SelecTEV™ Protease
Contents
1. 100 uL Recommended Conditions for Cleavage of a Fusion Protein An example of a cleavage experiment with 10 units of SelecTEV Protease is shown below Optimization of the cleavage conditions may be necessary depending on the protein of interest While cleavage occurs optimally in the provided SelecTEV Protease Buffer at 30 C SelecTEV Protease is active between 4 30 C and pH 6 0 8 5 Note A precipitate may be observed after thawing of the 20X SelecTEV Buffer Warm to 37 C and vortex to bring back into solution prior to use 1 Add the following to a microcentrifuge tube Final Concentration Volume uL Component Amount X Fusion Protein 30 ug 5 Selec TEV 20X Buffer 1X 1 DTT 100mM 1 mM 1 SelecTEV Protease 10 U uL 10 U Y Water N A 100 Total Volume 2 Incubate the reaction at 30 C for at least 1 hour 3 Stop the reaction by adding SDS sample buffer 62 5mM Tris HCl pH 6 8 2 SDS 5 B mercaptoethanol or 0 1M DTT 25 glycerol 0 01 Bromophenol Blue and heating at 95 C for 5 minutes 4 Analyze 10 uL from step 2 by SDS PAGE gel The percent protein cleavage is determined by analyzing the amount of uncleaved protein remaining after digestion After evaluating the initial results the cleavage reaction may be optimized for your specific protein by adjusting the amount of SelecTEV Protease incubation temperature and or reaction time Removing SelecTEV2. the cleavage reaction into buffer strips metal from IMAC without DTT or EDTA before loading sample on resin column SelecTEV There may be additional Examine your substrate s amino acid sequence for Protease is motifs in your fusion protein potential TEV Protease recognition sites Mutation demonstrating non that SelecTEV Protease of these sites may be required specific cleavage recognizes as cleavage as evidenced by the sites presence of more cleavage products than expected TEV Protease References Kapust R B T zser J Fox J D Anderson D E Cherry S Copeland T D and Waugh D S 2001 Tobacco etch virus protease Mechanism of autolysis and rational design of stable mutants with wild type catalytic proficiency Prot Eng 14 993 1000 Kapust R B T zs r J Copeland T D and Waugh D S 2002a The P1 specificity of tobacco etch virus protease Biochem Biophys Res Commun 294 949 955 Lundb ck A K Van Den Berg S Hebert H Berglund H Eshaghi S 2008 Exploring the activity of tobacco etch virus protease in detergent solutions Analytical Biochemistry 382 69 71 Mohanty A K Simmons C R Wiener M C 2002 Inhibition of tobacco etch virus protease activity by detergents Protein Expression and Purification 27 109 114 Phan J Zdanov A Evdokimov A G Tropea J E Peters H P K Kapust R B Li M Wlodawer A and Waugh D S 2002 St
3. the troubleshooting table below to address your potential issue Problem Probable Cause Potential Solution SelecTEV Protease is not cleaving my fusion The cleavage recognition site is not accessible by SelecTEV Protease Modify the reaction conditions Variables may include time temperature salt concentration and or detergent level protein See references below TEV Protease Reference section for additional information on possible modifications of the reaction conditions Add a linker between the TEV cleavage site and the protein of interest Cleavage reaction is Remove inhibitory components from sample using inhibited by components in dialysis or other methods prior to cleavage the sample Possible inhibitory components include imidazole or detergents SelecTEV Binding of the SelecTEV Dialyze your protein out of imidazole containing Protease is not captured on the IMAC resin Protease to the affinity resin is inhibited by the presence of imidazole buffer during SelecTEV cleavage of your fusion protein MA157 Rev B SelecTEV Protease Problem Probable Cause Potential Solution The capacity of the resinis Test larger amounts of resin exceeded Remember to take into consideration any His by SelecTEV cleavage which will also bind to the resin tagged fragments released from the fusion protein DTT or EDTA in the sample Dilute or dialyze
4. 093 1 1 0 mL 1 0mLx5 100 mM DTT F853091 1 500uL 500 uLx5 SelecTEV Protease Description SelecTEV Protease is an improved form of Tobacco Etch Virus TEV protease that has been engineered to be more specific active and stable than the native protease SelecTEV Protease recognizes the seven amino acid sequence Glu Asn Leu Tyr Phe GinvGily and closely related sequences SelecTEV protease cleaves between the Gin and Gly residues This seven amino acid sequence is rarely found in proteins making SelecTEV Protease an ideal choice for tag removal from fusion proteins The optimal temperature for cleavage is 30 C however the enzyme is active over a wide temperature range 4 30 C and pH 6 0 8 5 to accommodate your specific protein Following digestion SelecTEV Protease is easily removed from the cleavage reaction by affinity chromatography using the polyhistidine tag at the N terminus of the protease SelecTEV Protease is purified from E coli by affinity chromatography using the polyhistidine tag and is 90 pure when visualized on an SDS PAGE gel MA157 Rev B SelecTEV Protease Unit Definition One unit of SelecTEV Protease cleaves 285 of 3 ug of control substrate in 1 hour at 30 C Unit Assay Conditions The cleavage assay is performed in 1X SelecTEV Buffer 50 mM Tris HCl pH 8 0 0 5 mM EDTA and 1 mM DTT with 10 units enzyme and 30 ug control substrate at 30 C for 1 hour ina total volume of
