A colorectal antigen (IOR-C2) was characterized by a monoclonal antibody produced against the colon cancer cell line SW1116. By immunohistochemical staining the antigen was abundant and strongly expressed in epithelium of normal colon whereas colorectal carcinomas showed a more variable and heterogenous reactivity to the antibody (IOR-C2). Radioimmunoprecipitates of SW1116 cell homogenates showed a 160-200 kD band in SDS gels. Physicochemical characterization indicate that at least two IOR-C2 reactive sites are present on the antigen tested and that it is mainly an 0-linked glycoprotein carbohydrate chain which can also be N-linked to the protein. The expression of IOR-C2 mimics that of the colon associated antigen (CAA) and NCC-CO-450 antigen but is distinct from these with regard to its expression in carcinomas as well as its physicochemical characteristics.

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Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.

The FK506-binding proteins (FKBPs) perform an extensive variety of functions in numerous organisms from archaea to humans. The FKBPs are distinguished by their peptidyl-prolyl cis-trans isomerase (PPIase) activity and ability to bind the immunosuppressive drugs FK506 and rapamycin. Here, we report the isolation and characterization of FKBP45, a novel member of the FKBP family obtained from U1 small nuclear RNA (snRNA) binding assays using Bombyx mori nuclear extracts. The protein, an apparent orthologue of FKBP46 from the armyworm, Spodoptera frugiperda, was found to associate with U1 stem-loop I RNA in vitro. The FKBP45 cDNA was isolated and the genomic sequence was characterized, including the positions of exon/intron junctions and consensus splice sites. Using bioinformatics, transcription factor consensus binding sites were identified and subsequent Western blotting from developing eggs indicate that FKBP45 is differentially expressed during embryogenesis. A database was assembled using more than 1,800 available FKBP amino acid sequences and pairwise sequence alignments revealed several putative FKBP45 orthologues in various species. Analysis of these sequences revealed the position of an RNA binding domain within this new protein. In addition, FKBP45 possesses similar characteristics to several potential orthologues, including the presence of bipartite nuclear localization signals (NLSs) and phosphorylation sites.

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