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Synthetic antibody libraries are constructed from scratch using designed synthetic DNA. Precise control over design enables the use of highly optimized human frameworks and the introduction of defined chemical diversity at positions that are most likely to contribute to antigen recognition. We describe complete methods for the design, construction, and application of simplified synthetic antibody libraries built on a single human framework with diversity restricted to four complementarity-determining regions and two amino acids (tyrosine and serine). Despite the extreme simplicity of design, these libraries are capable of generating specific antibodies against diverse protein antigens. Moreover, the same methods can be used to build more complex libraries that can produce synthetic antibodies with affinities and specificities beyond the scope of natural antibodies. Most importantly, these simplified methods rely on standard supplies, equipment, and methods that are accessible to any molecular biology laboratory.

The homologous recombination systems of linear double-stranded (ds)DNA bacteriophages are required for the generation of genetic diversity, the repair of dsDNA breaks, and the formation of concatemeric chromosomes, the immediate precursor to packaging. These systems have been studied for decades as a means to understand the basic principles of homologous recombination. From the beginning, it was recognized that these recombinases are linked intimately to the mechanisms of phage DNA replication. In the last decade, however, investigators have exploited these recombination systems as tools for genetic engineering of bacterial chromosomes, bacterial artificial chromosomes, and plasmids. This recombinational engineering technology has been termed “recombineering” and offers a new paradigm for the genetic manipulation of bacterial chromosomes, which is far more efficient than the classical use of nonreplicating integration vectors for gene replacement. The phage λ Red recombination system, in particular, has been used to construct gene replacements, deletions, insertions, inversions, duplications, and single base pair changes in the Escherichia coli chromosome. This chapter discusses the components of the recombination systems of λ, rac prophage, and phage P22 and properties of single-stranded DNA annealing proteins from these and other phage that have been instrumental for the development of this technology. The types of genetic manipulations that can be made are described, along with proposed mechanisms for both double-stranded DNA- and oligonucleotide-mediated recombineering events. Finally, the impact of this technology to such diverse fields as bacterial pathogenesis, metabolic engineering, and mouse genomics is discussed.

Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I. Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences.

Bacteriophage T4 is the archetype of virulent phage. It has evolved very efficient strategies to subvert host functions to its benefit and to impose the expression of its genome. T4 utilizes a combination of host and phage-encoded RNases and factors to degrade its mRNAs in a stage-dependent manner. The host endonuclease RNase E is used throughout the phage development. The sequence-specific, T4-encoded RegB endoribonuclease functions in association with the ribosomal protein S1 to functionally inactivate early transcripts and expedite their degradation. T4 polynucleotide kinase plays a role in this process. Later, the viral factor Dmd protects middle and late mRNAs from degradation by the host RNase LS. T4 codes for a set of eight tRNAs and two small, stable RNA of unknown function that may contribute to phage virulence. Their maturation is assured by host enzymes, but one phage factor, Cef, is required for the biogenesis of some of them. The tRNA gene cluster also codes for a homing DNA endonuclease, SegB, responsible for spreading the tRNA genes to other T4-related phage.

The requirement of a DNA/RNA molecule to prime DNA synthesis imposes replication strategies to avoid the lost of the genetic information contained at the 5′-end of the lagging strand. Double-stranded (ds) DNA phages have developed different mechanisms to overcome such a problem. In most cases, phages produce head–tail concatemers, dependent on the presence of terminal redundancies, as in phages T4, T7, and SPP1, or on the circularization and further rolling circle replication, as it occurs in phage λ. Other phages, as ϕ29, have evolved to use a protein to prime DNA synthesis from each end, the priming protein becoming covalently linked to the 5′-ends of the genome. In other cases, as bacteriophage Mu, dsDNA replication depends on its capacity to be integrated into the host genome, replicating as a transposable element, yielding multiple copies of the viral genome at random locations of the bacterial chromosome. The 5′-replication quandary does not exist in circular single-stranded DNA phages, as ϕX174, which is replicated by a looped rolling circle to produce circular unit length genomes. In the case of ss- and dsRNA phages no such a problem exists since there is specific recognition by the RNA polymerase of the 3′-end of the template RNA.

The bacteriophage P22–based challenge system is a sophisticated genetic tool for the characterization of sequence‐specific recognition of DNA and RNA in vivo. The construction of challenge phage follows simple phage lysate preparations and detection of constructs by positive selection methods for plaques on selective strains. The challenge phage system is a powerful tool for the characterization of protein–DNA and protein–RNA interactions in vivo. The challenge phage has been further developed to characterize the interactions of multiple proteins in heteromultimeric complexes that are required for DNA binding. Under appropriate conditions, expression of the ant gene determines the lysis–lysogeny decision of P22. This provides a positive selection for and against DNA binding: repression of ant can be selected by requiring growth of lysogens, and mutants that cannot repress ant can be selected by requiring lytic growth of the phage. Thus, placing ant gene expression under the control of a specific DNA–protein interaction provides very strong genetic selections for regulatory mutations in the DNA‐binding protein and DNA‐binding site that either increase or decrease the apparent strength of a DNA–protein interaction in vivo. Furthermore, the challenge phage contains a kanamycin‐resistance element that can be used to either directly select for lysogeny or to determine the frequency of lysogeny for a given protein–DNA interaction to measure the efficiency of DNA binding in vivo. Selection for lysogeny can be used to isolate DNA‐binding proteins with altered or enhanced DNA‐binding specificities. The challenge phage selection provides a general method for identifying critical residues involved in DNA–protein interactions. Challenge phage selections have been used to genetically dissect many different prokaryotic and eukaryotic DNA‐binding interactions.

