ADAM-HCV, a new-concept diagnostic assay for antibodies to hepatitis C virus in serum.

We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides. Peptides that mimic several immunodominant epitopes of the virus were used to develop a novel type of diagnostic assay which efficiently detects antibodies to HCV in serum. This type of analysis provides a conclusive diagnosis for many patients identified as indeterminate according to presently available serological assays.

'In vitro evolution' of ligands for HCV-specific serum antibodies.

We developed a strategy to improve the properties of ligands selected from phage-displayed random peptide libraries. A site-directed mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties. We refer to this strategy as in vitro evolution' of ligands. Here we report the application of this in vitro evolution protocol to the identification of improved ligands for HCV-specific serum antibodies. A single clone or population of clones were processed to generate a secondary library. Screening of these libraries with sera from HCV-infected patients identified peptides with an enhanced and broadened ability to detect HCV-specific serum antibodies.

DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.

Factors limiting display of foreign peptides on the major coat protein of filamentous bacteriophage capsids and a potential role for leader peptidase.

Many small peptides can be displayed on every copy of the major coat protein in recombinant filamentous bacteriophages but larger peptides can only be accommodated in hybrid virions mixed with wild-type protein subunits. A peptide insert of 12 residues capable of display at high copy number in a hybrid virion was found to be incapable of supporting recombinant virion assembly, a defect that could not be overcome by over-expressing leader peptidase in the same Escherichia coli cell. In contrast, over-expressing leader peptidase did increase the copy number of two 9-residue peptides that were poorly incorporated into hybrid virions. The factors that limit peptide display are varied and not restricted to the early stages of viral assembly.

Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction.

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH--cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.

Spinach ferredoxin i: overproduction in Escherichia coli and purification.

Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.