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 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Rabinovsky
E D
ED
Department of Neurology, Baylor College of Medicine, Houston, Texas.
Browder
D P
DP
McManaman
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000096:biosynthesis; D002846:Chromatography, Affinity; D020934:Ciliary Neurotrophic Factor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D020934:Ciliary Neurotrophic Factor; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D002795:Choline O-Acetyltransferase