In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.
|Title of host publication||Protein Downstream Processing|
|Place of Publication||Totowa|
|Publication status||Published - 2014|
|Name||Methods in molecular biology|
Levisson, M., Spruijt, R. B., Winkel, I. N., Kengen, S. W. M., & van der Oost, J. (2014). Phage Display of Engineered Binding Proteins. In N. E. Labrou (Ed.), Protein Downstream Processing (Vol. 1129, pp. 211-229). (Methods in molecular biology). Humana Press. https://doi.org/10.1007/978-1-62703-977-2_19