Phage display is a process in which large libraries of protein or peptide variants are prepared as fusion proteins with one of several coat proteins on the surface of bacteriophage. Because each phage-displayed variant is both displayed on the surface of the phage particle (virion) and encoded by the DNA packaged within that virion, phage display provides a useful means of purifying rare members of the library with specifically selected binding properties, amplifying the selected members over multiple rounds of selection, determining the sequence of the selected protein, and expressing the protein as a soluble product.

Publisher Summary This chapter examines the various aspects of single-stranded DNA family shuffling. DNA shuffling is a technique that allows accelerated and directed protein evolution in vitro . This technique has been shown to be powerful in combining independently isolated mutations of a gene into a single progeny. By using the sense strand ssDNA of one gene and the antisense-strand ssDNA of another gene as starting materials for DNA family shuffling, high recombination efficiency can be expected, as only heteroduplex molecules are formed in the first round of polymerase chain reaction (PCR). Single-stranded DNAs can be prepared by cloning into a phagemid vector such as pBluescript. The phagemid replicates as a double-stranded plasmid in an Escherichia coli host. The PCR procedure used for ssDNA shuffling is basically carried out in two steps. The first PCR step is conducted without primers in order to reassemble the DNase I-digested fragments into longer pieces. It is suggested that an effective DNA shuffling method combined with a high-throughput screening method will provide the technical solutions for engineering proteins in basic and applied scientific applications.

Bacteriophages are used as the object of study to understand gene regulation. They are also used as vectors in recombinant DNA research. This chapter covers the calculations needed to determine phage titer, the number of infecting phage attacking each cell, the probability that any one cell will be infected (by using Poisson distribution analysis), and burst size.

Publisher Summary This chapter describes a recent in vitro strategy developed in the laboratory using the receptor for ferrichrome-iron in Escherichia coli (FhuA)–reconstituted liposomes as a simple system for analyzing phage DNA delivery into liposomes. Different methods to analyze DNA transfer with a special emphasis on cryotransmission electron microscopy, which has been demonstrated as a powerful tool, is also described. Receptor-containing proteoliposomes also represent an attractive model system to study the mechanism of DNA condensation and play a role as alternative gene vectors. Infection of the host cell by different kinds of bacteriophages mostly follows a similar scheme and occurs in three steps: (1) the phage binds reversibly to the cell surface, (2) then it recognizes and binds irreversibly to an outer membrane receptor, and (3) then, following binding to its receptor, the phage releases its DNA, which crosses the bacterial envelope linearly, base pair after base pair. Some phages require an energized membrane and/or additional components for DNA release, whereas for others the interaction with their cognate receptors is sufficient to trigger DNA ejection. Therefore, reconstituting the membrane receptor FhuA into liposomes allows in vitro analysis of the mechanism of phage infection and the subsequent transport of the phage genome into lipid vesicles.

Publisher Summary This chapter focuses on the application of random peptide phage display to the study of nuclear hormone receptors. The chapter is organized into four sections: (1) the construction of a peptide phage display library, (2) a step-by-step protocol for biopanning (the affinity selection process), (3) the discussion of a mammalian cell-based screening technology to validate and complement the in vitro phage display approach, and (4) the use of phage display selected peptides as antagonists of nuclear receptor function. The selection of peptides that bind to the target proteins of interest is achieved by incubating phage libraries containing small random peptide inserts with the target proteins that are immobilized on a solid support. Phage display has been widely used as a method to map protein–protein interactions, identify peptide ligands for cell surface receptors, and map antibody–antigen epitopes. By using phage display, random peptide libraries can be generated and specific target-interacting peptides can be identified. The random peptide libraries are created by inserting short random oligonucleotides within the coding sequence of the M13 bacteriophage capsid protein, with the subsequent display of random peptides on the outer surface of the phage. In addition to using phage display to study receptor conformation and SERM pharmacology, this powerful technique has been used to develop highly specific peptide antagonists that block receptor function by interfering with required protein–protein interactions. A very good alternative to immobilizing steroid–nuclear hormone receptors and other DNA-binding transcription factors is by using biotinylated oligonucleotides (corresponding to their cognate response element) pre-captured on streptavidin-coated plates.

Publisher Summary Lysogeny and transduction describes a type of phage/host interaction and a method of bacterial gene transfer (procaryotic sex), respectively. This chapter describes methods that have been found useful in studying lysogeny and transduction in the marine environment. Lysogeny occurs when a phage enters into a stable symbiosis with its host. The host (bacterium or algal cell) and phage capable of entering into such a relationship are termed a “lysogen and temperate phage,” respectively. The temperate phage genome becomes integrated into one of the replicons of the cell (chromosome, plasmid, or another temperate phage genome) and is termed a “prophage.” In transduction, the genes are originated in a bacterial host and are not a normal part of the phage genome. The detection of lysogeny in cultures or natural populations is usually through prophage induction by use of a mutagenic agent, usually mitomycin C. The methods described are all based on some derivative of this procedure. Bacteriophage-mediated transduction is one of three well-known mechanisms, along with conjugation and transformation, of horizontal gene transfer among prokaryotic organisms. In transduction, bacterial DNA or plasmid DNA is encapsulated into phage particles during lytic replication of the phage in the donor cell and is transferred to the recipient cell by infection. This donor DNA either undergoes recombination with the host chromosome to produce a stable transductant or remains extrachromosomal as a plasmid.

