Abstract

Peptide synthesis is widely used for the production of small proteins and peptides, but producing uniformly isotopically labelled peptides for NMR and other biophysical studies could be limited for economic reasons. Here, we propose a use of a modified pGEV-1 plasmid to express neurotensin (NT 1–13), pGlu 1-Leu 2-Tyr 3-Glu 4-Asn 5-Lys 6-Pro 7-Arg 8-Arg 9-Pro 10-Tyr 11-Ile 12-Leu 13-OH, as a C-terminal fusion protein with the GB1 domain of streptococcal protein G. The free carboxyl-terminus is important for the function of several peptide hormones, including neurotensin. Therefore, for the pGEV-NT 1–13 construct, the C-terminal pGEV-encoded 6×His tag was removed and an N-terminal 8×His tag was introduced for affinity purification. To facilitate removal of tags using CNBr cleavage, a methionine was introduced at the N-terminal of the peptide. Furthermore, this pGEV-NT 1–13 plasmid was used as a template to include a Pro-7 to Met mutation for CNBr cleavage, giving NT 8–13, the sub-fragment crucial for the biological activity of this peptide. These two constructs are being used to produce uniformly labelled NT 1–13 and NT 8–13 in high yield and in a cost effective way, using cheap 15N and/or 13C source. The modification proposed here using the pGEV-1 plasmid could be an alternative option for the high expression of other isotopically labelled and unlabelled short peptides, including hormones and hydrophobic membrane peptides.

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