Combining yeast ER retention effect and high-throughput sequencing in YESS approach for engineering Next-generation protease


Meng Mei;Yu Zhou;Chan Yu;Wenfang Peng;Chao Zhai;Guimin Zhang;Li Yi;


Proteases make up a very large protein family that has broad potential applications ranging from biomedical research to therapeutics.To expand its application,protease specificity and activity have to be engineered against specific substrates.However,limited proteases have been successfully engineered so far,mainly due to lacking of effective biological methods.Recently,a novel high-throughput screening method,named the yeast endoplasmic reticulum(ER)sequestration system(YESS),was successfully established by our group,in which exogenous protease,such as Tobacco etch virus protease(TEV-P),was successfully engineered with high activity against new and specific substrate sequences(PNAS,2013).A key feature of the YESS approach is that target protease and its substrates can be largely retained in the yeast ER through a C-terminal fused ER retention sequence,FEHDEL,facilitating the subtle proteolysis between the protease and its substrates.Inhere,the known yeast ER retention sequence,FEHDEL,was fully analyzed and engineered to obtain an artificial ER retention sequence.This engineered artificial ER retention sequence significantly enhanced the ER retaining time of the target protein when fused at its C-terminal end,which efficiently expanded the dynamic range of the YESS approach,enabling the detection of very weak proteolytic reactions caused by human Matrix metalloproteinase 7(MMP7)against its substrates in yeast cells.In addition,the YESS platform is also combined with Next-Generation sequencing(NGS)to enable a comprehensive survey of protease specificity.In this approach,a combinatorial substrate library is targeted to the yeast ER and transported through the yeast secretory pathway,interacting with any protease(s)residing in the ER.Multi-color FACS screening is used to isolate cells labeled with fluorophore-conjugated antibodies,followed by NGS to profile the cleaved substrates.Proteolysis in the yeast secretory pathway was mapped for the first time revealing a major cleavage pattern of Ali/Leu-X-Lys/Arg-Arg.The YESS-NGS method was also successfully applied to profile the sequence specificity of the wild-type and an engineered variant of the TEV-P,proving its efficiency in deciphering the substrate specificity of a target protease.Through combining the engineered ER retention sequence and NGS technology with the newly established YESS approach,certain proteases now is able to be engineered through high-throughputmanner.




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Total: 6 articles

  • [1] Li,Q;Yi,L;Marek,P;Iverson,B.L, FEBSLett, FEBS Letters (FEBS Letters)
  • [2] Yi,L;Li,Q;Taft,J.M;Gebhard,M.C;Georgiou,G;Iverson,B.L, Methods Mol.Biol,
  • [3] Yi,L;Gebhard,M.C;Li,Q;Taft,J.M;Georgiou,G;Iverson,B.L, PNAS, Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America)
  • [4] Li,Q;Yi,L;Marek,P;Iverson,B.L, FEBSLett, FEBS Letters (FEBS Letters)


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