Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1992-04-02
1994-09-20
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
530399, 530413, C12P 2100, A61K 3736, C07K 310, C07K 1300
Patent
active
053488636
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the enzymatic preparation of basic fibroblast growth factor (bFGF).
bFGF was originally isolated from brain and pituitary as a polypeptide of 146 amino acids (Esch et al, PNAS USA 82, 6507-6511, 1985). The gene for bovine bFGF has been cloned (Abraham et al, Science, 233, 545-548, 1986). The nucleotide sequence predicted a 155 amino acid bFGF translation product. Further work has shown that a 154 amino acid bFGF can be extracted together with a 146 amino acid bFGF from normal pituitary tissue upon addition of enzyme inhibitors (Ueno et al, Biochem. Biophys. Res. Comm. 138, 580-588, 1986) and that acid proteases in brain and hepatoma cells cleave bFGF (Klagsbrun et al, PNAS USA 84, 1839-1843, 1987).
Protein engineering techniques have allowed the availability of recombinant growth factors for therapeutic use. However, once they have been expressed these growth factors could be processed into a mixture of different forms. FGFs are no exception in this respect (Barr et al, J. Biol. Chem. 263, 31, 16471-16478, 1988).
We have now devised a process for the preparation of a bFGF which is truncated at its N-terminus. This process can be applied to obtain the 146-amino acid form of bFGF from longer forms and to produce a single form of bFGF from a mixture of bFGFs. The resulting form is pure and is not contaminated by other forms of bFGF.
Accordingly, the present invention provides a process for the preparation of a bFGF, which process comprises: has the 9-10 Leu-Pro bond; said bond; and
A mixture of two or more bFGFs which each have the 9-10 Leu-Pro bond, which have the same amino acid sequence from position 11 to their C-termini and which have different N-terminal amino acid sequences may be employed in step (i). Their amino acid sequences may differ only by having different N-termini. Typically, the only difference between the sequences of the bFGFs is that the number of N-terminal amino acid residues is different. One bFGF may have one or more additional N-terminal amino acid residues than another bFGF. Alternatively, the N-terminal amino acid sequences before position 9 of each bFGF may comprise different amino acid residues.
It is therefore possible to apply the present process to any mixture of bFGFs where the amino acid sequence of each bFGF in the mixture starts with a N-terminal amino acid residue numbered lower than 9 and the bFGFs have different N-termini. The mixture employed in step (i) may therefore be composed of bFGFs having the general formula: the amino acid sequence of (11-155)bFGF. Full length bFGF has 155 amino acid residues and can be designated 155-bFGF or (1-155)bFGF. The amino acid sequence of human (1-155)bFGF is shown in SEQ ID NO: 1. The invention can therefore be applied to one of, or a mixture of two or more of, (1-155)bFGF to (8-155)bFGF in which case x in the formula above is an integer of from 8 to 1 respectively and (AA).sub.x denotes sequence shown one of SEQ ID NOS: 2 to 6, IleThrThr, ThrThr or Thr. In particular, the invention can be applied to a mixture of 154 amino acid residue bFGF[(2-155)bFGF] and 153 amino acid residues bFGF[(3-155)bFGF].
The bFGF or the mixture of bFGFs is generally obtained by recombinant DNA techniques. Different forms of bFGF are obtained in such mixtures due to processing of the translation product at its N-terminus. The or each bFGF may be a human, murine or rodent bFGF.
An adduct may be formed between a protecting agent for bFGF selected from heparin and heparan sulphate, and the mixture of bFGFs in any convenient fashion. The protecting agent and bFGFs are typically provided in an aqueous solution. This solution may be buffered. An antioxidant such as dithiothreitol may be present to prevent protein oxidation. The ratio of bFGF:protecting agent may be from 0.5:1 to 10:1 (w/w), for example from 1:1 to 5:1 (w/w). The protection of the bFGFs as adducts prevents further unwanted hydrolysis when pepsin A is added.
Pepsin A (EC 3.4.23.1) or cathepsin D (EC 3.4.23.5) is next contacted with the adduct. This results in speci
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Betbeder Didier
Monsan Pierre
Paul Francois
Sarmientos Paolo
Farmitalia Carlo Erba S.r.l.
Robinson Douglas W.
Weber Jon P.
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