5. Protease after Substrate Cleavage SelecTEV Protease contains a polyhistidine tag at its N terminus After cleavage of the fusion protein you may remove SelecTEV Protease from the cleavage reaction by affinity MA157 Rev B SelecTEV Protease chromatography on an immobilized metal affinity chromatography IMAC resin such as Ni NTA Qiagen TALON Clontech or His Select Sigma Perform the binding and elution as described in the resin manufacturer s protocol The cleaved native protein will be in the flow through fractions as long as the cleaved protein does not contain a histidine tag and the protease will remain on the resin Notes e Imidazole remaining in the sample could prevent the polyhistidine tag on SelecTEV from binding to the IMAC resin Remove or dilute imidazole prior to IMAC purification e Many IMAC resins do not tolerate 1 mM DTT Dilute the sample in column binding buffer or remove DTT prior to purification SelecTEV Cleavage During Dialysis The cleavage reaction can be performed during buffer exchange by dialysis Conditions may be adjusted from those recommended above In general use 1 uL SelecTEV 10 U for every 10 ug of fusion substrate If dialysis is carried out at 4 C an overnight 16 hours reaction time is recommended SelecTEV Protease Cleavage Troubleshooting There are a variety of reasons that SelecTEV Protease may not cleave a particular substrate as expected Refer to
6. covered by other patents issued and applicable in the United States and certain other countries Because purchase of this product does not include a license to perform any patented application users of this product may be required to obtain a patent license depending upon the particular application in which the product is used It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties Limited Label License Lucigen does not encourage or support the unauthorized or unlicensed use of third party intellectual property It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties If the purchaser is not willing to accept these use limitations Lucigen Corporation is willing to accept return of the product for a full refund For information on obtaining a license to use this product for purposes other than those permitted above contact Lucigen Corporation 2905 Parmenter St Middleton WI 53562 Email Lucigen lucigen com Phone 608 831 9011 Fax 608 831 9012 The consideration paid for this product grants a Limited License to use the product pursuant to the terms set forth in this Limited Label License Academic Not for Profit and For Profit institutions acquire certain limited nontransferable rights with the purchase of this product see below By use of this product you accept the terms and conditions of the L
7. imited Label License The purchase price of this product includes limited nontransferable rights to use only the purchased amount of the product and only as described in the product s User s Manual This limited license specifically excludes manufacture of SelecTEV Protease or any derivatives thereof The buyer cannot modify this product for any purpose without express written consent of Lucigen Corporation Lucigen Corporation reserves all other rights in particular the purchaser of this product may not transfer or otherwise sell this product or its components or derivatives to a third party and no rights are conveyed to the purchaser to use the product or its components or derivatives for commercial purposes The buyer may transfer information or materials made through the employment of this product or its components to a scientific collaborator provided that such transfer is not for MA157 Rev B SelecTEV Protease commercial purposes and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for commercial purposes Commercial purposes includes any activity for which a party receives consideration and may include but is not limited to 1 use of the product or its components or derivatives in manufacturing 2 use of the product or its components or derivatives for diagnostic purposes 3 use of this
