Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli.
The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/l per A600 unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography. Purified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.
McDonald
J R
JR
Synergen, Inc., Boulder, CO 80301.
Ko
C
C
Mismer
D
D
Smith
D J
DJ
Collins
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D020934:Ciliary Neurotrophic Factor; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
D020934:Ciliary Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins