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doi:10.1016/j.bmc.2004.06.047 
Copyright © 2004 Elsevier Ltd All rights reserved.
A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein
Thomas J. Tolbert†, Dirk Franke and Chi-Huey Wong
, 
Received 22 June 2004;
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Abstract
We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6–133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.
Article Outline
- 1. Introduction
- 2. Results and discussion
- 3. Conclusion
- 4. Experimental
- 4.1. General
- 4.2. Cloning of GPRT-C37-H6
- 4.3. Production and purification of GPRT-C37-H6
- 4.4. Cloning of His-tagged interleukin-2 (6–133)
- 4.5. Production and purification of His-tagged interleukin-2 (6–133)
- 4.6. TEV protease cleavage of GPRT-37-H6
- 4.7. TEV protease cleavage of His-tagged interleukin-2 (6–133)
- 4.8. Synthesis of H-Asn(GlcNAc)-Gly-Gly-thioester
- 4.8.1. Synthesis of 1 (Boc-Asn(GlcNAc)-Gly-Gly-OH)
- 4.8.2. Synthesis of 2 (H-Asn(GlcNAc)-Gly-Gly-thioester)
- 4.9. Ligation of H-Asn(GlcNAc)-Gly-Gly-thioester to C37-H6
- 4.10. Ligation of H-Asn(GlcNAc)-Gly-Gly-thioester to interleukin-2 (6–133)
- Acknowledgements
- Supplementary data
- References