8. nnnsensnnnneen 7 Technical Support Lucigen is dedicated to the success and satisfaction of our customers Our products are tested to assure they perform as specified when used according to our recommendations It is imperative that the reagents supplied by the user are of the highest quality Please follow the instructions carefully and contact our technical service representatives if additional information is necessary We encourage you to contact us with your comments regarding the performance of our products in your applications Thank you Lucigen Technical Support Email techserv lucigen com Phone 888 575 9695 Product Guarantee Lucigen guarantees that this product will perform as specified for one year from the date of shipment Please avoid using reagents for longer than one year from receipt MA157 Rev B SelecTEV Protease Product Designations SelecTEV Protease is supplied with SelecTEV 20X Buffer and 100 mM DTT The catalog numbers are listed below Product Kit Size Catalog Number 1 000 Units 30810 1 SelecTEV Protease 5 000 Units 30810 2 Components and Storage SelecTEV Protease and the supplied components must be stored at 20 C y 5 C max 25 C min 1 SelecTEV Protease consists of the following components Component Part Number 1 000 Units 5 000 Units SelecTEV Protease 10 U uL F833167 100 uL 100 uLx 5 SelecTEV 20X Buffer F883
9. product or materials made therefrom to provide a service information or data to a third party in return for a fee or other consideration or 4 resale of the product or its components or derivatives whether or not such product or its components or derivatives are resold for use in research Lucigen Corporation will not assert a claim of infringement against the buyer of this product provided that none of this product or any of its components or any claim in the foregoing patent or patent applications was used in the manufacture of a product for commercial purposes Academic Not for Profit and For Profit institutions must obtain a separate license from Lucigen Corporation to use this product for any purpose other than those permitted above MA157 Rev B
10. ructural basis for the substrate specificity of tobacco etch virus protease J Biol Chem 277 50564 50572 Tozser J Tropea J E Cherry S Bagossi P Copeland T D Wlodawer A and Waugh D S 2005 Comparison of the substrate specificity of two potyvirus proteases FEBS J 272 514 523 Van den Berg S Lofdahl P A Hard T and Berglund H 2006 Improved solubility of TEV protease by directed evolution J Biotechnol 121 291 298 MA157 Rev B SelecTEV Protease Online resources http meli nciferf gov waugh tech fag tev pdf http www cardiff ac uk biosi staffinfo ehrmann tools Recognition htm Notice of Limited Label License Copyright Patents Warranties Disclaimers and Trademarks Copyright 2015 by Lucigen Corp All rights reserved Lucigen and SelecTEV are registered trademarks of Lucigen Corporation Lucigen s products are sold for research use only and are not to be used in humans or for medical diagnostics Lucigen s liability with respect to any SelecTEV product is limited to the replacement of the product No other warranties of any kind expressed or implied including without limitation any implied fitness for any particular use are provided by Lucigen Lucigen is not liable for any direct indirect incidental or consequential damages arising out of or in connection with the use or inability to use any of its products Some applications in which Lucigen enzymes can be used may be
11. xls Lucigen Simplifying Genomics SelecTEV Protease IMPORTANT 20 C Storage Required Immediately Upon Receipt FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE Lucigen Corporation 2905 Parmenter St Middleton WI 53562 USA Toll Free 888 575 9695 608 831 9011 FAX 608 831 9012 lucigen lucigen com www lucigen com SelecTEV Protease Table of Contents Technical SU aca EEE 2 Product DeSIQNatlONS irronneen eo cdee cee nn rene OTTE TEETE EEEE TS TERE 3 Components and ca nae 3 SelecTEV Protease Description cccccccceeeeeeeeeeeeeeeeeeeeeaeeeeeeceeeeeeeceaeeeeseaeeeesecaeeeeesenaeeeeseeieeeesesaees 3 Unit Detit sisie EE 4 Unit Assay Conditions vcd 4 Recommended Conditions for Cleavage of a Fusion Protein s2r44044H00nnnon Hrn non nnnnnnnnnnnn nn 4 Removing SelecTEV Protease after Substrate Cleavage 444u44424400nnnnnnnnnnnnnnnnnnnnnnnannnnnnnnn 4 SelecTEV Cleavage During Dialysis ccccccceeeeeeeeeeeeeneeeeeeeneeeeeeeneeeeeseaeeeeseeaeeeeseenaeeeesenieeeesenaees 5 SelecTEV Protease Cleavage Troubleshooting 4444444444000nnnnnnnonnnnnnnnnnnnnnnnnnnnnnnnannnnnnnnn 5 TEV Protease References 4444444224400nnnnnnnnnnnnnnnnnnnannnnnnnnnnnnnnnnnnnnnnnnannnnnnnnnnnnsnnnnnnnnnnnnnnnsannn nn 6 Online resources uusessessssnnsnnnnnnennnnnnnnnnnnnnnnnnnnnnnnnonnnnsonnnnnnnnnnnensonnnnsnnnnnnnnnsnnnnsnnnnnn
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