Navigation Links
Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini

Recombinant Enterokinase for highly specific digestion of purified proteins or proteins in crude extracts

Ed Marsh * Johanna Claycomb * ChaoFeng Zheng * Denise Wyborski * John C. Bauer * Joseph A. Sorge * A.K. Bernardino * Peter Vaillancourt
Stratagene Cloning Systems, Inc.

Stratagene introduces recombinant Enterokinase, an efficient, sitespecific protease that cleaves at the carboxy terminus of its recognition target. Cleavage of fusion proteins that contain the enterokinase (EK) recognition sequence positioned between an Nterminal purification tag and the Cterminal polypeptide of interest results in Cterminal fusion partners that have no extraneous amino acids derived from the cleavage site. Thus, recombinant proteins with native amino acid sequence are produced. Stratagene's purified Enterokinase is free of contaminating proteases, functions efficiently over a wide range of reaction conditions and is provided with Soybean Trypsin Inhibitor Agarose (STI Agarose) for rapid and quantitative removal of EK from digests.

figure 1

Site-specific proteases and their cognate target amino acid sequences are often used to remove the fusion protein tags that facilitate enhanced expression, solubility, secretion or purification of the fusion protein. The serine protease enterokinase (EK) is particularly attractive because it cleaves after the C-terminal amino acid of its recognition site, (aspartic acid)4lysine. This cleavage produces fusion proteins that have native amino termini following removal of N-terminal tags by cleaving with EK (figure 1). In the past, the use of EK for fusion protein cleavage was hampered by the generally poor quality of the commercially available enzyme preparations that had been purified from native sources. Such enzyme preparations exhibited low specific activity and contamination with other less specific serine proteases, which often resulted in degradation of target proteins.

Recently, the catalytic subunit of bovine EK has been cloned and expressed in E. coli and purified to homogeneity.1, 2 Stratagene's Enterokinase has high specific activity and is virtually free of contaminating proteases. Purified recombinant Enterokinase is highly active over wide ranges in pH (pH 4.5-9.5) and temperature (4-45C) and efficiently cleaves fusion proteins in partially purified protein preparations.2

Cleavage of CBP-JNK with Enterokinase

figure 2

Coding sequence for the protein c-Jun N-terminal kinase (JNK)3 was PCR amplified using a 5'-oligonucleotide primer designed to fuse the 5-amino-acid EK target site to the natural amino terminus of the JNK protein. The PCR product was inserted into the affinity pCALnEK expression vector4 such that the EK target peptide-JNK fusion protein was fused in frame, downstream of the calmodulin-binding peptide (CBP) purification tag. The resulting fusion protein, CBP-JNK, was expressed in Epicurian Coli BL21(DE3) cells and purified from crude cell lysates to apparent homogeneity, as determined by visual inspection of silver-stained PAGE gels after a single pass over calmodulin (CaM) affinity resin4 (data not shown). A series of reactions containing various amounts of recombinant EK was incubated at room temperature for 21 hours, and portions of the reactions were analyzed by SDS-polyacrylamide gel electrophoresis ((SDS-PAGE).). As the results in figure 2 indicate, after a 21-hour incubation, all of the fusion protein is cleaved in the absence of any detectable nonspecific proteolytic activity.

Figure 3

Following purification of fusion proteins and proteolytic removal of the affinity peptide tags, recombinant EK reactions contain a mixture of the protein of interest, the free affinity tag, recombinant EK and, occasionally, small amounts of uncleaved fusion protein. Recovery of the mature cleavage product from this mixture is often desired. Figure 3 (Panel A) shows the results of a simple and efficient one-step protocol for removing both recombinant EK and products that contain the CBP affinity tag. In the first experiment, the fusion protein was briefly digested with recombinant EK to yield a mixture of uncleaved fusion protein, mature JNK cleavage product and the free CBP tag (lane 2). The reaction was absorbed for 30 minutes with a mixed slurry of CaM affinity resin and STI Agarose (the latter is for the removal of recombinant EK, not visible on the gel). In this experiment, all of the detectable fusion protein and CBP were bound to the resin (lane 4), while the vast majority of the JNK cleavage product remained in the unbound fraction (lane 3). In a separate experiment, (Panel B) a reaction containing an excess of recombinant EK (200 ng) was split and absorbed with either CaM affinity resin alone (STI, +CaM) or with a mixed slurry of CaM affinity resin and STI Agarose (+STI, +CaM), and the unbound material was assayed for the presence of recombinant EK activity using a fluorescent peptide substrate (see legend to Figure 3 ). As the results in panel B show, the recombinant EK was quantitatively removed when the STI Agarose was added to the absorption step.

Conclusions

Stratagene's pure, high-specific-activity, recombinant EK allows efficient, specific cleavage of fusion proteins over a wide range of conditions. Recombinant EK can be used in conjunction with the Affinity and ESP protein expression and purification systems and, in particular, with the new E. coli fusion vector, pCALnEK, to produce unmodified cloned proteins in high yield. With the introduction of STI Agarose, cleaved recombinant proteins with native amino acid sequence can be efficiently recovered from recombinant EK digests without buffer changes or long incubation times.

REFERENCES
  1. LaVallie, E.R., et al. (1983) J. Biol. Chem. 268: 2331123317.

  2. CollinsRacie, L.A., et al. (1995) Biotechnology 13: 982987.

  3. Zheng, C.F., and Simcox, T.G. (1996) Strategies 9: 2021.

  4. Wyborski, D.L., Bauer, J,C., McGowan, B., Sorge, J.A. and Vaillancourt, P. (1997) Strategies 10: 1517.

  5. Hirel, P.H., et al. (1989) Proc. Nat. Acad. Sci. USA 86: 82478251.


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
2. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
3. Efficient Transfection of Neurospora Crassa
4. Efficient Transfection of Neurospora Crassa
5. Efficient Recovery of Ultrapure Plasmid DNA
6. Efficient and Reliable PCR Setup Using Eppendorf MasterMix
7. Efficient DNA transfection of primary CNS neurons using TransMessenger Transfection Reagent
8. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
9. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
10. Efficient and Reliable Linear Amplification of cRNA
11. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Efficient Cleavage Fusion Proteins Yield Native Amino Termini

