Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1996-08-05
1997-11-04
Tsang, Cecilia J.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530303, A61K 3828, C07K 1400
Patent
active
056839804
DESCRIPTION:
BRIEF SUMMARY
This is a 371 of PCT/SE 94/01076 filed Nov. 14, 1994.
FIELD OF THE INVENTION
The present invention relates to the use of insulin-like growth factor binding protein (IGF-BP) for refolding of Insulin-like growth factor (IGF) and to a process for the production of biologically active and native IGF-I, characterised in that IGF-I in a reduced or misfolded form is subjected to treatment with IGF-BP to obtain disulphides bridges between cysteine residues 6-48, 18-61 and 47-52, respectively.
BACKGROUND OF THE INVENTION
Human insulin-like growth factor I (IGF-I) is a single-chain peptide growth factor of 70 amino acids, originally isolated from serum. IGF-I is positively regulated by growth hormone (GH) and shows mitogenic effects on many cell types. Therefore, IGF-I is thought to mediate many of the growth promoting effects of GH. In the regions of hornology, IGF-I and insulin are 49% homologous, including the six cysteine residues, furnishing three disulphide bridges. The three dimensional structure of IGF-I has been modelled based on the x-ray structure of insulin, and this model has recently been confirmed in the disulphide bridge regions by distance constraints obtained by 2-D NMR spectroscopy of IGF-I (for a review on. IGF, see: Insulin-like growth factors I and II, Humbet R. E, Eur. J. Biochem 190, 445-462,1990).
Human recombinant IGF-I has been produced as a secreted product in both Escherichia coli and Saccharomyces cerevisiae. In isolated material from both species, IGF-I is found mainly as misfolded forms with intermolecular disulphides. Surprisingly, two distinct monomeric forms, with differences in theft disulphide bond patterns, have been identified. One of these two forms contains the disulphide bond topology expected from the insulin structure, and this form (disulphides 6-48, 47-52 and 18-61) is biologically active. The other monomeric form, designated `mis-matched` (disulphides 6-47, 48-52 instead of the native 6-48, 47-52), lacks IGF-I receptor affinity. In addition, in vitro refolding of reduced IGF-I by oxygen, has demonstrated that native, mis-matched and aggregated IGF-I accumulate, even under dilute refolding conditions (Iwai, M., et al (1989) J. Biochem. Vol. 106, Page 949; Samuelsson, E., et al (1991) Bio/Technology Vol. 9, Page 363).
In serum, and in other body fluids, IGF-I, IGF-II, and variants of these two IGFs are often bound to specific carrier proteins which have been designated IGF binding proteins (IGF-BPs). To date, six distinct, but homologous, IGF-BPs have been characterised. There are a number of reports, some of them contradictory, concerning the biological significance of the different IGF-BPs; to protect IGFs from clearance and proteolytic degradation, to transport IGFs to different tissues, to play a part in hormone regulation, to prevent hypoglycaemia by inhibiting binding of IGF-I to the insulin receptor, to increase the potency of IGFs by interacting with cell surfaces, to remove IGFs from tissue and to inhibit the biological activity of IGFs (for a recent review on IGF-BPs, see Shimasaki and Ling (1992) Progress in Growth Factor Research, Vol. 3, Page 243).
An expression system for production of the complex between IGF and IGF-BP53, is disclosed in WO 89/09268 (Genentech). The host cell is a CHO-cell. The complex is proposed to be useful for metabolically affecting the circulatory system in mammals.
A major problem when recombinant proteins are overproduced in efficient bacterial expression systems is related to the folding of the protein products into their native conformations. Many high level expression system in Escherichia coli results in the production of aggregates of denatured proteins, so called inclusion bodies, which in some cases may be refolded into the wanted native protein. In this refolding process, the inclusion body must be dissolved e.g. with a denaturant, such as guanidine or urea. If needed, reduction of disulphide bonds are also performed. By dilution or dialysis, the protein can be refolded into its native three dimensional conformation. H
REFERENCES:
patent: 5158875 (1992-10-01), Miller et al.
patent: 5407913 (1995-04-01), Sommer et al.
Biosci. Biotech. Biochem., vol. 56, No. 1, 1992, Yasamusa Marumoto et al, "Purification and Refolding of Recombinant Human IGF II from Silkworms Infected with Recombinant Bombyx mori Nuclear Polyhedrosis Virus" pp. 13-16.
Bio/Technology, vol. 9, Apr. 1991, Elisabet Samuelsson et al, "Facilitated In Vitro Refolding of Human Recombinant Insulin-Like Growth Factor I Using a Solubilizing Fusion Partner" pp. 363-366.
Hober Sophia Elisabeth
Nilsson Bjorn
Celsa Bennett
Pharmacia & UpJohn AB
Tsang Cecilia J.
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