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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.