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:10/10/2017)... 10, 2017 SomaGenics announced the receipt of ... develop RealSeq®-SC (Single Cell), expected to be the first ... (including microRNAs) from single cells using NGS methods. The ... to accelerate development of approaches to analyze the heterogeneity ... "New techniques for measuring levels of mRNAs in individual ...
(Date:10/10/2017)... PA (PRWEB) , ... October 10, 2017 , ... ... year’s recipients of 13 prestigious awards honoring scientists who have ... presented in a scheduled symposium during Pittcon 2018, the world’s leading conference and ...
(Date:10/9/2017)... ... 2017 , ... The award-winning American Farmer television series will feature 3 Bar ... Tuesdays at 8:30aET on RFD-TV. , With global population estimates nearing ten billion ... continue to feed a growing nation. At the same time, many of our valuable ...
(Date:10/9/2017)... ... October 09, 2017 , ... The ... medical marijuana products targeting the needs of consumers who are incorporating medical marijuana ... place in Phoenix, Arizona. , As operators of two successful Valley dispensaries, The ...
Breaking Biology Technology:
(Date:3/30/2017)... HONG KONG , March 30, 2017 ... developed a system for three-dimensional (3D) fingerprint identification by adopting ground ... technology into a new realm of speed and accuracy for use ... applications at an affordable cost. ... ...
(Date:3/29/2017)... , March 29, 2017  higi, the health IT ... North America , today announced a ... the acquisition of EveryMove. The new investment and acquisition ... of tools to transform population health activities through the ... data. higi collects and secures data today ...
(Date:3/27/2017)... CENTRE, N.Y. , March 27, 2017 /PRNewswire-USNewswire/ ... Healthcare Information and Management Systems Society (HIMSS) Analytics ... Outpatient EMR Adoption Model sm . In addition, ... 12% of U.S. hospitals using an electronic medical ... CHS for its high level of EMR usage ...
Breaking Biology News(10 mins):