Publisher Summary This chapter presents the design and discusses the use of phage display libraries for the selection of antibodies and enzymes. Phage antibody technology—that is, the display and use of antibody repertoires on the surface of bacteriophages—is a simple and inexpensive methodology that reliably yields specific monoclonal antibody fragments in 1–2 weeks of limited experimental work provided that a small amount of pure antigen is available. The attraction of phage display relies on the fact that a polypeptide can be displayed as fusion protein on the phage surface by inserting the gene coding for the polypeptide in the phage genome. On purification of a phage particle by virtue of the binding specificity displayed on its surface from a large repertoire of phage particles, the genetic information coding for the binding protein can be isolated and can amplify the corresponding phage by bacterial infection. Phage remains infective when treated with acids, bases, denaturants, and even proteases, allowing a variety of selective elution protocols. Several rounds of selections can be performed. As a consequence, even very rare binding specificities present in large repertoires can be selected and amplified from a background of phages with irrelevant binding specificities.

Publisher Summary This chapter describes a selection technology related to phage display, the selectively infective phage (SIP) technology. In both SIP and phage display, libraries of proteins or peptides of interest are displayed on the tip of a filamentous phage fused to the gene 3 protein (g3p). However, in contrast to the conventional phage display in which separation of binders with an immobilized ligand is required, in SIP technology the selection step is carried out in solution and directly coupled to the infection event. Therefore, no elution of binders is ever necessary, and incomplete elution of the most tightly binding molecules is not a limitation. This is achieved by taking an advantage of the modular structure of g3p, which occurs probably in five copies on the tip of the filamentous phage and consists of the N-terminal domains N1 [68 amino acid (aa)], N2 (131 aa), and the C-terminal domain CT (150 aa), connected by the glycine-rich linkers G1 (18 aa) and G2 (39 aa). However, this system, which is more powerful but also less controllable, can be used only with proteins and peptides for both partners. This chapter provides several general protocols that apply for in vitro and in vivo SIP and compares in more detail the use of both methods by the means of a few examples. Two other names, direct rescue interaction (DIRE) and selection and amplification of phage (SAP), have also been used for the SIP technology.

This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10 13 pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage 3 . Since the synthetic VIII fusion gene can be customized, this system should provide the flexibility required to construct phage libraries displaying a variety of different peptides and proteins and to select variants possessing the highest affinity for target molecules of a diverse chemical nature.

This chapter discusses the requirements for a substrate phage experiment illustrated by reference to protease substrate phage. Substrate phage is a powerful adaptation of phage display which makes it possible to determine substrate specificity by screening peptide phage libraries. The method has been successfully applied to determining the substrate specificity of several different proteases but could also be applied to determining peptide sequence specificity for a wide variety of peptide post-translational modifications. In the case of protease substrate phage, a randomized peptide coding sequence is inserted between an affinity domain and a truncated form of M 13 gene III. For protease substrates, in vitro modification involves simply cleaving the peptide substrates by incubation with protease under appropriate conditions. This selection may be employed to generate information on both protease-sensitive and protease-resistant sequences. The amount of protease used will depend on its specificity and catalytic efficiency, and usually must be determined empirically. Following phage sorting, it is useful to perform a rapid assay to confirm that sequences isolated from the phage library are indeed good substrates. It is found that in the case of protease substrate phage, it is important that cleavage occurs preferentially in the linker between the affinity domain and gene III.

This chapter describes screening phage-displayed random peptide libraries. Screening of phage-displayed random peptide libraries represents a powerful means of identifying peptide ligands for targets of interest. Fundamentally, the screening of phage-displayed random peptide libraries for binding peptides includes the steps such as obtaining a suitable library and source of target, and incubating the library with target and capturing binding phage. It is found that in addition to being used as a vector for accessible expression of peptide libraries, M 13 has been used to display mutant libraries of entire proteins, including antibodies and growth hormone. To minimize problems arising from instability of phage-displayed peptides, phage should be protected from proteolytic degradation. Displayed peptides are susceptible to degradation during phage propagation in a bacterial culture. Elution of phage from the target:phage complex is an important aspect of screening phage-displayed random peptide libraries. One round of affinity purification typically affects the recovery of approximately 1% of PFU representing any given binding clone. The number of phage particles representing any given binding clone that are recovered from the first round is reduced to the point that binding clones may be lost if subjected to a second round of purification without intervening amplification. It is found that enrichment of binding phage may be assessed by doping a library aliquot with an equal number of nonbinding phage which is distinguishable from library phage.

This chapter discusses various aspects of multicombinatorial libraries and combinatorial infection. The antibody phage display can provide direct and easy access to human antibodies and the possibility of generating anti-self antibodies. The lox -Cre system of bacteriophage P1 has already been tested for the assembly of two different vectors. Using this approach, antibody libraries with a size more representative of the natural immune repertoire have been obtained. A circular form of bacteriophage h integrates into the bacterial chromosome during the establishment of lysogeny. Two sequences are involved in intermolecular recombination, a 240-bp phage-encoded attP-specific site and a 23-bp attB site in the bacterial chromosome. Upon recombination, one can obtain a new phagemid (pM835) carrying both the antibody chains or encoded peptides and characterized by the functional association of the cat resistance gene with its promoter. Different aspects of the recombination process were successively analyzed, and the functionality was assessed in terms of recombination efficiency, infectivity, and ability to express soluble molecules. The att system has been proven to be a very efficient means of inducing the irreversible association between two vector partners. It is suggested that the association of variable polypeptides encoded on two different vectors gives access to unexpected experiments such as the improvement of binding affinity of heterodimeric receptors, and many other experiments involving the coexpression of two polymorphic molecules.

