EnterokinaseMax (EKMax ) - Thermo Fisher Scientific
Contents
1. MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes2. cleavage sequence is present in many expression vectors available from Invitrogen contact Technical Service for more information Genes cloned into the multiple cloning site of these vectors express recombinant N terminal fusion proteins The native proteins can be released from the N terminal fusion peptide or protein by digesting with enterokinase EnterokinaseMax EKMax is a specially prepared recombinant enzyme consisting of the catalytic subunit of the holoenzyme This subunit is expressed and purified from the yeast Pichia pastoris yielding an enzyme with higher specific activity This results in more efficient cleavage using less enzyme Enterokinase enteropeptidase EC 3 4 21 9 is the physiological activator of trypsinogen and a serine protease that exhibits specificity for the sequence Asp Lys Anderson et al 1977 The bovine holoenzyme is a heterodimer consisting of a 115 kDa structural subunit and a 35 kDa catalytic subunit The larger subunit acts as a membrane anchor and positions the catalytic subunit on the luminal side of the brush border membrane The catalytic subunit is homologous to other serine proteases and is inhibited by chemical modification of the serine and histidine active site residues Grant and Hermon Taylor 1977 Light and Liepnieks 1979 Maroux et al 1971 It has been proposed that the active center of enterokinase possesses a distinctive cationic subsite that binds Asp Enterokinase is
3. highly specific and tolerates very few changes to its recognition site If the ionic charge of the recognition site is preserved enterokinase will recognize the site but the rate of hydrolysis of the peptide bond will be reduced Light and Janska 1989 The four aspartyl residues act as a signal for enterokinase cleavage It has been reported that with only three aspartyl residues the rate of hydrolysis is reduced Two aspartyl residues preceding the lysyl residue are the minimum number of acidic residues needed to maintain specificity Maroux et al 1971 Non specific cleavage by enterokinase may occur in the cases described above but this is usually alleviated by reducing the amount of enzyme used EKMax is a clone of the catalytic subunit of enterokinase LaVallie et al 1993 expressed in the yeast Pichia pastoris EKMax is secreted into the medium purified and migrates at 43 kDa on an SDS PAGE gel The calculated molecular weight of the protein is 26 3 kDa but it contains three sites for asparagine linked glycosylation The apparent molecular weight of 43 kDa is consistent with previous observations LaVallie et al 1993 and is assumed to be because of N linked glycosylation EKMax Digestion Introduction Additional Materials Needed Important You will need to have pure or partially pure fusion protein This section describes how to digest the fusion protein with EKMax to cleave your protein from the fusion
4. no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose continued on next page References Anderson L E Walsh K A and Neurath H 1977 Bovine Enterokinase Purification Specificity and Some Molecular Properties Biochemistry 16 3354 3360 Barratti J Maroux S and Louvard D 1973 Effect of Ionic Strength and Calcium Ions on the Activation of Trypsinogen by Enterokinase A Modified Test for the Quantitative Evaluation of the Enzyme Biochem Biophys ACTA 321 632 638 Grant D A W and Hermon Taylor J 1977 Hydrolysis of Artificial Substrates by Enterokinase and Trypsin and the Development of a Sensitive Specific Assay for Enterokinase in Serum Biochem J 155 243 254 LaVallie E R Rehemtulla A Racie L A Diblasio E A Ferenz C Grant K L Light A and McCoy J M 1993 Cloning and Functional Expression of a cDNA Encoding the Catalytic Subunit of Bovine Enterokinase J Biol Chem 268 23311 23317 Light A and Janska H 1989 Enterokinase enteropeptidase Comparative Aspects TIBS 14 110 112 Light A and Liepnieks J J 1979 The Preparation and Purification of Bovine Enterokinase J Biol Chem 254 1677 1683 Maroux S B
5. aratti J and Desnuelle P 1971 Purification and Specificity of Porcine Enterokinase J Biol Chem 246 5031 5039 Schagger H and von Jagow G 1987 Tricine Sodium dodecyl sulfate Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa Anal Biochem 166 368 379 1998 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
6. eir respective affinity columns releasing the desired protein and leaving the fusion partner bound to the column For methods to digest Xpress fusions bound to TM TM ProBond and thioredoxin fusion proteins in situ on ThioBond contact Technical Service see page 7 continued on next page EKMax Digestion continued Obtain Purified Fusion Protein Note Dialysis of the Fusion Protein Recommendation Purify at least 120 ug of your fusion protein using your system of choice following the manufacturer s instructions e To purify thioredoxin fusion proteins refer to the ThioBond manual TM included with the ThioBond resin or contact Invitrogen for information TM TM e To purify Xpress fusion proteins refer to the ProBond Purification System manual or contact Invitrogen for information Note Both manuals may be downloaded from www invitrogen com EKMax will digest fusion proteins in crude cell lysates Note however that you will lose your fusion tag and will have to develop a separate protocol for purification of your protein TM It may be necessary to dialyze your fusion protein against 1X EKMax buffer see Recipes page 6 before digesting it with EKMax EKMax is inhibited by high ionic strength 250 mM NaCl reduces EKMax activity to 75 of normal and 2 M NaCl almost completely inhibits enzyme activity Barratti et al 1973 Also EKMax is known to be i
