Panning and selection of proteins using ribosome display.
Eukaryotic ribosome complexes can be used as a means to display a library of proteins, and isolate specific binding reagents by screening against target molecules. Here we present, as an example, a method for the display of a library of immunoglobulin variable-like domains (VLDs) for the production of stable mRNA/ribosome/protein complexes. These complexes are produced by the addition of specific in vitro transcriptional promoter elements and translation control sequences to the template DNA. Furthermore, an appropriate spacer (anchor) domain is included for efficient folding of the nascent translated protein, which remains attached to the ribosome complex. Ribosome complexes are panned against hen egg lysozyme-conjugated magnetic beads and genes encoding specific, binding, V-like domains are recovered by RT-PCR and cloned into an Escherichia coli expression vector.
Coia
G
G
CRC for Diagnostic Technologies, CSIRO Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia. greg.coia@hsn.csiro.au
Pontes-Braz
L
L
Nuttall
S D
SD
Hudson
P J
PJ
Irving
R A
RA
D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D009113:Muramidase / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D012270:Ribosomes / Q000378:metabolism*
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011506:Proteins; D009113:Muramidase