Expression and purification of recombinant neurotensin in Escherichia coli.
An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.
Williamson
P T
PT
Biomembrane Structure Unit, Biochemistry Department, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Roth
J F
JF
Haddingham
T
T
Watts
A
A
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D009496:Neurotensin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011993:Recombinant Fusion Proteins; D009496:Neurotensin; D005982:Glutathione Transferase