Preparation and affinity purification of a novel, biologically active, CNTF fusion protein

A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N-terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub-cloned into the pFLAG-1 vector and transfected into the JM 109 strain of E. coli. The transfected cells expressed high levels of the fusion protein (FLAG-CNTF) following induction by isopropyl β-D-thiogalactoside (IPTG). FLAG-CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno-affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG-CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG-CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N-terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR-32 human neuroblastoma cell line. Treatment of IMR-32 cells with FLAG-CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2–3-fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG-CNTF and recombinant human CNTF on IMR-32 ChAT activity showed that both factors exhibited similar potencies. Our studies demonstrate the use of simple, commercially available, recombinant technology to efficiently generate large quantities of purified, biologically active CNTF. © 1994 Wiley-Liss, Inc.