7. ermine the optimal units of EKMax needed for complete digestion use five different amounts of EKMax 4 units 1 unit 0 1 unit 0 01 unit and 0 001 unit For 4 units of EKMax use 4 ul of undiluted EKMax Use 1X EKMax buffer to make serial 10 fold dilutions of the enzyme 2 Set up 6 reactions including a reaction without EKMax to control for proteases in your protein solution Fusion Protein 20 ug 10X EKMax Buffer 3 ul EKMax 1 4 ul Deionized Water to 29 ul use 30 ul for the no EKMax control Final Volume 30 ul 3 Mix well and incubate at 37 C overnight 16 hours If your protein is unstable and degrades at 37 C try incubation at 22 C 16 C or 4 C It is not necessary to increase the time of digestion to compensate for the decrease in temperature 4 Prepare and load 1 20 ul on an SDS PAGE gel see next section For western blotting load 1 ug of your fusion protein and for Coomassie stained gels load 10 ug If you wish to digest your fusion protein in a crude lysate be sure to dialyze to remove any inhibitors of EKMax Set up your pilot reactions as described above TM to determine the amount of EKMax needed to digest your fusion protein Use an SDS PAGE gel that will allow you to differentiate between undigested and digested fusion protein The type of gel and concentration of acrylamide depends on the size of your fusion protein and the fusion partner Some fusion partner
8. invitrogen EnterokinaseMax EKMax Catalog nos E180 01 E180 02 Version H 16 June 2006 25 0110 11 Table of Contents Table of Contents tete un br I ie es iii Important Informa th On cias ii caves ds haces A doula lea A AAA E TE iv aa a aaa LPAR EE PENE A I EE E AEA E EA A EEE E A A A A A EA A ET isencdasisenevec 1 D aT Wanda 1 ERMAX B A E A ES 2 App dik ea naer e Ea E EES EC r EE EE EA EE E EEEE EEES TECE E E ORENEAN 6 RECIPES E E E E E o ea 6 Technical Service a T a R A R A orang spe se os so ats A ne A A A Aal 7 References N O dona O A I E E denen a ones 8 iii Important Information Shipping Storage EnterokinaseMax and the 10X EKMax reaction buffer are shipped on blue ice and should be stored at 20 C Contents EKMax in 50 mM potassium phosphate pH 8 0 500 mM NaCl and 50 glycerol is supplied as follows Catalog no E180 01 Item Amount Volume EKMax 250 units 1 unit ul 250 ul 10X EKMax Reaction Buffer 500 mM Tris HCI pH 8 0 22 C 10 mM CaCh 1 Tween 20 1 5 ml Catalog no E180 02 Item Amount Volume EKMax 1000 units 1 unit ul 1 0 ml 10X EKMax Reaction Buffer 500 mM Tris HCl pH 8 0 22 C 10 mM CaCh 1 Tween 20 3x1 5 ml Additional Materials Needed Unit Definition Limited Label License No 62 EKMax Enterokinase iv You will e 37 C need to have the following materials water bath or heat block e Deio
9. n Buffer Appendix 500 mM Tris HCI pH 8 0 10 mM CaCl 1 Tween 20 v v 1 Zo 3 4 For 1 liter dissolve 60 5 g Tris base in 950 ml deionized water Adjust pH to 8 0 with concentrated HCI Add 1 47 g CaCL 2H20 and 10 ml Tween 20 and mix Adjust the volume to 1 liter Store at room temperature Technical Service Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Limited Warranty
10. nhibited by gt 2 M urea gt 20 mM B mercaptoethanol P ME gt 0 1 SDS gt 50 mM imidazole and pH values below 6 and above 9 Use the table below to determine if you need to dialyze your fusion protein prior to digestion with EKMax If your purified protein contains gt 2 M urea gt 250 mM NaCl gt 20 mM ME 0 1 SDS or gt 50 mM imidazole dialyze to remove the inhibitors is in a buffer where the pH is lower than 6 or higher than 9 dialyze to adjust the pH to between 6 and 9 is free from inhibitors and the pH is between 6 and 9 do not dialyze Proceed straight to Preparation of Pilot Reactions below If you are not sure whether you should dialyze your protein or not dialyze a small volume of your protein solution and test both dialyzed and undialyzed samples in a pilot EKMax digestion continued on next page EKMax Digestion continued Preparation of Pilot Reactions Note SDS PAGE Analysis Recommendation for Thioredoxin Fusion Proteins You will need at least 120 ug of your fusion protein for the pilot reactions The ratio of enzyme to fusion protein to achieve complete digestion may vary depending on the protein expressed It is very important to use only the minimal amount of EKMax necessary to completely digest the fusion protein Excess TM EKMax may cause non specific cleavage of your fusion protein in some cases 1 To det
11. nized water e 1 5 ml microcentrifuge tubes e SDS PAGE apparatus and buffers see page 4 for guidelines TM e EK Away Resin and buffers Invitrogen Catalog no R180 01 e 15 ml polypropylene tubes optional e Chromatography columns optional Catalog no R640 50 e Rocker or rotator 1 unit is defined as the amount of EKMax TM that will digest 20 pg of BioEase Mogl multicopy suppressor of GSP1 1 fusion protein to 90 completion in 20 minutes at 37 C in 50 mM Tris HCl pH 8 0 1 mM CaCh and 0 1 Tween 20 1X EKMax Buffer For comparison purposes 1 Invitrogen unit of EKMax 190 trypsinogen activation units Use this conversion only as a rough estimate of how much TM EKMax to use It is important to empirically determine the optimal amount of EKMax to digest your fusion protein see page 2 This product is sold under patent license from Genetics Institute Inc for Research Use Only Licenses for commercial manufacture or use may be obtained from Genetics Institute Inc Overview Introduction Description of Enterokinase Specificity of Enterokinase Expression of the Recombinant Catalytic Subunit Methods Enterokinase is a highly specific serine protease that can be used to digest fusion proteins to release the fusion partner or tag from the desired protein The enzyme recognizes the sequence Asp Lys and cleaves after the lysine residue This
12. partner This requires setting up a series of pilot reactions to determine empirically the best conditions for digestion The efficiency of cleavage of EKMax will differ with each fusion protein Also if you are accustomed to using EK3 Biozyme it is still necessary to test different concentrations of EKMax to determine the right amount for complete digestion The table below outlines the steps needed to digest your fusion protein and obtain pure protein Stage Description 1 Obtain purified fusion protein at a concentration of gt 0 1 mg ml 2 Dialyze if necessary into 1X EKMax Buffer 3 Set up pilot reactions using different amounts of EKMax and digest overnight 4 Assay reactions on an SDS PAGE gel and analyze Optimize digestion conditions by adjusting amount of enzyme or the temperature as needed 6 Scale up digestions to produce more of your native protein Purify your native protein away from the fusion partner and EKMax You will need to have the following materials e 37 C water bath or heat block e Deionized water e 1 5 ml microcentrifuge tubes e SDS PAGE apparatus and buffers see page 4 for guidelines e EK Away Resin and buffers Invitrogen Catalog no R180 01 e 15mlsnap cap polypropylene tubes optional e Chromatography columns optional Catalog no R640 50 e Rocker or rotator We have found that EKMax will digest fusion proteins bound to th
13. s will be too small to resolve conveniently i e the Xpress tag You should be able to distinguish the removal of the fusion partner from your protein either by Coomassie staining or by using antibody detection methods Choose the dilution TM of EKMax that gives you complete digestion of your recombinant fusion protein A Tricine gradient gel may be necessary to visualize the 14 6 kDa thioredoxin fusion partner Schagger and von Jagow 1987 Alternatively a western blot using the Anti Thio Antibody Catalog no R920 25 may be used to detect the accumulation of the thioredoxin fusion partner and or subsequent loss of signal from the native protein continued on next page EKMax Digestion continued Recommendation for Xpress Fusion Proteins Optimizing EKMax Cleavage Scale Up of EKMax Reaction Removal of EKMax Removal of Fusion Partners Since the Xpress peptide is less than 4 kDa a shift in the size of your protein may be undetectable A western blot using the Anti Xpress Antibody Catalog no R910 25 or the Anti Xpress HRP Antibody Catalog no R911 25 may be necessary to visualize the cleavage and removal of the Xpress fusion peptide from fusion proteins Perform a western blot and look for the loss of the signal from your protein The Xpress peptide is so small it may not transfer well to nitrocellulose or nylon making it difficult to detect In some cases increa
14. sing the calcium chloride concentration to 10 mM or the amount of Tween 20 to 1 in the digestion reaction increases the activity of EKMax There is a possibility that EKMax may recognize sites that are similar to its recognition site however the rate of hydrolysis will be reduced This is usually TM alleviated by decreasing the amount of EKMax After you have optimized the EKMax reaction you may scale up your digestion reaction in a linear manner You may need a concentrated solution of fusion protein in order to scale up your digestion Use standard ultrafiltration methods to concentrate your protein solution After digestion EKMax may be removed by affinity chromatography on soybean trypsin inhibitor STI resin For easy removal of EKMax EK Away Resin Catalog no R180 01 is available from Invitrogen EK Away Resin consists of soybean trypsin inhibitor immobilized on 4 beaded agarose For a protocol to use EK Away Resin refer to the EK Away manual The manual is available at www invitrogen com or contacting Technical Service see page 7 Fusion partners may be removed by the same affinity resin as was used to purify the fusion protein For protocols to remove the Xpress tag or the thioredoxin fusion partner after EKMax digestion contact Technical Service see page 7 For other fusion proteins consult the manufacturer of your particular system Recipes 10X EKMax Reactio
Download Pdf Manuals
Related Search