Polynucleotide constructs for secreted glycosylated plasminogen

Details Polynucleotide constructs for secreted glycosylated plasminogen Polynucleotide constructs for secreted glycosylated plasminogen

1991-05-06

1994-03-29

Hill, Jr., Robert J.

Chemistry: molecular biology and microbiology

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

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

435 692, 4351723, 4352401, 4352402, 4353201, C12N 1515, C12N 1503, C12N 1506, C12P 2102

Patent

active

052984001

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The invention relates to enhanced secretion of PAI-2, to recombinant polynucleotide constructs suitable for providing enhanced secretion of PAI-2, to the expression products of those constructs and to their uses, as well as a 414 amino acid form of PAI-2, a synthetic signal peptide used in the preparation of glycosylated, secreted PAI-2 and 60 kD glycosylated, secreted, recombinant PAI-2.


DEPOSITION OF MICROORGANISMS

BTA 1445, an E. coli strain harbouring a PAI-2 encoding construct was deposited with the American Type Culture Collection of 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under the provisions of the Budapest Treaty on 11 Feb. 1987 under accession number ATCC 53585.


BACKGROUND ART

Plasminogen activator (PA) inhibitor Type 2 (PAI-2) is one of four immunologically distinct groups of PA inhibitors. PA inhibitors are members of the serpin gene family.
PAI-2, also termed minactivin, has been purified from the human monocytic cell line U937 (International Patent Application PCT/AU85/00191 published as W086/01212 and PCT/AU87/00068 published as W087/05628). It has also been detected in pregnancy plasma and in the conditioned medium of several human cells including peripheral blood monocutes, HT1080 fibrosarcoma cells and HEp3 laryngeal carcinoma cells.
Distinct molecular weight species of PAI-2 have been identified:
A 46 kD form is non-glycosylated and primarily cell associated. This form has been found in lysates of U937 cells and purified from the conditioned medium of phorbol ester (PMA) stimulated U937 cells (International Patent Application No. PCT/AU87/00068).
A 60 kD glycosylated form has been found to be secreted by U937 cells (Genton et al., 1987) as well as being present in the maternal plasma of pregnant women (Lecander and Astedt, 1986).
The non-glycosylated 46kD intracellular form accounts for 80-90% of PAI-2 synthesized in U937 (Genton et al., 1987) and HT1080 cells (Medcalf et al., 1988)
Examination of the primary amino acid sequence of PAI-2 reveals that the molecule lacks the characteristic transient signal peptide usually found at the amino (NH.sub.2)-terminus of secreted proteins. Although the NH.sub.2 - terminus of the purified 46 kD form of PAI-2 from U937 cells has not been determined, apparently because it is blocked (Kruithof et al, 1986), the NH.sub.2 - terminus of the 60 kD glycosylated version has been reported to be the initiator methionine (residue 1, Ye et al., 1988). This implies that secretion occurs without cleavage of a signal peptide from PAI-2. The mode of translocation of PAI-2 through the cell is not understood, however an internal signal has been proposed (Ye et al., 1988).
As is the case with most potent biologically active proteins, PAI-2 is produced in very small amounts in vivo and as such is difficult to purify and characterise by conventional biochemical approaches. The recent cloning of the gene for PAI-2 and its expression in bacterial cells (International Patent Application No. PCT/AU87/00068) now allows the production of significant quantities of purifed 46 kD PAI-2 which is needed to evaluate its biological efficacy in clinical applications. However this bacterial material is not glycosylated, nor modified post-translationally in a manner analogous to that secreted by human cells. Therefore it is desirable to produce glycosylated forms of PAI-2 using transfected mammalian cells, since the two forms of PAI-2 may differ in their biological activities e.g. binding affinity for urokinase, PA specificity, immunogenicity, in vivo half-life etc.
The native PAI-2 gene has previously been expressed in a number of heterologous mammalian expression systems (International Patent Application No. PCT/AU87/00068). Although PAI-2 is synthesized in these systems, expression levels are low, and the majority of the product (approx. 90%) is non-glycosylated and intracellular. PAI-2 produced in this form is a suitable molecule for prophylactic, therapeutic and/or diagnostic uses but its use is limited by the quantities obtainable and limited glyc

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