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F F Clerc

Publications and source records attributed to F F Clerc.

5 recordsLinked to original sources
PubMed1994

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

Amino Acid Sequence↗
PubMed1993

Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.

Amino Acid Sequence↗
PubMed1991

Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides.

The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant. In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.

Amino Acid Sequence↗
PubMed1988

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

Algorithms↗