Process for the preparation of biologically active NGF by genetic engineering

摘要

For the preparation of biologically active beta -NGF (nerve growth factor) by genetic engineering by expression of an appropriate DNA sequence in prokaryotes as host cells, a DNA sequence is used which is composed in the 5'-3'-direction of sequences which code for (a) methionine as a starting amino acid, (b) 3 to 5 amino acids of an n-terminal sequence of a protein which can be expressed in the host cells used with a high expression rate, (c) 4 to 10 amino acids from the pro sequence of the beta -NGF precursor and (d) the 118 amino acids of the mature beta -NGF which, after expression of this DNA sequence in insoluble aggregates of inactive beta -NGF obtained from E. coli, are converted into a soluble active form by (1) solubilisation, (2) if appropriate dialysis and/or derivatisation of the resulting beta -NGF with the oxidised form of a compound, whose reduced form contains sulphydryl groups and whose oxidised form is a dimer of the compound bonded via a disulphide bridge, to give the mixed disulphide, and (3) naturation with the aid of a redox system consisting of a compound in oxidised and/or reduced form, whose reduced form contains sulphydryl groups and whose oxidised form contains a dimer bonded via a disulphide bridge, and removing, if appropriate subsequently with the aid of a protease, the amino acids of the sequences (a) and/or (b) and/or (c) and/or up to 9 N-terminal amino acids of the sequence (d). An active beta -NGR is obtained which contains less than 5% of beta -NGF degradation products or beta -NGF modified by a Met radical at the N terminus and is preferably free of other mammalian cell proteins and can be shortened or lengthened at the N terminus. <IMAGE>