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Expression cloning of farnesylated proteins

Elsevier Science & Technology
DOI: 10.1016/s0076-6879(01)32202-4
  • Section Ii. Screening Analyses
  • Biology
  • Chemistry
  • Medicine


Publisher Summary The identification of novel prenylated proteins has most often involved the labeling of cultured cells with [3H] mevalonolactone that is enzymatically converted to [3H] farnesyl pyrophosphate (FPP) and [3H] geranylgeranyl pyrophosphate (GGPP) prior to being incorporated into protein. Cellular proteins can then be analyzed by gel electrophoresis and autoradiography. However, this procedure is inefficient, because of slow [3H] mevalonate uptake, and is therefore limited to the detection of abundant proteins. A second limitation of [3H] mevalonate labeling is that it does not distinguish between farnesylated and geranylgeranylated proteins. This chapter outlines an enzymatic screening protocol to identify novel farnesylated proteins from bacterial expression libraries. This method allows the systematic screening of large cDNA libraries in a manner that is quicker and simpler than the conventional approach of purifying radiolabeled proteins, obtaining protein sequence, and cloning by degenerate oligonucleotides. This technique helps in screening of human and rat retina and total mouse embryo expression libraries. With the advances in the genome initiative, particularly the availability of large numbers of expressed sequence tags (ESTs), expression cloning of novel prenylated proteins may not be the most effective approach in identifying these proteins. Instead, the biochemical characterization of ESTs containing putative consensus C-terminal prenylation motifs may prove more satisfactory. However, the extension of this expression cloning technique to additional organ systems and particularly to organisms that are currently not included in the genome initiative should allow the isolation of additional novel isoprenylated proteins.

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