(Date:2/27/2015)... FamilyFarms Group is pleased to announce its ... Farms, received the 2015 Tomorrow’s Top Producer Horizon Award. ... of 35, who has demonstrated excellence in the business ... the Tomorrow's Top Producer Horizon Award. Every year I ... Award and learning from their experiences. It is truly ...
(Date:2/26/2015)... -- On behalf of biotech client GenScript USA, ... & Spitzer, P.A. (Wilentz) secured a significant jury verdict ... excess of $10 million. The jury determined that Genewiz ... it improperly hired one or more of GenScript,s employees.  ... notwithstanding the verdict, a new trial, and remittitur. ...
(Date:2/26/2015)... 2015 Protiviti ( www.protiviti.com ), a global ... Governance Portal , designed to increase the efficiency ... tasks across multiple business units and projects. The Governance ... better align their controls with the COSO ( Committee ... In response to client feedback, Protiviti has also included ...
(Date:2/26/2015)... Calif. , Feb. 26, 2015 The healthcare ... this ever-changing arena, Frost & Sullivan will host its ... Sullivan Executive MindXchange , March 8 to 10, at the ... Diego, Calif. This event is a ... healthcare leaders to discuss industry disruptions and how to succeed ...
Breaking Biology Technology:Matt Sims Receives the 2015 Tomorrow’s Top Producer Horizon Award 2Wilentz Client GenScript Awarded $10 Million in Trade Secrets Case Against Genewiz 2Wilentz Client GenScript Awarded $10 Million in Trade Secrets Case Against Genewiz 3Protiviti Governance Portal Helps Companies Drive Governance, Risk and Compliance Productivity Across Multiple Business Units 2Protiviti Governance Portal Helps Companies Drive Governance, Risk and Compliance Productivity Across Multiple Business Units 3Embrace the Future of Medical Technologies with Frost & Sullivan 2Embrace the Future of Medical Technologies with Frost & Sullivan 3
... spray-on films developed by UC San Diego chemists will ... reveal trace amounts of nitrogen-based explosives. , Contaminated fingerprints ... under ultraviolet light. One of the films can distinguish ... could provide evidence to help solve a crime, or ...
... piracy and terrorism at sea are on the rise, ... some governments and international organizations that pirates and terrorists ... a RAND Corporation study issued today. , The objectives ... aimed at financial gain while the goal of terrorism ...
... Severe brain injury due to blunt force trauma could ... glycol or PEG, mixed in sterile water and injected ... Central,s Journal of Biological Engineering . , ... performed experiments in rats which showed that PEG was ...
Cached Biology Technology:Glowing films developed by UC San Diego chemists reveal traces of explosives 2RAND study finds increase in piracy and terrorism at sea 2RAND study finds increase in piracy and terrorism at sea 3
(Date:2/23/2015)... 23, 2015 NXT-ID, Inc. (NASDAQ: ... biometric authentication company focused on the growing mobile commerce ... 62114357 for DISTRIBUTED VOICE DIRECTED PAYMENT METHOD ... a new groundbreaking payment method. Payment accounts may be ... only be accessed if both the speech (the word ...
(Date:2/12/2015)... MINNETONKA, Minn. , Feb. 12, 2015 /PRNewswire/ ... technology company specializing in clinical study management systems, ... Partner Program , further distinguishing iMedNet ... for Clinical Research Organizations (CROs) and healthcare consultants.  ... sales support, prospective customer referrals and numerous co-marketing ...
(Date:2/9/2015)... , Feb. 9, 2015  Lintec of America ... methods for carbon nanotube (CNT) macrostructures, including sheets, yarns ... Dallas (UTD). Leveraging the vast industrial resources ... Tokyo, Japan , Lintec of America is forming ... Richardson, TX , focusing on scaling up the ...
Breaking Biology News(10 mins):NXT-ID Patents Voice Directed Payments 2NXT-ID Patents Voice Directed Payments 3MedNet Solutions Announces Key Enhancements To Its iMedNet Partner Program 2Lintec announces exclusive license to commercialize innovative carbon nanotube technology from the University of Texas at Dallas 2Lintec announces exclusive license to commercialize innovative carbon nanotube technology from the University of Texas at Dallas 3Lintec announces exclusive license to commercialize innovative carbon nanotube technology from the University of Texas at Dallas 4
... Society for Medical Oncology (ESMO), the European Society ... Society of Thoracic Surgeons (ESTS) and the European ... first European Multidisciplinary Conference on Thoracic Oncology (EMCTO). ... and a multidisciplinary perspective of state-of-the-art on ...
... already having a detectable impact on birds across Europe, ... their findings to create the world,s first indicator of ... scale. Published in the journal PLoS ONE ... for the Protection of Birds have shown a strong ...
... Scientists trying to understand why oaks are starting to ... just below the surface to find some answers. ... small rodents that live underground, prefer oak roots to ... the rodents as a possible factor leading to high ...
Cached Biology News:European Multidisciplinary Conference on Thoracic Oncology 2European Multidisciplinary Conference on Thoracic Oncology 3Climate change affecting Europe's birds now, say researchers 2Climate change affecting Europe's birds now, say researchers 3Danger lurks underground for oak seedlings 2
... Reusable, press-to-seal gasket and cover slip ... disassembled for hybridizing and imaging thick or ... up to a 13 mm diameter. Build ... specimen handling, eliminate evaporation and leaking even ...
Comp Dim: chamber diam. depth 20 2.8 mm chamber maximum volume 720 μL...
...
... for in vitro diagnostic assays, all EIA/RIA ... for uniform binding, high optical clarity and ... are part of the Certified Surface Chemistry ... be within QA release specifications. Linkage: This ...
Biology Products: