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Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coil cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coil BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.
Sperm proteins of marine sessile invertebrates have been extensively studied to understand the molecular basis of reproductive isolation. Apart from molecules such as bindin of sea urchins or lysin of abalone species, the acrosomal protein M7 lysin of Mytilus edulis has been analyzed. M7 lysin was found to be under positive selection, but mechanisms driving the evolution of this protein are not fully understood. To explore functional aspects, this study investigated the protein expression pattern of M7 and M6 lysin in gametes and somatic tissue of male and female M. edulis. The study employs a previously published monoclonal antibody (G26-AG8) to investigate M6 and M7 lysin protein expression, and explores expression of both genes. It is shown that these proteins and their encoding genes are expressed in gametes and somatic tissue of both sexes. This is in contrast to sea urchin bindin and abalone lysin, in which gene expression is strictly limited to males. Although future studies need to clarify the functional importance of both acrosomal proteins in male and female somatic tissue, new insights into the evolution of sperm proteins in marine sessile invertebrates are possible. This is because proteins with male-specific expression (bindin, lysin) might evolve differently than proteins with expression in both sexes (M6/M7 lysin), and the putative function of both proteins in females opens the possibility that the evolution of M6/M7 lysin is under sexual antagonistic selection, for example, mutations beneficial to the acrosomal function that are less beneficial the function in somatic tissue of females.Mol. Reprod. Dev. 79: 517-524, 2012.
The generation of antibodies with designated specificity requires cost-intensive and time-consuming screening procedures. Here we present a new method by which hybridoma cells can be selected based on the specificity of the produced antibody by the use of antigen-toxin-conjugates thus eliminating the need of a screening procedure. Initial experiments were done with methotrexate as low molecular weight toxin and fluorescein as model antigen. Methotrexate and a methotrexate-fluorescein conjugate were characterized regarding their toxicity. Afterwards the effect of the fluorescein-specific antibody B13-DE1 on the toxicity of the methotrexate-fluorescein conjugate was determined. Finally, first results showed that hybridoma cells that produce fluorescein specific antibodies are able to grow in the presence of fluorescein-toxin-conjugates.