Expression system for producing apolipoprotein AI-M

Details Expression system for producing apolipoprotein AI-M Expression system for producing apolipoprotein AI-M

1995-08-25

1998-02-24

Wax, Robert A.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4353201, 4352523, 530359, 536 235, C12P 2106, C12N 1500, C12N 120, C07H 2104

Patent

active

057211140

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to an expression system yielding high levels of protein in the culture medium of Escherichia coli to produce Apolipoprotein AI Milano (Apo AI-M). The product may be used for the treatment of atherosclerosis and cardiovascular disease.


BACKGROUND OF THE INVENTION

The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. While CM constitute a short-lived product of intestinal fat absorption, VLDL and, particularly, LDL are responsible for the cholesterol transport into tissues, among these, also into the arterial walls. In contrast, HDL are directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as "reverse cholesterol transport" (RCT).
The "protective" role of HDL has been confirmed in a number of studies (e.g. Miller et al. (1977) Lancet 965-968 and Whayne et al. (1981) Atherosclerosis 39: 411-419). In these studies, the elevated levels of LDL, less so of VLDL, are associated with an increased cardiovascular risk, whereas high HDL levels seem to confer cardiovascular protection. The protective role of HDL has been further strongly supported by the in vivo studies, showing that HDL infusions into rabbits may hinder the development of cholesterol induced arterial lesions (Badimon et al. (1989) Lab. Invest 60: 455-461) and/or induce regression of these same (Badimon et al. (1990) J. Clin. Invest. 85: 1234-1241).
Recent interest in the study of the protective mechanism(s) of HDL has been focused into apolipoprotein AI (Apo AI), the major protein component of HDL. High plasma levels of Apo AI are associated with reduced risk of CHD and presence of coronary lesions (Maciejko et al. (1983) N. Engl. J. Med. 309: 385-389, Sedlis et al. (1986) Circulation 73: 978-984).
Human apolipoprotein AI-Milano (Apo AI-M) is a natural variant of Apo AI (Weisgraber et al. (1980) J. Clin. Invest 66: 901-907). In Apo AI-M the amino acid Arg173 is replaced by the amino acid Cys173. Since Apo AI-M contains one Cys residue per polypeptide chain, it may exist in a monomeric or in a dimeric form. These two forms are chemically interchangeable, and the term Apo AI-M does not, in the present context, discriminate between these two forms. On the DNA level the mutation is only a C.fwdarw.T transition, i.e. the codon CGC changed to TGC. However, this variant of Apo AI is one of the most interesting variants, in that Apo AI-M subjects are characterized by a remarkable reduction in HDL-cholesterol level, but without an apparent increased risk of arterial disease (Franceschini et al. (1980) J. Clin. Invest 66: 892-900). By examination of the genealogic tree, these subjects appear to be "protected" from atherosclerosis. Human mature Apo AI and Apo AI-M consist of 243 amino acids. They are synthesized as precursor proteins, preproApo AI and preproApo AI-M of 267 amino acids. The 18 amino acid prepeptide is cleaved off in the secretion machinery leaving a proprotein with an extension of 6 amino acids. ProApo AI and proApo AI-M are then converted to the mature forms by a plasma proteolytic activity.
Attempts have been made to produce human Apo AI by way of recombinant DNA technology. In the European patent publication No. 0267703 the preparation of Apo AI from E. coli is described. The process describes a chimeric polypeptide where the Apo AI moiety is fused to the N-terminal amino acid residues of .beta.-galactosidase or to one or more IgG-binding domains of Protein A, or to the prosequence of human Apo AI

REFERENCES:
Franceschini et al., A-I.sub.Milano Apoprotein, J. Clin. Invest., vol. 66, (1980), pp. 892-900.
Badimon et al., Regression of Atherosclerotic Lesions by High Density Lipoprotein Plasma Fraction in the Cholesterol-fed Rabbit, J. Clin. Invest., vol. 85 (1990), pp. 1234-1241.
Moguilevsky et al., Production of Human Recombinant Proapolipoprotein A-I in Escherichia coli; Purification and Biochemical Characterization, DNA, vol. 8, No. 6 (1989), pp. 429-436.
Isacchi et al., Mature apolipoprotien AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli, Gene, vol. 81 (1989), pp. 129-137.

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