ABSTRACT
Advances in the field of antibody engineering, and the emergence of powerful screening technology such as filamentous phage display allowed to generate fully human antibodies with high affinities against virtually any desired target from immune or even naIve human repertoires. As a result, the immunogenicity problems related to applications of non-human based recombinant antibodies as therapeutic reagents in human were bypassed. In this study, we constructed large human immunoglobulin libraries from the lymph nodes of breast carcinomas patients in two different formats of single-chain fragments of variable domains [scFv] of antibodies. The heterogeneity of the libraries were tested by restriction enzyme analysis and sequencing on DNA samples of randomly selected colonies. Functional expression of the selected scFv molecules in E. coli was demonstrated by Western blotting. Phage rescue and panning of these libraries against the candidate tumor antigens will lead to the identification of novel human scFvs for tumor detection and pave the way towards the generation of a fully human IgG with desired effector functions for possible future tumor therapy
Subject(s)
Humans , Female , Carcinoembryonic Antigen , Genetic Engineering , Peptide LibraryABSTRACT
Display of peptides on the surface of bacteria offers many new and exciting applications in biotechnology. Fimbriae is a good candidate for epitope display on the surface of bacteria. The potential of CS3 fimbriae of enterotoxigenic E. coli as a display system has been investigated. A novel cell surface display system with metal binding property was developed by using CS3 fimbriae. Short metal binding peptide, Gly-Cys- Gly-Cys-Pro- Cys- Gly- Cys- Gly as a cysteine rich peptide, was inserted into CS3 fimbriae and displayed on the surface of E. coli. Bacteria expressing hybrid pili with cysteine rich peptide could adsorb 392.5, 510 and 905 nmol of Ni2+, Cd2+ and Pb2+ per mg [dry weight] of cells, respectively, which are five-fold [nickel] and three-fold [cadmium] more than E. coli expressing native pili. Thus, expression of Cys-rich peptide enables bacteria to act as a metalloaffinity adsorbent. These results open the possibility for biosorption of heavy metal ions using engineered microorganisms
Subject(s)
Cadmium , Nickel , Escherichia coli Proteins , Metals, HeavyABSTRACT
A random 12 mers phage library was used to screen a pool of immunoglobulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELISA for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phagedisplayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen