Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library.

A novel molecular analysis of genes encoding catalytic antibodies.

Among the numerous questions remaining opened about catalytic antibodies (abzymes), the understanding of the origin of the genes encoding them is of vital significance. An original statistical analysis of genes encoding abzymes is described in the present report. Results suggested that these genes display a high conservation degree with their germline counterpart and a limited number of amino acid changes. Hence, on the contrary with high-affinity antibodies, maturation process by accumulation of somatic hypermutations is not required for the catalytic function. We demonstrated that despite a weak somatic mutation rate, the physicochemical properties of mutated amino acid (AA) are predominantly dissimilar with that of the germline AA. Further, we developed a novel approach in order to analyze the nature of genes encoding catalytic antibodies. For the first time, an unexpected and significant high level expression of rare gene subgroups was noticed and emphasized. The data described in this paper would lay the foundation for future studies about origin of genes encoding catalytic antibodies.