NSTI Nanotech 2009

Chromato-panning: a chromatographic biopanning procedure for phage display libraries in supermacroporous cryogel

W. Noppe, I. Yu Galaev, B. Mattiasson, H. Deckmyn
Katholieke Universiteit Leuven Campus Kortrijk, BE

Keywords: phage display, chromato-panning, supermacroporous cryogel

Abstract:

Classical biopanning procedures used to select high affinity phages from phage display libraries are rather time consuming procedures. We designed and evaluated a more compact, milder, less time consuming procedure for phage selection by using supermacroporous cryogels. The supermacropores in the cryogel (1-100m) allow passage of particulate matter such as phages and cells. The target was covalently coupled to an epoxy-activated polyacrylamide cryogel. After blocking,the column was washed and the phage library was loaded on the column, non-bound phages were washed out. Next the column was thermostated at 37C and E.coli cells were applied for direct on-column infection by the bound phages, after which the infected E.coli cells were recovered by elution with 1M NaCl. The suspension of eluted cells was directly diluted in LB buffer to keep the viability of the E.coli cells, that were consequently out-plated on agar plates for amplification. Phages were recovered after overnight growth. When using the classical biopanning we needed 3-5 rounds to select phages with comparable affinity. In an alternative setting, we used selected high affinity phages bound themselves to the cryogel matrix as an affinity ligand for purification of the target protein from crude feedstocks
 
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