This chapter discusses a cDNA cloning system based on filamentous phage. Established cDNA cloning systems make use of a variety of vectors and screening methods to isolate clones encoding genes of interest. It is found that in the pJuFo phage system both the cDNA and its product can be selected by a specific ligand and the phage amplified in E. coli . For the construction of pJuFo, the phagemid pComb3 was used as a starting vector. The pComb3 vector contains two lac -promoter/operators. Each promoter is followed by the pel B leader/signal sequence. The cloning sites chosen for the construction of libraries in pJuFo are those restriction enzymes which infrequently cleave eukaryotic genes. The restriction sites BglII/XbaI/KpnI were selected to facilitate the transfer of cDNA libraries from kZap vectors to pJuFo. The most important consideration of phage screening using the pJuFo system is that the ligand used to screen the phage is tagged or immobilized in such a way that the ligand is still biologically functional. It is mandatory to test the bioactivity of the ligand after immobilization or tagging. The most attractive feature of the pJuFo cDNA cloning system is without doubt the fact that the recombinant cDNA product and the corresponding cDNA are physically linked and packed as one entity into a filamentous phage particle. It is found that the pJuFo system appears to be approximately 10 10 times more sensitive than established cDNA cloning systems.

Publisher Summary This chapter is divided into two main sections. The first describes the various aspects of displaying RNA- binding proteins (BPs) on filamentous phage: assessing and optimizing export, the production and purification of fusion phage, the preparation of the RNA target, and the selective binding of fusion phage. The second section describes how to use phage display for the in vitro genetic analysis of RNA-BPs: the synthesis of a combinatorial phagemid library, the production of the corresponding fusion phage library, and the genetic selection of RNA-BP variants, with certain binding properties. In both sections, the studies of the U1A protein are used to illustrate these methods. The U1A protein is a component of the U1 small nuclear ribonucleo-protein particle that forms a part of the spliceosome. It is a representative of the largest family of RNA-BPs that binds RNA through an RNA recognition motif (RRM) domain. The various RRM domains of this protein family enable the different family members to bind to a wide variety of RNA sequences and structures, with a high degree of specificity. The U1A protein contains two RRM domains, the N-terminal of which is responsible for binding the protein to hairpin II of U1 RNA (UlhpII). This chapter describes the display of the U1A N-terminal RRM domain on phage, the construction of a combinatorial library of fusion phage displaying U1A mutants, and the selection of U1A variants that bind tightly to U1hpII RNA.

Publisher Summary A variety of vectors have been used for the presentation of antibodies on the phage surface. These include phage vectors that encode the pill fusion and all the functions required for replication, packaging, and infection of bacteria and phagemid vectors that require “rescue” with a helper phage. Phagemid vectors comprise the pill fusion, plasmid, and phage origins of replication and antibiotic resistance markers; the helper phage provides the other functions for single-stranded replication and packaging. Helper phages are poorly packaged, in competition with phagemids, due to a defective origin (Vieira and Messing). This chapter describes the protocols suitable for helper VCS M13 (Stratagene, La Jolla, CA) and M13 K07, both of which confer kanamycin resistance on their hosts. Because the phagemid pIII fusion competes with helper phage pill for incorporation into the phage coat, majority of the phage particles display none or only one antibody molecule. Proteolytic cleavage of antibody, from the surface of phage, also reduces the number of antibody fragments displayed.

This chapter discusses various aspects of phagemid-displayed peptide libraries. The phagemid system has been used in bacteriophage display libraries for the purpose of presenting a modified bacteriophage protein. Employing the phagemid approach, a phage display system is designed that utilized phagemid technology and in vitro mutagenesis to generate libraries of random peptides on the surface of bacteriophage. The advantages of this system include the ability to control the expression level of peptides on the phage surface through induction of the lac promoter, facile, large-scale production of ssDNA, which serves as a universal template from which many different peptide libraries can be prepared, and elimination of restriction digests during preparation of libraries. A final modification in the pill.PEP vector was introduced by the insertion of a Not l linker, which replaces the spacer and adds a unique Not l restriction site between the Xho l and Spe l sites. The single-stranded, uracil-containing DNA packaged in the phagemid particles serves as the template for second-strand synthesis. The random oligonucleotide pool is used collectively to prime second-strand synthesis. It is found that the synthesis reaction is purified and restricted with Not I in the appropriate digestion buffer.

Publisher Summary Recently developed techniques allow the isolation of peptide ligands for any of a variety of receptor molecules. The most widely used technique for constructing and screening very large peptide libraries involves the display on the surface of filamentous phage. Phage libraries of greater than 1010 different compounds can be screened efficiently, because the phage particle makes a connection between the peptide, fused to a coat protein on the outside of the virion, and the genetic material, encoding that the peptide contained within the virion. This connection allows the isolation of the phage of interest through the binding properties of the peptides, and then the subsequent identification of the peptides through the ability of the genetic material to replicate and yield sequence information. This chapter describes the protocols for the construction and screening of peptides-on-plasmids libraries and for initial characterization of the resulting clones. The protocols that are used here have improved somewhat since the publication of the original paper, describing the technique. The peptides-on-plasmids method is technically more demanding than phage library screening. The procedures described in the original paper and those described here allow efficient enrichment of the receptor-specific peptides.

