Navigation Links
New Yeast Cloning System for Producing Proteins with Native Amino Acid,,,Sequences

fusion protein gave approximately 4 mg per liter (data not shown). The GST-chicken calmodulin recombinant protein produced approximately 12 mg per liter of fusion protein. Figure 3 shows the one-column purification of the GST-calmodulin fusion protein and subsequent digestion with enterokinase.

Conclusions

The pESP LIC cloning and protein purification kits allow efficient and directional cloning of high-fidelity PCR products immediately downstream of the GST purification tag and the recognition site for enterokinase. Single-step purification of the recombinant GST fusion protein and removal of the purification tag by enterokinase cleavage yield polypeptides with completely native amino acid sequences. The ESP LIC cloning and protein purification kit includes all the necessary components for LIC cloning, expression in S.pombe and GST affinity purification.

Materials and Methods

For protein induction, the expression strain was inoculated into 10 ml of YES media and incubated at 30C overnight. This culture was used to inoculate another 10 ml of YES media and was incubated at 30C until the culture reached mid-log phase. The cells were collected by centrifugation, washed once with 50 ml of sterilized water, resuspended in 10 ml of EMM media with 25 M thiamine (repressed) or without thiamine (induced) and incubated at 30C for 18 to 20 hours. Crude lysates were prepared by washing cells in PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 1% Triton X-100), then lysing cells by vortexing with glass beads in PBST buffer containing protease inhibitors (1 mM PMSF, 1 g/ml aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin). Soluble protein was bound to glutathi
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Epitope-Tagging Vectors for Functional Analysis in Yeast
2. Yeast Protein Production System Features High Yields and One-Step Purification
3. Electroporation of Yeast Artificial Chromosomes
4. New Automated Yeast Cell Counter Ends Tedium & Errors of Manual Counting
5. High Yield Protein Production from Pichia pastoris Yeast: A Protocol for Benchtop Fermentation
6. Enhanced PCR Cloning System
7. pfuturbo DNA Polymerase: A High-Performance, High-Fidelity Enzyme Ideal for PCR Cloning
8. Functional Cloning Using ViraPort Retroviral cDNA Expression Libraries
9. Mammalian Expression Vector for Efficient Cloning of PCR Fragments
10. QIAGEN PCR CloningPlus Kit
11. Cloning Based on Efficient Three-Fragment Assembly DNA Ligation
Post Your Comments:
(Date:7/31/2014)... Boston, MA (PRWEB) July 31, 2014 ... ) finds itself flush with innovative adult stem cell ... patents and has three additional patent applications ... Office. , The patented inventions address two of the ... and regenerative medicine. Adult stem cells are difficult ...
(Date:7/30/2014)... MD (PRWEB) July 30, 2014 ... today the release of the REVELERIS® Prep ... organic chemists, preparative chromatographers, and other researchers to ... using a single unit. , With the REVELERIS® ... flash and preparative chromatography modes with a simple ...
(Date:7/30/2014)... July 30, 2014  Replikins Ltd. today released new ... Ebola virus. Before the current outbreaks, the mean Ebola ... amino acids) between 1995 and 2002 was 1.1; the ... 2012, and sixteen-fold in 2013, thus predicting the current ... technology has been shown to be able to predict ...
(Date:7/30/2014)... 2014   Wyle has been named ... Mission Support Services (FILMSS) contract awarded by the ... terms of the contract, Wyle will provide mission ... Calif. In particular, Wyle will provide program and ... on the International Space Station and collaborative science ...
Breaking Biology Technology:The Adult Stem Cell Technology Center, LLC Launches A Marketing Campaign To License Adult Stem Cell Biotechnologies 2The Adult Stem Cell Technology Center, LLC Launches A Marketing Campaign To License Adult Stem Cell Biotechnologies 3Grace Launches New REVELERIS® Prep Purification System 2Wyle Wins NASA's Fully Integrated Lifecycle Mission Support Services Contract 2
... traditional August break, the Senate may help decide the future ... hope a scientifically sound and ethical resolution is reached - ... ,Just as Republicans in the Wisconsin Legislature are divided over ... President Bush suffered a rare political defeat in May when ...
... crazy? , ,Theres a question that every technology entrepreneur ... families, friends, employees, customers and, yes, investors. When youre ... that new-to-the-world and must-have tech breakthrough, your sanity will ... even more interesting than the question is how tech ...
... Waukesha, Wis. - ProHealth Care , a ... deployed an application delivery system from NetScaler, Inc. ... the system to better coordinate application usage and response ... and two regional hospitals, 13 primary care clinics, home ...
Cached Biology Technology:Stem cell debate in Washington grows hotter as summer recess nears 2Stem cell debate in Washington grows hotter as summer recess nears 3Psychologist - technology entrepreneurs frequently experience hypomania 2Psychologist - technology entrepreneurs frequently experience hypomania 3
(Date:7/30/2014)... points is a straight line, and now Harvard researchers ... axiom to heart. , Though competition among individual ... racing for the chance to fertilize the egg, Harvard ... that allow them to take a straighter path to ... Heidi Fisher, a post-doctoral student working in the lab ...
(Date:7/30/2014)... A group of researchers from the University of Helsinki ... experimentally to reproduce in mice morphological changes which have ... gradual modifications in the embryonic development of mice teeth, ... morphologically are very similar to those observed in the ... millions of years ago. , To modify the ...
(Date:7/30/2014)... Press (OUP) is pleased to announce its new relationship ... 2015, OUP and CINP will partner to publish ... a fully open-access journal. , Alan Frazer, Editor-in-Chief of ... size and expertise of OUP will further facilitate the ... of the very leading journals within neuropsychopharmacology. The conversion ...
Breaking Biology News(10 mins):When cooperation counts 2When cooperation counts 3Scientists reproduce evolutionary changes by manipulating embryonic development of mice 2OUP to partner with the International College of Neuropsychopharmacology 2
... CITY, Aug. 31, 2009 Researchers at Huntsman Cancer Institute ... on Ewing,s sarcoma, an often deadly bone cancer that typically ... with poor outcomes have tumors with high levels of a ... chemotherapy. The research is published online today in the journal ...
... its usefulness in counting copies of duplicated genome sequences and ... study to be published Aug. 30 in Nature Genetics ... differ from one person to the next. The researchers ... Search Tool. The study is titled, "Personalized Copy Number and ...
... on the neural mechanisms involved in social behavior, neuroscientists ... the brain structure responsible for our sense of personal ... 30 issue of the journal Nature Neuroscience , ... social distance is an issue. The structure, the ...
Cached Biology News:New hope for deadly childhood bone cancer 2Counting duplicated genome segments now possible 2Counting duplicated genome segments now possible 3Caltech neuroscientists find brain region responsible for our sense of personal space 2Caltech neuroscientists find brain region responsible for our sense of personal space 3Caltech neuroscientists find brain region responsible for our sense of personal space 4
...
... PlusOne Silver Staining Kit, Protein, 1. ... and Dernick (1), the kit gives ... electrophoresis systems.Silver Staining Kit, Protein provides ... proteins in polyacrylamide gels in less ...
Mouse monoclonal [IFR0104] to Salmonella enteriditis ( Abpromise for all tested applications)....
Cyno macaque plasma and serum - various quantities available...
Biology Products: