ABSTRACT
This chapter discusses a two-step purification scheme to obtain functional Neurotensin from rat (NTR) based on immobilized metal affinity chromatography followed by an agonist ligand column step. E. coli has been used successfully for the expression of G protein-coupled receptors in functional, membrane-inserted form, despite being very different from eukary-otic expression systems. The great attraction of E. coli as an expression host is the simplicity of its use. The time required for the construction of recombinant expression vectors and the initial expression experiment is very short in comparison to other host systems. Progress in purification of NTR is indicated by an increase of the specific binding expressed as “pmol/mg of protein.” The methods described above for the expression in E. coli and purification of functional NTR fusion protein have been developed in view of simple procedures to allow the isolation of sufficient receptor protein for crystallization trials and further scale-up.
