Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1994-05-11
2002-04-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C530S300000, C530S324000, C530S395000
Reexamination Certificate
active
06372886
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to methods for the expression of human apolipoprotein(a) Kringle domains in bacterial cell hosts, to vectors useful for expression of the kringle domains and to the production of antibodies to human lipoprotein(a). Other aspects of the invention include methods of determining levels of serum lipoprotein(a) using these antibodies to define the antigenic distribution within the kringles 4 and 5 domains of human apo(a).
2. Description of Related Art
Human lipoprotein (a) has generated considerable interest because of its apparent correlation in blood with high risk of coronary heart disease (Scanu and Fless, 1990). Exactly how lipoliprotein (a) contributes to increased risk of heart disease is not known; however, according to some clinical studies, there appears to be a positive correlation between lipoprotein (a) blood levels and atherosclerosis. Lipoprotein (a) may favor the process of plaque buildup in the blood vessel wall. Indeed, lipoprotein (a) has been found at high levels in segments of coronary arteries after bypass surgery as well as in segments of peripheral vessels (Lawn, 1992).
Lipoprotein (a) is formed from the association of apo B100 and apolipoprotein (a) (apo (a) via a disulfide bond. Apo (a) is a large glycoprotein with extensive sequence homology to plasminogen (McLean, et al., 1987). Apo(a) exhibits size heterogeneity (300-800 KDa), the functional significance of which is not well understood. However, it is known that this size heterogeneity results from variation in the number of Kringle 4 domains in the molecule. Kringle 4 domains comprise on the average 78 amino acid residues with three highly conserved intramolecular disulfide bonds. It has been estimated that the number of Kringle 4-encoding repeats in the apo (a) gene can range from 9 to 35 (Lackner, et al., 1991). Kringle domains are so termed because of their resemblance to Danish pastries which have a comparable twisted structure (Lawn, 1992).
Kringle domains are found in other large proteins, most typically tissue plasminogen activator (Wilhelm, et al., 1990) and plasminogen (Mehnhart, et al., 1991). While there is extensive homology between apo (a) kringle 4
2
and kringle 4 of plasminogen, there are significant differences in the amino acid sequences, potentially causing changes in function. It has been suggested that physiologically lipoprotein (a) may effect the transport of cholesterol to damaged vessels, thus delivering a material critical for cell repair. On the other hand, excess lipoprotein (a) at the vessel wall site may favor accumulation of material, leading to atheroscelerotic plaque buildup (Lawn, 1992).
The Kringle 4 domains of apo (a) can be divided into 10 subtypes differing from plasminogen Kringle 4 by 12 to 23 amino acids (Morisett, et al., 1990). Like plasminogen, apo (a) shows high affinity for lysine-like ligands (Eaton, et al., 1987) and may also bind proline and hydroxyproline (Trieu, et al., 1991). However, the structural determinants of ligand binding for this protein are unknown. Individual Kringle domains appear to be independent structural units with autonomous functions (Trexler, et al., 1983). Kringle 2 of TPA and kringle 1 of plasminogen have been expressed in
E. coli
and found to bind to lysine, but kringle 4 and 5 domains from apo (a) have neither been expressed in
E. coli
nor characterized in specific binding properties.
In addition to kringle 4, apo (a) also contains a single Kringle 5 domain and a protease domain, both of which show high homology to the corresponding plasminogen domains (MacLean, et al., 1987).
Kringle domains of apo (a) may be the recognition sites for antibody binding. If such sites were identified, and were unique, a valuable method for specifically determining blood apo (a) levels would be available. At present, however, specific recognition sites on kringle 4 domains have not been identified and no truly immunospecific methods of determining apo (a) levels are available.
SUMMARY OF THE INVENTION
The present invention addresses one or more of the foregoing problems associated with the expression and purification of kringle apo (a) domains. The invention in particular includes the expression of recombinant kringle 4 and 5 domains from gram-negative bacteria, for example,
E. coli
. Recombinant kringle domains may be employed to generate antibodies which interact with apo (a) and lipoprotein (a). Such antibodies form the basis for various immunoassays designed to detect lipoprotein (a) and provide reproducible methods to detect this protein in human plasma.
One aspect of the present invention concerns the construction of an
E. coli
expression vector useful for producing recombinant kringle domains. These vectors include an inducible promoter sequence, a repressor gene sequence, a fusion protein and a polylinker gene sequence into which a DNA segment encoding a kringle polypeptide is cloned. The repressor gene is positioned upstream of an inducible promoter sequence which in turn is upstream of a selected fusion protein. The polylinker sequence is located between the fusion protein and the kringle polypeptide encoding DNA segments. The polylinker may be constructed with one or more restriction sites into which the kringle gene segment is inserted.
In preferred embodiments of the present invention, vectors incorporate maltose binding protein (MBP) as the fusion protein although other fusion partners might also be employed. MBP protein includes an amino acid sequence sensitive to Fx
a
protease. The expressed recombinant polypeptide includes three extraneous amino acids from the polylinker when the construct as shown in
FIG. 1
is employed. MBP facilitates fusion protein isolation by virtue of its binding to amylose sepharose columns.
Kringle polypeptide expression has been demonstrated in
E. coli
but other prokaryotes or even eukaryotic host cells might be employed. Turning to the expression of the disclosed human apo (a) kringle polypeptides, once a suitable (full length if desired) clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system for the recombinant preparation of the various regions or domains. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that either eukaryotic or prokaryotic expression systems may be employed in the expression of kringle apo (a) domains; however, vector constructs useful for such expression have not been heretofore available, nor has a recombinant apo (a) kringle been successfully expressed in a gram negative host cell until the present invention.
Human apo (a) kringle 4 and 5 domains have now been successfully expressed in bacterial expression systems with the production of correctly folded structures. The cDNA for apo (a) kringle and kringle 5 domains has been separately expressed in
E. coli
systems, with the encoded proteins being expressed as fusions with maltose binding protein (MBP), a most preferred embodiment. Other fusion proteins with such fusion partners as &bgr;-galactosidase, ubiquitin,
Schistosoma japonicum
glutathione S-transferase, and the like are also envisioned. It is believed that bacterial expression has numerous advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
If, however, an eukaryotic expression system is chosen, it is believed that almost any eukaryotic expression system may be utilized for the expression of human apo (a) kringles, e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed. However, in preferred embodiments, it is contemplated that vectors constructed analogously to pIH821, will be employed incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5. Examples of host cells commonly
Fless Gunter M.
Scanu Angelo M.
Achutamurthy Ponnathapu
Arch Development Corp.
Fulbright & Jaworski
Rao Manjunath N.
LandOfFree
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