This chapter describes various aspects of the biology of the filamentous bacteriophage. A number of filamentous phages have been identified which are able to infect a variety of gram negative bacteria. They have a single-stranded, covalently closed DNA genome which is encased in a long cylinder approximately 7 nm wide and by 900 to 2000 nm in length. The genome of the Ff phage is a single-stranded covalently closed DNA molecule. Dimers of pV bind single-strand DNA in a highly cooperative manner. It is this binding that prevents the conversion of the newly synthesized single-stranded DNA. The products of the next group of genes, III, VI, VII, VIII, and IX, constitute the capsid proteins of the phage particle. All reside in the cytoplasmic membrane until they are assembled into a phage particle. The product of gene IV is synthesized with a 21-residue amino-terminal signal sequence. It is secreted across the cytoplasmic membrane in a manner dependent on the Sec system of the host. Polyphage produced in the absence of pVI is 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 pIII.

This chapter discusses various aspects of phage display. Phage display is proving to be a very powerful means of selecting specific binders in a library from nonbinders or low-affinity binding clones. The efficiency of the system in recovering rare binders from repertoires is affected by a number of parameters including the affinity of the displayed clone and the display level which is achieved on phage particles. Clones that are well expressed have a selective advantage. Phage vectors give greater display levels and binding characteristics than phagemid vectors rescued with wild-type helper virus such as VCSM 13 helper phage. It is found that regardless of which system is used, there can be a significant decrease in display level by proteolysis of the fusion. Incubation of low amounts of binding phage with immobilized antigen reveals that up to 34% of these particles are capable of binding and infecting upon recovery. As the input is increased, however, there is a reduction in the proportion binding. One consequence of the reduced binding with richer mixes is that there will be a significant proportion of binders which have been unable to associate during the binding step. It is suggested that depletion of unwanted binders by prior deselection using immobilized antigen can be an inefficient process.

Publisher Summary A “fusion phage” is a filamentous virion, which displays a foreign peptide fused to a coat protein and harbors the gene for the fusion protein within its genome. This chapter discusses the basic method of fusion-phage technology and describes an application for which these surface expression vectors are particularly well suited—the construction of epitope libraries. In such a library, the phages display “random” foreign peptides encoded by degenerate synthetic oligonucleotides spliced into the coat protein gene, the library as a whole representing up to billions of peptide sequences. If a phage displays a peptide that is a strong ligand for an antibody or other binding protein, it can be readily purified out of a library, even one containing a vast excess of nonbinding clones. This chapter further describes complex libraries, in which the displayed ligands are whole-folded domains. A recombinant coat protein gene can be transplanted from the phage genome into a type of plasmid called a phagemid. Phagemids contain the filamentous phage intergenic region comprising the origins of plus- and minus-strand synthesis and all other cis-acting elements needed for the synthesis of ssDNA and packaging it into virions.

Publisher Summary Lambda ZAP vectors are designed to combine specific features from the λ phage and filamentous phage and plasmid vectors to produce a single cloning vector, which is optimized for efficiency, ease of use, and versatility. Although primarily developed for the construction of complementary DNA (cDNA) libraries, they have also proven uses for genomic, libraries, antibody expression libraries, eukaryotic expression libraries, and subtraction libraries. The primary distinguishing features of these vectors is the ability to convert the DNA insert within the vector from λ phage to phagemid without the requirement for subcloning procedures. This greatly increases the rate at which DNA clones can be characterized. There is a variety of potential approaches that can accomplish the excision of a plasmid molecule positioned within a λ vector. The simplest is the insertion of rare restriction sites flanking the plasmid vector. Following isolation of the desired λ clone, the phage can be grown and then cut with the appropriate restriction sites and religated under conditions permitting circularization of the plasmid vector. This DNA can then be transformed into competent Escherichia coli or other bacterial cells. The partial filamentous phage origins used in λ ZAP to mediate in vivo excision are also present in the excised piasmid portion to allow for eventual isolation of single-stranded DNA from the excised phagemid.

Publisher Summary Cloning of complementary DNA (cDNA) has become an indispensable tool in molecular biology for the analysis of gene expression. Directional cloning of the cDNA is superior to bidirectional cDNA libraries for a variety of applications, including the preparation of strand-specific probes, expression of cDNA inserts, or subtractive hybridization. Various strategies have been used to generate directional cDNA libraries. To produce complex cDNA libraries, these methods generally require cloning into bacteriophage λ vector. This chapter describes a strategy that allows the construction of highly complex, directional cDNA libraries directly into a phagemid vector, thereby circumventing the limitations imposed by cloning into bacteriophage λ. Vector DNA is prepared for cloning by digestion with SfiI and is subsequently purified. The preparation of the bacteria for electroporation is much less complex than any other method available for high-efficiency transformation. Phagemid-based cloning system is more efficient than λ-based methods. Like other methods, this has the disadvantage of requiring cleavage of the cDNA product with a restriction endonuclease.

Publisher Summary pBluescriptll phagemid vectors are multifunctional cloning vectors designed to simplify and expedite gene cloning and analysis. The vectors contain polylinkers that are designed to optimize a number of cloning procedures including directional cloning, gene mapping, unidirectional deletions of predictable length, T3 and T7 polymerase-mediated transcription, blue/white color selection for clones with insert, double-stranded DNA sequencing, and β-galactosidase fusion protein expression. pBluescript vectors contain an ampicillin resistance gene. pBluescript vectors replicate at a high copy number owing to the lack of the pBR322 rom gene. The versatility of pBluescript vectors alleviates the need for successive subcloning. With 21 restriction sites, in two orientations, most cloned genes can be inserted directionally into the pBluescript polylinkers. Polymerase chain reaction-amplified fragments from RNA, cloned DNA, or genomic DNA can be inserted directly into pBluescript vectors, bypassing the need to construct large libraries. The restriction enzyme sites in the pBluescript polylinkers are arranged in a manner that allows unidirectional deletions of inserts with exonuclease III.

Publisher Summary Oligonucleotide-directed mutagenesis allows the introduction of almost any precisely defined mutation into a cloned, sequenced gene. The mutation may comprise single or multiple mismatches or it may involve the insertion or deletion of a large number of bases. The chapter describes a number of methods for the efficient production of site-directed mutations. The gapped duplex, uracil-containing template, and coupled primer approaches have all been used to improve the basic method described in detail. However, these methods are limited to protocols using single-stranded vectors, involve the transfection of heteroduplex DNA, and very often require plaque purification steps. The original phosphorothioate-based mutagenesis method has undergone a number of improvements and has several advantages over the other methods addressed in the chapter. Finally, the phosphorothioate approach is not limited to the use of single-stranded or phagemid DNA but has been extended to double-stranded DNA vectors.

Publisher Summary This chapter describes a comprehensive review of the recent advances in this newly recognized field. It should be noted that the concepts discussed here extrapolate well to nonpeptide strategies. The mass screening of collections of diversity is a major topic unto itself. Around 1984, the field of chemically generated molecular diversity began when Geysen demonstrated that peptides could be synthesized in a reusable form in number orders of magnitude greater than before. The format allows 96 individual peptides (or mixtures) to be synthesized simultaneously at the ends of the rods. The technique utilizes conventional solid phase peptide synthesis (SPPS) methods, readily available materials and instrumentation, and has been used, for example, to synthesize more than 3,000 peptides in a week. The development of a method to prepare large numbers of peptides rapidly led naturally to strategies to map peptide epitopes. An epitope can be classified as both continuous and discontinuous. The combination of epitope mapping, with other structure–activity relationship (SAR) methods, made possible by the multi-pin or alternative MPS methods, has been used to enhance the tumor necrosis factor (TNF) activity. Powerful biological methods have been described for the generation of peptide libraries displayed on the surface of a bacteriophage or phagemid. Such libraries contain tens to hundreds of millions of filamentous phage clones, each displaying a unique peptide sequence on the phage surface. The amino acid sequences of binding peptides displayed by the phage are identified by sequencing the corresponding coding region of the viral DNA. Millions, approaching billions, of peptides can be surveyed rapidly for binding. Ideally, a range of peptide ligands are identified that recognize all the available sites in and around a binding pocket. The combination of chemical and biological diversity techniques has been contemplated for various purposes. For example, encoded combinatorial chemical libraries have been proposed as a means of identifying compounds bound to the receptors.

Publisher Summary This chapter discusses a strategy that allows the construction of highly complex (> 10 7 members), directional complementary DNA (cDNA) libraries directly into a phagemid vector, thereby circumventing the limitations imposed by cloning into bacteriophage λ. Cloning of cDNA has become an indispensable tool in molecular biology for the analysis of gene expression. Directional cloning is necessary for a variety of applications; expression cloning, antisense inhibition of gene function, and subtractive hybridization. Other methods have been described for directional cDNA cloning, but these are often technically difficult, have inserts that are not removable intact, or require cloning into phage λ. The procedure outlined above generally circumvents these problems. Preparation of the vector DNA requires only digestion with Sill and dephosphorylation; no critical tailing steps are involved. The preparation of the cDNA for cloning is straightforward and can be accomplished with commercial reagents except for the three oligonucleotides, which can readily be obtained from custom synthesis services. The preparation of the bacteria for electroporation is much less complex than any other method available for high-efficiency transformation.

Publisher Summary pBluescript vectors are multifunctional vectors. Many tests can be performed on the cloned insert with minimal effort and without requiring cumbersome subcloning steps into “specialized” vectors. Most of the features described in this chapter are available in kit format from commercial sources, which makes the experiments easier, more efficient, and reliable. pBluescriptII are phagemid vectors are multifunctional cloning vectors designed to simplify and expedite gene cloning and analysis. The vectors contain polylinkers that have been designed to optimize a number of cloning procedures, including directional cloning, gene mapping, unidirectional deletions of predictable length, T3 and T7 polymerase-mediated transcription, blue/white color selection for clones with insert, double-stranded DNA sequencing, and fl-galactosidase fusion protein expression. The fl filamentous phage origin of replication allows preparation of single-stranded DNA for mutagenesis and DNA sequencing. The ColE1 origin of replication and the ampicillin resistance gene allow propagation and selection of clones in plasmid form.

