Figure 4. Effect of different concentrations of Triton X-100 in the column and elution buffers on the contaminating NADH oxidation activity and native thioesterase activity of recombinant CpTE1. (Click to enlarge)
Figure 5. Removal of Triton X-100 and glycerol from MBP-fused CpTE1 protein purified by the alternative protocol. (Click to enlarge)
In summary, we have observed contaminating NADH oxidation activity present in some MBP fusion proteins expressed in E. coli and purified by conventional amylose resin-based affinity chromatography. The contamination could be removed by either addition of 0.1% Triton X-100 and 2% glycerol in the homogenization and wash steps to the conventional purification system for MBP-fusion protein, or by Ni-NTA resin-based purification protocols if a His-tag is also engineered into the fusion proteins. It is possible that some MBP-fusion proteins may not bind well to amylose in the presence of this much detergent, in which case adding a His-tag to facilitate Ni-NTA resin-based purification protocol may be a better choice, or different concentrations of detergent may be tested to identify the best protein conditions. Because MBP fusion systems are commonly used to express foreign proteins in E. coli, and a large number of enzymes use NADH as a cofactor, it is necessary for the research community, particularly for those who study enzymes that use NADH as a cofactor, to be aware of this problem and the alternative protocols to eliminate any contamination.
We thank Drs. Haili Zhang and Xicheng Ding for technical assistance and Dr. S. Dean Rider for critical reading of the manuscript. This study was supported by grants from the National Institutes of Health (NIH), National Institute of Allergic and Infectious Diseases (NIAID; R01 AI44594 and R21 AI80022 to G.Z.). This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Address correspondence to Guan Zhu, Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX, USA. e-mail: [email protected]
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