Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties.

Phage display screening in low dielectric media.

Here we report the first application of phage display screening in low dielectric media. Two series of phage clones with affinity for alpha-chymotrypsin (CT) were selected from a Ph.D.(TM)-C7C library, using either a buffer or acetonitrile in buffer (50%, v/v). The affinity of lysates, individual clones or selected cyclic peptides for the enzyme was studied by examining their influence on CT activity. Peptides displayed on phage selected in buffer provided significant protection from enzyme autolysis resulting in marked increase in CT activity (>100%). Phage selected in ACN provided some, albeit weak, protection from the detrimental influence on CT from ACN. In conclusion, the results demonstrate the potential for the application of phage display screening protocols to targets in media of low dielectricity.

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature.

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C-D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.

Configurations of the N-terminal amphipathic domain of the membrane-bound M13 major coat protein.

The M13 major coat protein has been extensively studied in detergent-based and phospholipid model systems to elucidate its structure. This resulted in an L-shaped model structure of the protein in membranes. An amphipathic alpha-helical N-terminal arm, which is parallel to the surface of the membrane, is connected via a flexible linker to an alpha-helical transmembrane domain. In the present study, a fluorescence polarity probe or ESR spin probe is attached to the SH group of a series of N-terminal single cysteine mutants, which were reconstituted into DOPC model membranes. With ESR spectroscopy, we measured the local mobility of N-terminal positions of the protein in the membrane. This is supplemented with relative depth measurements at these positions by fluorescence spectroscopy via the wavelength of maximum emission and fluorescence quenching. Results show the existence of at least two possible configurations of the M13 amphipathic N-terminal arm on the ESR time scale. The arm is bound either to the membrane surface or in the water phase. The removal or addition of a hydrophobic membrane-anchor by site-specific mutagenesis changes the ratio between the membrane-bound and the water phase fraction.

High copy display of large proteins on phage for functional selections.

We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (K(d)>1 microM). The display of a tetrameric protein, streptavidin (approximately 50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.

The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide.

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endo-genous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group.

Complementary DNAs covering the entire RNA genome of soybean dwarf luteovirus (SDV) were cloned and sequenced. Computer analysis of the 5861 nucleotide sequence revealed five major open reading frames (ORFs) possessing conservation of sequence and organisation with known luteovirus sequences. Comparative analyses of the genome structure show that SDV shares sequence homology and features of gene organisation with barley yellow dwarf virus (PAV isolate) in the 5' half of the genome, yet is more closely related to potato leafroll virus in its 3' coding regions. In addition, SDV differs from other known luteoviruses in possessing an exceptionally long 3' terminal sequence with no apparent coding capacity. We conclude from these data that the SDV genome represents a third variant genome type in the luteovirus group.

Photobiological activity of sulphur and selenium analogues of psoralen.

Eight psoralen analogues, in which sulphur or selenium replaces one or both intracyclic oxygen atoms, were synthesized. Photoreaction with M13mp19 RF DNA in the presence and absence of oxygen (wavelength, greater than 320 nm) was studied. The damaged viral DNA was transfected into Escherichia coli and scored for infectivity towards Ca-treated wild-type E. coli. This allowed a comparative evaluation to be made of the heteropsoralens in terms of the photoreaction with DNA and the photodynamic effect. Most of the seleno- and thio-psoralens show very high photoactivity towards DNA compared with psoralen and 8-methoxypsoralen (8-MOP). Their photoreactivity is due mainly to a [2 + 2] photoreaction, since only a minor influence of molecular oxygen could be detected. Some of the studied seleno- and thio-psoralens are very efficient DNA photoinactivating agents and show great promise in photochemotherapy.

Cloning of several cDNA segments coding for human liver proteins.

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.