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:4/24/2015)... April 24, 2015 Seoul Semiconductor ... for various applications in the lighting market. ... installations of this technology in emerging markets such ... a wide range of applications such as streetlight ... decorative lighting. , First launched in 2005 the ...
(Date:4/23/2015)... , April 23, 2015 BioTE Medical President ... book "Age Healthier Live Happier - Avoiding Over-Medication through ... Age Management Medicine Group Conference at JW Marriott / ... ",Age Healthier Live Happier, is a welcomed ... over-medication and searching for a higher quality of life ...
(Date:4/23/2015)... LAKE FOREST, Calif. , April 23, 2015 ... provider of advanced cryogenic logistics solutions for the ... has further established its cryogenic logistics solutions within ... customer agreement with Yancey Farms to support the ... top German Sport horse Stallions. ...
(Date:4/23/2015)... April 23, 2015  Metanome, Inc., an industry ... company has changed its name to Diversigen, Inc.  ... as the single commercial source for the myriad ... on the diverse opportunities of the microbiome.  From ... accepts – including those that are difficult to ...
Breaking Biology Technology:Acrich Technology from Seoul Semiconductor Adopted in Installations Worldwide 2Release and Book Signing of BioTE Medical President Gary Donovitz M.D. 'Age Healthier Live Happier' at Age Management Medicine Group (AMMG) Conference 2Release and Book Signing of BioTE Medical President Gary Donovitz M.D. 'Age Healthier Live Happier' at Age Management Medicine Group (AMMG) Conference 3Cryoport to Support the Equine Animal Husbandry Market Through Agreement with Yancey Farms 2Cryoport to Support the Equine Animal Husbandry Market Through Agreement with Yancey Farms 3Metanome Announces Corporate Name Change to Diversigen 2
... Wis. - Lucigen Corp . and ... giving Lucigen exclusive worldwide rights to manufacture and sell ... , ,Under the agreement, Imaxio will retain the right ... applications, but Lucigen will provide the cells to commercial ...
... - It has yet to turn a profit, and it ... scope, but the decision by Madison research tool manufacturer NimbleGen ... to $75 million is the latest bit of evidence that the ... continues a pattern of growth and visibility for NimbleGen, which ...
... Alfalight , a manufacturer of high-power diode lasers, has ... million from the U.S. Army Research Laboratory . ... suitable for pumping high-power, fiber-laser systems, developed in conjunction ... commercial products. , ,Manoj Kanskar, vice president of research ...
Cached Biology Technology:Lucigen Corp. enters global licensing agreement 2IPO-bound NimbleGen traveling in fast company 2IPO-bound NimbleGen traveling in fast company 3IPO-bound NimbleGen traveling in fast company 4
(Date:4/14/2015)... , April 14, 2015 NXT-ID, Inc. (NASDAQ: ... authentication company focused on the growing mobile commerce market, ... for the 2015 New York Design Awards under the ... New York City Design Awards are part of a ... over 2,500 ratings from the marketplace, industry and judging ...
(Date:4/9/2015)... Calif. , April 9, 2015 Synaptics ... interface solutions, announced today that it will report financial ... Thursday, April 23, 2015, after the close of market. ... analysts and investors at 2:00 p.m. PT (5:00 p.m. ... To participate on the live call, analysts ...
(Date:4/2/2015)... , April 2, 2015 At its 2015 ... Lake City , the American College of Medical ... new directors to its Board.  Members of the ACMG ... and for forming and advancing its policies and programs. ... profession. "It,s an eventful time in medical ...
Breaking Biology News(10 mins):NXT-ID's Wocket Smart Wallet Nominated for 2015 New York Design Awards in 'Product Design- Technology' 2NXT-ID's Wocket Smart Wallet Nominated for 2015 New York Design Awards in 'Product Design- Technology' 3Synaptics to Report Third Quarter Results on April 23 2American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 2American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 3American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 4American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 5American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 6American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 7American College of Medical Genetics and Genomics Announces New Board Members: Dr. Louanne Hudgins is ACMG President-Elect 8
... Dana-Farber Cancer Institute researchers have identified a molecular mechanism ... how consuming foods rich in saturated fats and trans-fatty ... and increases one's risk of heart disease and certain ... scientists led by Bruce Spiegelman, PhD, report that the ...
... patients with high-risk breast cancer treated with radical mastectomy ... to better survival outcomes with few long-term toxic effects, ... which appears in the January 19 issue of the ... Columbia randomized radiation therapy trial was designed to determine ...
... recent U.S. study, investigators in Japan found no association ... of high blood pressure, as reported in the March ... available online. , The researchers reported that they were ... known as the Kaposi's sarcoma virus, in the lungs ...
Cached Biology News:Scientists ID molecular 'switch' in liver that triggers harmful effects of saturated and trans fats 2Scientists ID molecular 'switch' in liver that triggers harmful effects of saturated and trans fats 3Novel Asthma Study Shows Multiple Genetic Input Required; Single-gene Solution Shot Down 2Association of herpesvirus with lung disorder questioned 2
... White solid. PROTECT FROM LIGHT. ... water soluble pyrimidinone compound that acts as ... of PARP (IC 50 = 200 ... rmPARP2, respectively) with little activity against the ...
... speed, timer, SB3, Fully adjustable mixing angle, ... holders to hold a number of different ... ideal for aerating cultures, keeping biological samples ... The rotator can be used in incubators ...
...
... The DNA Engine Opticon 2 real-time ... of LEDs for fluorescence excitation (470-505 nm) ... (523-543 nm, 540-700 nm). The Opticon 2 ... thermal cycler, which includes a thermal gradient ...
Biology Products: