Production of the neuronal growth-associated protein GAP-43 in a bacterial expression system.
GAP-43, a major protein of neuronal growth cones and certain presynaptic terminals, is a candidate for important functions in both axon growth and synaptic plasticity. To facilitate studies that may elucidate these functions, we have efficiently generated large quantities of GAP-43 by introducing a GAP-43 cDNA into a bacterial expression system driven by T7-RNA polymerase. Two constructs were expressed in this system: one (pT7Ava-GAP) produces a fusion protein in which the first 16 amino acids of GAP-43 are replaced by 11 amino acids of the phage T7 capsid protein; the other (pT7FL-GAP) produces full length GAP-43. After the bacteria were lysed, both products were soluble, and could be efficiently purified by HPLC chromatography on a C4 reversed-phase column. One liter of bacterial culture yielded 50 mg of purified fusion protein or 10 mg of complete GAP-43. When it was incubated with protein kinase C, the fusion protein was phosphorylated at the same single site (serine 41) that is phosphorylated in cultured neurons. The ability to produce large quantities of GAP-43 by this procedure should expedite future studies investigating its structure, posttranslational modification, and function.
Schuh
S M
SM
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110.
Spencer
S
S
Willard
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D019922:GAP-43 Protein; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D016900:Neurofilament Proteins / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D011493:Protein Kinase C