can be identified quickly. It is interesting to note that the idea of addressable tags is also now being adopted in some combinatorial chemical libraries Needels et al.

Within a few years of George Smith's experiments the first phagedisplayed random peptide libraries were assembled Cwirla et al.