کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1935854 | 1050676 | 2008 | 5 صفحه PDF | دانلود رایگان |

We have examined the potential of displaying a protease species in vitro using ribosome display and demonstrate specific capture on the basis of its catalytic activity. Using a model bacterial cysteine protease, sortase A (SrtA), we show that this enzyme can be functionally expressed in vitro. By overlap PCR we constructed ribosome display templates with the SrtA open reading frame fused to a C terminal glycine–serine rich flexible linker and a tether derived from eGFP. Using the broad range cysteine protease irreversible inhibitor E-64 linked to acrylic beads, we show that we can isolate SrtA ribosome display ternary complexes, and recover their encoding mRNA by RT-PCR. This recovery was lost when applied to a SrtA catalytically inactive mutant, or could be alleviated by competition with free inhibitor. This sensitive technique could be further developed to allow the screening of proteases against putative inhibitors and/or the identification of novel proteolytic species.
Journal: Biochemical and Biophysical Research Communications - Volume 370, Issue 1, 23 May 2008, Pages 77–81