Nils B. Adey

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

Characterization of phage that bind plastic from phage-displayed random peptide libraries

During routine screenings of random peptide libraries displayed at the N terminus of the pIII coat protein of M13 bacteriophage, clones were isolated that bound directly to the polystyrene (PS) surface used to immobilize the target protein. The plastic-binding phage (P-b phi) bind to both unblocked plastic (PS and polyvinyl chloride, PVC) and plastic blocked with bovine serum albumin (BSA) but require non-ionic detergent to bind to plastic blocked with milk. Comparison of the P-b phi to antibody-binding phage (Ab-b phi) indicates that similar numbers of phage particles are bound, but fewer P-b phi the recovered by acid elution. Sequence determination of the displayed peptides reveals they lack amino-acid sequence similarity yet are highly enriched for the Tyr and Trp residues. However, because not all phage that display peptides rich in Tyr and Trp residues bind to plastic, and other methods of screening random peptide libraries have identified different classes of plastic-binding peptides, the relative abundance of Tyr and Trp residues should not be considered diagnostic of plastic-binding. In summary, these results help characterize one of the most common methods used to screen random peptide libraries and suggest strategies to avoid isolating P-b phi. Furthermore, while it is generally believed that proteins bind to plastic by non-specific interactions, these results show that a bias in aa composition can exist.

Nucleotide sequence of the BALB/c mouse β-globin complex

Abstract The nucleotide sequence of 55,856 base-pairs containing all seven β-globin homologous structures from chromosome 7 of the BALB/c mouse is reported. This sequence links together previously published sequences of the β-globin genes, pseudogenes and repetitive elements. Using low stringency computer searches, we found no additional β-globin homologous sequences, but did find many more long interspersed repetitive sequences (L1) than predicted by hybridization. L1 is a major component of the mouse β-globin complex with at least 15 elements comprising about 22% of the reported sequence. Most open reading frames greater than 300 base-pairs in the cluster overlap with L1 repeats or globin genes. Polypurine, polypyrimidine and alternating purine/pyrimidine tracts are not evenly dispersed throughout the complex, but they do not appear to be excluded from or restricted to particular regions. Several regions of intergenic homology were detected in dot-plot comparisons of the mouse sequence with itself and with the human β-globin sequence. The significance of these homologies is unclear, but these regions are candidates for further study in functional assays in erythroid cell lines or transgenic animals.

CHAPTER 13 – Screening Phage-Displayed Random Peptide Libraries

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.

CHAPTER 16 – Preparation of Second-Generation Phage Libraries

This chapter describes the preparation of second-generation phage libraries. Once recombinants have been characterized from a phage display library, it is often useful to construct and screen a second-generation library that displays variants of the original sequence. In making these second-generation libraries, it is helpful to mark the vector differently from the original isolated recombinant. One of the most widely used forms of recombinant DNA-based mutagenesis is known as oligonucleotide-mediated site-directed mutagenesis. An oligonucleotide is designed such that it can base-pair to a target DNA, while differing in one or more bases near the center of the oligonucleotide. A convenient means of introducing mutations at a particular site within a coding region is by cassette mutagenesis. When oligonucleotides are used as the method of introducing mutations into a cassette, the oligonucleotides can be designed several ways. The codons at the randomized positions can be synthesized as NNK or NNS, with N, K (G+T), and S (C+G) representing different combinations of incorporated bases to encode all 20 residues. In DNA shuffling, genes are broken into small, random fragments with DNase I, and then reassembled in a PCR-like reaction, but without any primers. It is suggested that the process of reassembling can be mutagenic in the absence of a proofreading polymerase, generating up to 0.7% error when 10- to 50-bp fragments are used.

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library.

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OlambdaR, WRAAV or WRXXAAAL, where +, -, O, lambda and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025-23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides.

CHAPTER 3 – Vectors for Phage Display

This chapter discusses various aspects of the vectors for phage display. A range of vectors is available for exogenous expression on the surface of bacteriophage M13 virus particles. The display sites most commonly used are within genes III or VIII, although there have been attempts at cloning in genes VII and IX. In several vectors, the displayed element is separated from the remainder of pill or pVIII, by short linkers. To reduce the level of nonrecombinants in phage display libraries, the reading frames of genes III or VIII have been intentionally destroyed in the stuffer sequence of some vectors. This has been accomplished by frame-shifting the gene when it was engineered with restriction sites. A mean for selecting for recombinant versus parental phage is to include a stop codon in the stuffer fragments of genes III or VIII. Under tetracycline selection, the fd-tet vector can be grown in bacterial cells as a plasmid, independent of phage production. This permits the propagation of engineered M 13 genomes which normally would not yield viable phage as plasmids inside bacteria. Suppressed stop codons have also been placed between displayed protein domains and domain II of pill. It is found that when such recombinants are propagated in a suppressor carrying a bacterial strain, virus particles are produced incorporating chimeric protein domain pill into the capsids. It is suggested that monovalent display can be a useful tool for the display of peptides and proteins that may not be well tolerated by M 13 in 5 or 2800 copies.

Construction of Random Peptide Libraries in Bacteriophage M13

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.

CHAPTER 4 – Microbiological Methods

This chapter discusses various aspects of microbiological methods. Most aspects of phage display require the use of basic microbiological methods. Heat sterilizes a platinum inoculating loop and it is cooled by waving the loop in the air or plunging it into sterile culture medium. The glass spreader can be sterilized by dipping it into a beaker containing 95% ethanol and then holding it in the flame of a Bunsen burner to ignite the ethanol. In some cases, lacZ carrying viral or phagemid vectors can be followed by monitoring the expression of 13-galactosidase activity. The number of plates should be equal to the number of dilutions that will be tested, although a few extra plates can be added to the incubator in order to anticipate any ad hoc increases in the size of the experiment. For many phage-display experiments, phagemid vectors are used in place of phage. One important difference between the two vector systems is that quantitation of the number of phage particles is done by monitoring their ability to form colonies in the presence of an antibiotic. In some cases, it is advantageous to purify phage from the polyethylene glycol using CsCI equilibrium gradients. It is suggested that trace amounts of PEG can interfere with the binding of phage to various targets.

CHAPTER 5 – Construction of Random Peptide Libraries in Bacteriophage M13

This chapter discusses construction of random peptide libraries in bacteriophage M I3. Bacteriophage M 13 has been adapted for the expression of diverse populations of peptides in a manner that affords the rapid purification of active peptides by affinity selection. A variety of strategies have been used to produce clonable degenerate DNA fragments that are appropriate for encoding random peptides. Synthesis of long oligonucleotides often results in a crude product containing a large fraction of contaminating n-I and smaller products. In addition to preparing an adequate quantity of vector DNA, steps should be taken to limit the number of parental clones among the recombinants in the library. Several strategies have been employed to minimize the recovery of parental clones by vector reclosure, including the use of two different restriction enzymes that produce noncompatible cohesive ends, the treatment of digested vector with alkaline phosphatase, and the gel purification of digested vector from undigested vector and parental insert fragments. Electroporation produces high efficiencies of transformation by subjecting a cell/DNA mixture to a brief but intense electrical field of exponential decay. It is suggested that because of the strong electrical fields used in this procedure, the cell/DNA mixture must be free of salt to avoid arcing during electroporation.