A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein.
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.
Tolbert
Thomas J
TJ
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC 483A, La Jolla, CA 92037, USA.
Franke
Dirk
D
Wong
Chi-Huey
CH
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D006020:Glycopeptides; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease