Navigation Links
Purification of rIL-13 Using an UNO Q1 Column


Interleukin 13 is a recently described cytokine produced by activated T cells. This protein is responsible for both positive and negative responses to regulatory molecules within the immune system. rIL-13 has been shown to modulate macrophage functions as well as play a direct role in regulation of proliferation and differentiation of hematopoietic stem cells. In addition, a recent study has indicated that rIL-13 inhibits human immunodeficiency virus type I production in blood derived macrophages. For this application, recombinant rIL-13 was overexpressed in E. coli. in the form of inclusion bodies. This report describes the isolation and purification of rIL-13 using an UNO Q1 (Bio-Rad catalog # 720-0001) column.


Experimental
rIL-13 was obtained from the cell pellet in a two part process, sample preparation and chromatographic separation. See Figure 1 for a flow chart of sample preparation. Sample preparation involved suspending the cell pellet in 50 mM Tris, pH 8.0, 20 mM DTT with 350 l of bacterial protease inhibitor (Sigma P8465). The suspension was then passed through a Microfluidics cell disruptor at 12,000 psi five times. The lysed cell suspension was then centrifuged, washed, and the rIL-13 was solubilized with 8 M urea. It was found that rIL-13 will only solubilize with a high concentration of chaotrope under reducing conditions. The final solubilized preparation was then filtered through a 0.45 m filter and applied to an UNO Q1 column.

The rIL-13 was eluted by a linear gradient as described in Figure 2. Due to the viscosity of the buffers used in the experiment, the UNO Q1 column was run at 3 ml/min.


Results
Figure 2 shows the elution profile from the UNO Q1 column. rIL-13 was found to elute from this column in the conductivity range of 1.75-3.55 ms/cm.

SDS-PAGE (Figure 3) analysis using a 15% Tris-HCl Ready Gel (Bio-Rad catalog #161-1157) indicated fractions 1820 contained rIL-13 of at least 90% purity, while fraction 21 contained a small amount of impurities. A western blot of the gel was performed to identify the rIL-13 (data not shown). The apparent heterogeneity in the elution profile may arise from protein-protein interaction or folding intermediates, since by SDS-PAGE, the rIL-13 was nearly homogeneous.


References
1. Doherty, T. M., Kastelein R., Menon S., Andrade S., and Coffman, R. L., J. Immunol., 151, 7151, (1993).

2. Montaner, L. J., Doyle, A. G., Collin, M., Herbein G., Illei, P., James W., Minty, A., Caput D., Ferrara P., and Gordon, S., J. Exp. Med., 178, 743, (1993).

3. Jacobsen, S. E., Okkenhaug, C., Veiby, O. P., Caput D., Ferrara P., and Minty A., J. Exp. Med., 180, 75, (1994).

4. Lai, H. Y., Heslan, J. M., Poppema, S., Elliot, J. F., and Mosmann, T. R., The Journal of Immunology, 156, 3166, (1996).


We gratefully acknowledge receipt of rIL-13 containing E. coli cell pellet constructed by Madeleine Huey working under Dr. John Roberts at UCSF liver transplant.


back to top
'"/>

Source:


Page: All 1 2

Related biology technology :

1. High-Level Protein Expression, One-Column Purification, and FLAG Epitope Tagging in E. coli
2. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
3. Yeast Protein Production System Features High Yields and One-Step Purification
4. Expression and Purification of Recombinant Proteins That Have Native Amino Acid Sequence
5. Easy Cosmid DNA Purification with StrataPrep Kits
6. MinElute PCR Purification Kit
7. QIAquick PCR Purification Kit
8. Microarray Target Purification Kit and the Microarray Target Synthesis Kit (T7)
9. Double-Affinity Purification of GST-His Tagged Proteins Using Vivapure 8-to-96 Well Cobalt-Chelate Kit
10. Ultra-Fast Purification of Small Nucleic Acids
11. RNAClean - RNA & cDNA in vitro Reaction Purification
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/20/2017)... ... April 20, 2017 , ... ... scientific and clinical research community’s growing body of knowledge during its Eighth ... Gracie Theatre and the adjacent Darling Atrium. During the event, undergraduates, graduate students, ...
(Date:4/20/2017)... , April 20, 2017 /PRNewswire/ - Prometic Life Sciences Inc. (TSX: ... results at the International Liver Congress ("ILC") 2017 of the ... Amsterdam on the positive effects of ... model of obesity and metabolic syndrome. ... According to Dr. Lyne Gagnon, Vice-President of R&D ...
(Date:4/20/2017)... ... April 20, 2017 , ... Parallel6™ , the leader in ... today that they were named one of the 2017 Top 10 eClinical Trial ... in the pharmaceutical industry. , “We take pride in honoring Parallel6 as one of ...
(Date:4/20/2017)... Dutch philosopher Koert van Mensvoort - founder of the Next Nature Network ... - has written a ,Letter to Humanity, in support of International ... a slave and victim to its own technology, but to employ technology to ... ... – founder of the Next Nature Network and Fellow of ‘Next Nature’ at ...
Breaking Biology Technology:
(Date:3/23/2017)... report "Gesture Recognition and Touchless Sensing Market by Technology (Touch-based and Touchless), Product ... by MarketsandMarkets, the market is expected to be worth USD 18.98 billion by ... Continue Reading ... ...      (Logo: http://photos.prnewswire.com/prnh/20160303/792302) ...
(Date:3/22/2017)... VILNIUS, Lithuania , March 21, 2017 /PRNewswire/ ... identification and object recognition technologies, today announced the ... development kit (SDK), which provides improved facial recognition ... safety cameras on a single computer. The new ... algorithms to improve accuracy, and it utilizes a ...
(Date:3/22/2017)... 21, 2017 Vigilant Solutions , a ... enforcement agencies, announced today the appointment of retired FBI ... public safety business development. Mr. Sheridan brings ... including a focus on the aviation transportation sector, to ... position, Mr. Sheridan served as the Aviation Liaison Agent ...
Breaking Biology News(10 mins):