Heterologous high yield expression and purification of neurotensin and its functional fragment in Escherichia coli.
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 6xHis tag was removed and an N-terminal 8xHis 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 (15)N and/or (13)C 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.
Tapaneeyakorn
Satita
S
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Ross
Simon
S
Attrill
Helen
H
Watts
Anthony
A
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D009496:Neurotensin / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013291:Streptococcus / Q000235:genetics
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D009496:Neurotensin