Publisher Summary The λ ZAP vector system, although originally designed for the efficient construction and characterization of complementary DNA (cDNA) clones has proved useful for a broad range of applications. With an insert capacity of over 10 kb, the phage has also been used successfully for the construction of plant and bacterial genomic libraries. The construction of genomic libraries with smaller insert size is frequently beneficial in cloning a larger percentage of the genome in instances where large DNA clones containing unusual structure or secondary modifications are unstable. The requirement for producing a larger library to compensate for smaller insert size in order to obtain genome representation is frequently offset by the speed at which λ ZAP clones can be characterized. The chapter provides information about the modified λ ZAP vector system for the expression of antibody variable regions cloned via polymerase chain reaction (PCR) amplification. The chapter also discusses the introduction of a modified λ ZAP vector into the chromosome of transgenic mice for the purpose of generating a λ shuttle vector system capable of measuring spontaneous and induced tissue specific mutation rates in whole animals. The in vivo excision features of the mutagenesis λ ZAP vector permit rapid recovery of the target gene from the h phage for sequence identification of selected mutations.

Publisher Summary This chapter discusses the special uses of λ phage for molecular cloning. Bacteriophage λ is used as a vector for cloning foreign DNA. Its chromosome is a linear DNA molecule of approximately 50 kilobases (kb), with nearly 40% of the genome being unnecessary for the propagation of the phage. The combined use of known deletions and substitutions together with the ready selection of mutants lacking particular restriction targets has led to the generation of many vectors, while the technology of packaging its DNA in vitro has promoted the efficient use of λ vectors for cloning the smallest DNA fragments to those in excess of 20 kb. The λ genome is encapsidated in an icosahedral head, which projects a tail ending in a single tail fiber that adsorbs to a receptor site in the outer membrane of the bacterium. The receptor sites, encoded by the E. coli lamB gene, are stimulated by growth in medium containing maltose and adsorption is facilitated by magnesium ions.

Publisher Summary This chapter describes protocols that enable the synthesis and functional analysis of synthetic messenger RNAs (mRNAs) in microinjected Xenopus oocytes. These procedures also address the requirements for and the ability to introduce a 5′ terminal cap structure and a 3′ poly (A) tail into synthetic mRNAs. Functional mRNAs can be synthesized in vitro from complementary DNAs (cDNAs) of interest cloned into a variety of plasmid or phagemid vectors which contain promoters recognized by the highly specific bacteriophage SP6, T3, and T7 DNA dependent RNA polymerases. The ability to generate relatively large amounts of specific transcripts in combination with the efficient expression of these synthetic mRNAs in microinjected Xenopus oocytes provides a powerful and straightforward approach to address diverse problems ranging from the function and localization of a variety of proteins to the posttranscriptional and translational regulation of maternal mRNAs. The chapter discusses choice of transcription vectors and bacteriophage DNA–dependent RNA polymerases.

Publisher Summary Bacteriophage P4 is a small temperate bacteriophage that grows lytically only in the presence of a helper bacteriophage, such as P2. Each phage carries the genes necessary to assure its own DNA replication and integration into the host chromosome, but only P2 has genes that encode proteins needed to produce phage particles and lyse the cell. P4 activates the expression of the P2 structural proteins and is encapsidated within a phage particle that is similar to the P2 particle with the exception that the capsid is smaller. P4 can also exist as an integrated prophage or as a plasmid, and exploiting this dexterity leads to many strategies for manipulating DNA in vitro and in vivo. The major advantage of P4-derived vehicles is that they permit plasmids to be transferred efficiently from cell to cell without DNA isolation and with few constraints on the genotype of bacterial strains. P2 forms plaques on many enteric bacteria, including isolates of Klebsiella , Salmonella , Serratia , Shigella , and Yersinia , and it can be used to introduce encapsidated plasmid DNA into other gram-negative bacteria such as Rhizobium and Pseudomonas .

Publisher Summary This chapter describes the biochemistry of the interaction of phage coat protein with RNA and attempt to provide a molecular understanding of its high specificity. Coat protein binding is believed to serve two functions in the life cycle of the phage: 1) it acts as a translational repressor of the replicase gene early in infection, and 2) as an initiation site of phage assembly late in infection. This interaction has been extensively a prototype of sequence specific RNA-protein interactions. It is now clear that the phage coat proteins can be considered an example of a class of RNA hairpin binding proteins that are quite common in prokaryotes and eukaryotes. In case of bacteriophage coat protein, the coat protein assembles into phage-like capsids that can be purified by differential centrifugation and ion-exchange chromatography. Most coat proteins can be successfully renatured by the transfer from storage buffer directly into a variety of neutral buffers of moderate ionic strength. In many cases, these renatured proteins are fully active in both RNA binding and capsid assembly.

Publisher Summary This chapter discusses in vivo genetic engineering with bacteriophage Mu. Bacteriophage Mu was discovered as a temperate phage, which upon lysogenization, generated mutations in the host with a high frequency. Phage Mu is a temperate phage with a linear, double-stranded DNA genome. Like other temperate phages, when Mu infects a sensitive host it can enter either the lytic cycle or the lysogenic state. The difference between Mu and other temperate phages is that the Mu genome integrates in the host chromosome whether it enters the lytic cycle or the lysogenic state. Even Mu DNA isolated from phage particles is found in an integrated state, joined to segments of the host chromosome. Mu has become the subject of study of several groups and the starting material for a series of derivatives and strategies for in vivo genetic engineering. The chapter reviews Mu biology, highlighting the particular features of phage Mu that have been crucial for the development of the different tools and techniques for in vivo engineering..

Publisher Summary The development of oligonucleotide-directed site-specific mutagenesis on single-stranded phage template has provided a powerful new technique in the study of structure-function relationships of proteins. This chapter describes a simplified procedure for oligonucleotide-directed site-specific mutagenesis on M13 single-stranded phage deoxyribo nucleic acid (DNA) templates. The rationale behind this approach is to make use of the DNA repair mechanism present in the E. coli host to repair the gapped M13 phage DNA into the covalently closed circular form before replication of the DNA molecules. The mutagenesis procedure described in this chapter provides a rapid method for the modification of cloned genes. It takes advantage of the ability of E. coli to efficiently repair gapped circular DNA molecules, to shorten the overall time of the mutagenesis, from initiation of the mutagenesis reaction to the identification of the mutant phage plaque. Although this faster method is less efficient in the conversion of parent DNA to mutant DNA molecules, the ability to use the same oligonucleotide primer as a probe to identify the mutant phage plaques effectively compensates for this inefficiency.

The restriction endonuclease Eco RI splits the phage τ imm 434 DNA at 6 sites. The smallest of the formed fragments Eco RI-G contains the operator OR and promoters PR and Prm, gene cro and N-terminal part of the gene CI of the phage 434. τ DNA portion of the fragment contains the sequences of Po promoter, the gene CII and of the gene O N-terminal part. The complete primary structure of the Eco RI-G fragment was determined. It consists of 1287 base pairs of which 361 belong to the immunity region of the phage 434. The amino-acid sequences of the phage 434 protein cro and N-terminal part of the CI repressor were deduced from the nucleotide sequence. The proposed sequences of the phage τ CII protein and of the N-terminal part of O protein was also obtained. The sequence of the phage 434 promoter-operator region is quite different from that of the phage τ.

Publisher Summary This chapter describes two series of studies of the physiology and genetics of bacteriophage fl, with implications beyond the phage itself. It discusses the regulation of filamentous phage DNA synthesis and the unexpected participation of thioredoxin in phage morphogenesis. Phages in a lytic cycle do not need to limit their overall expression— their main interest is precisely to gain maximum expression at as great an expense to the host as possible. Any control they exhibit is meant to manage their own course of development and streamline the exploitation of the hapless host. Filamentous phages (fl, M13, and fd), on the other hand, are nonlytic; instead they grow continuously as long-term parasites (8-l0) and so must consider the appropriate balance of host and phage metabolism. These phages are particularly adept at this kind of control. Thioredoxin is a heat stable, ubiquitous protein containing active-site sulfhydryl groups that readily and reversibly form a disulfide on oxidation. In addition to extensive biochemical studies, thioredoxin has also been studied by X-ray crystallographic methods. Thioredoxin is also a constituent of phage T7 DNA polymerase, an enzyme that, when isolated, contains two polypeptide chains, one is the product of T7 gene 5 and the other is thioredoxin.

The protein that forms the connector of phage φ29, p10, binds to DNA. Apparently, p10 binding is not sequence specific. Nevertheless, the presence of the terminal protein (p3) covalently attached to the ends of φ29 DNA produces a significant increase of p10 molecules bound to the DNA ends, thus suggesting a terminal protein-mediated recognition of DNA ends by the phage connector. As the p3-DNA complex is the substrate for phage φ29 DNA packaging, these results may reflect a direct implication of the phage connector in the packaging process.

DNA replication of single-stranded phage fd requires E. coli DNA polymerase III holoenzyme and E. coli DNA binding protein I. Complementary strand synthesis is initiated by E. coli RNA polymerase, and viral strand synthesis is initiated by phage fd gene 2 protein. For unwinding of the double strands, viral strand replication also requires EU coli rep protein. The initiation sites have been localized in the intergenic region of the phage. Due to their orientation the replication of the complementary strand is initiated after almost a complete round of viral strand synthesis.

Publisher Summary This chapter describes the enzyme studies of φx174 DNA rep1ication. Replication of the single-stranded DNA of phage φx174 can be divided into three stages. In stage I, the single-stranded circle is converted into a duplex replicative form (RF-I) by the action of the DNA replication enzymes of the Escherichia coli host cell. In the second stage, multiple copies of RF-I are generated using the newly synthesized RF-I as a template; this process requires two additional proteins, the phage-encoded gene A protein and host rep protein. In the final stage, further production of RF-I ceases and only the viral strand is synthesized for envelopment into the capsid of a progeny phage. This last stage requires the gene A, rep proteins and also the products of the phage genes B, D, F, G, and H. The enzymology of E. coli replication proteins employed in φxDNA replication is a guide to their action in replicating the host chromosome. The chapter discusses the six repriming proteins that are required for SS––>RF on SSB-Coated φX DNA. It also presents that protein n’ recognizes a specific sequence of φX DNA. The chapter concludes with reconstitution of the RF ­––>RF system from purified proteins.

Phage infection of minicells provides a simple system to analyze the polypeptide (or RNA) products of a phage genome. In terms of this volume it is particularly important, as it provides a means to express in vivo the fragments of DNA cloned in λ vectors.39 Phage infection of minicells, as opposed to expression of plasmids in minicells, has the advantage of a zero time and that the minicells can be handled in a variety of ways, e.g., inducing or noninducing media, different temperatures, etc., before being required to express the phage DNA. In addition it is not necessary to construct a new minicell strain for each experiment as is the case in the expression of DNA cloned on plasmids. In terms of analysis of phage development, however, it should be remembered that significant differences exist between an infected cell and an infected minicell. Minicells are generally incapable of supporting significant amounts of phage DNA replication or producing progeny phage.17,28,37,40,41