Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-07
2003-11-11
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S236100, C435S091400, C435S219000, C435S252300, C435S320100, C435S348000, C435S069100, C435S069300, C436S546000, C530S384000, C530S390100, C530S391500, C530S391700
Reexamination Certificate
active
06645724
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to recombinant Factor C (rFC) of a horseshoe crab, produced in an insect cell system. The invention also relates to vectors for producing the protein by recombinant DNA methods and to methods for using the recombinant Factor C to detect endotoxins in a sample or for removal of endotoxins from a sample by affinity methods.
THE RELATED ART
The amoebocytes of horseshoe crabs contain an efficient coagulation cascade system which is activated by endotoxin, also known as lipopolysaccharide (LPS) from Gram negative bacteria. The enzymatic components of the coagulation cascade and the molecular events responsible for the subsequent gelation of the amoebocyte lysate have been characterized in
Tachypleus tridentatus
1
and
Carcinoscorpius rotundicauda
2,3,4
. Factor C has been shown to be the intracellular endotoxin-sensitive serine protease that initiates the coagulation cascade system
5
.
By spiking, the Limulus amoebocyte lysate (LAL) test detects femtogram levels of LPS
6
. Owing to its extreme sensitivity, the amoebocyte lysate, in particular, the LAL has been developed into a commercial assay for widespread use in the detection of pyrogenic LPS in drugs and other pharmaceutical products
7,8
. This assay is based on the LPS-induced coagulation reaction of the lysate, culminating in formation of a gel clot. However, (a) the possible lack of specificity due to 1-3 &bgr;-D glucan and (b) the batch-to-batch variation in the sensitivity of commercial lysate to LPS, due to seasonal and geographical differences in the starting material
9
has prompted our laboratory to employ recombinant DNA technology to genetically-engineer Factor C as an alternative source of novel “limulus lysate” for endotoxin detection.
cDNAs encoding Factor C have been cloned
1,10,15
. There are six potential glycosylation sites in the amino acid sequence of the Factor C from
Carcinoscorpius rotundicauda
(CrFC)
10,15
. Cloned cDNA encoding CrFC has been expressed in
E. coli
11
and also in yeast expression systems
12, 24
. The rFC obtained from yeast was found to be immunoreactive and capable of binding LPS, although only limited amounts of rFC produced in yeast were soluble
13,14
. Also, it was found that LPS could not activate the enzymatic activity of yeast rFC, thus, a direct enzyme-based LPS detection is not possible using rFC produced in yeast
14
.
Since the early 1970s, the diagnostic potential of LAL for endotoxemia has been recognized to be extremely important for timely and effective treatment. Thus, many studies have been conducted to improve the sensitivity of endotoxin assays by changing the formulation of the LAL and assay methodology
25
. Since blood (plasma) components interfere with the test, various methods to remove inhibition and/or enhancement have been developed. Furthermore, the LAL gelation clot method had been criticized as being subjective, semiquantitative and prone to variations in interpretations by different workers. Thus, advent of an improved fluorimetric assay for using LAL in LPS detection of plasma would be desirable. Even more desirable would be the compatibility of this assay for both LAL and a recombinant Factor C (rFC) in their sensitivity to LPS. Furthermore, this demonstrates the possible diagnostic utility of rFC for endotoxemia.
SUMMARY OF THE INVENTION
The present inventors believed that expression in insect cells rather than in a prokaryotic or simple eukaryotic expression system is suitable for producing rFC with full biological activity. Furthermore, horseshoe crabs and insects belong to the same phylum, Arthropoda, and so insect cells might more closely resemble the cells of the horseshoe crab than yeast cells in their physiology and biochemistry. Thus, rFC produced in insect cells might more closely resemble the protein as purified from the horseshoe crab and retain the bioactivity of having a serine protease activity activated by LPS.
The present invention relates to genetic engineering of a bioactive rFC, which unequivocally exhibits full biological functionality. It is capable of specifically recognizing and binding LPS and lipid A in both free and immobilized forms. Interference from 1-3 &bgr;-D-glucan, which switches on the alternate pathway in the coagulation cascade in conventional LAL, is not anticipated in assays of the present invention that use only Factor C as the LPS-binding, serine protease enzyme. Both the LPS-activated enzymatic assays of rFC and the enzyme linked immunosorbent assay (ELISA) lipid A binding assay could be formulated into a rapid high throughput mass screening test for LPS. Thus, a novel generation of “limulus amoebocyte lysate” has been invented, being capable of rapid and sensitive diagnosis and removal of subpicogram levels of endotoxin. The invention provides a standardized and convenient source of enzyme-based diagnostic reagent for detection of the ubiquitously contaminating endotoxin in pharmaceutical products. This inexhaustible supply of genetically-engineered Factor C can be easily standardized to circumvent batch-to-batch variations in sensitivity to LPS, a problem faced by the conventional LAL industry. Furthermore, the ability of the rFC of the invention to protect mice from endotoxemia, as well as its bacteriostatic activity, adds to its value in in vivo applications. Furthermore, the availability of rFC obviates the need for routine harvesting of the horseshoe crab for procurement of their amoebocyte lysate, and therefore, conserves this endangered “living fossil”.
The present inventors have succeeded in expressing biologically active rFC using recombinant baculoviruses and other vectors appropriate for expression of heterologous DNA in insect host cells. The rFC obtained is enzymatically active. Thus, expression of rFC in insect cells is a convenient and economical source of rFC protein for use in rapid, sensitive, specific and quantitative determination of LPS in pharmaceutical products and other biological fluids.
Thus, the present invention comprises purified rFC that is enzymatically active. The phrase “enzymatically active” means that the Factor C protein has the biological activity of binding LPS or lipid A, being activated as to its serine protease activity upon LPS or lipid A binding. Enzymatically active rFC will induce coagulation of an amoebocyte lysate and will also cleave synthetic substrates such as, but not limited to, Boc-Val-Pro-Arg-MCA, Mu-Val-Pro-Arg-AFC and Boc-Val-Pro-Arg-pNA.
The present invention is also embodied in a method for producing substantially purified, enzymatically active rFC. The method comprises expressing DNA encoding a Factor C protein having the enzymatic activity described above in a culture of insect cells, then isolating the enzymatically active Factor C protein. The isolation preferably includes an ultrafiltration step. The purification preferably also includes a step of gel-filtration chromatography on a matrix having an exclusion limit of 100 kilodaltons. The gel filtration is preferably applied after the ultrafiltration. The exclusion limit of the gel filtration matrix can vary substantially; an effective matrix will provide at least about a 4-fold increase in the serine protease activity of an ultrafiltered crude preparation as measured by the fluorometric assay described herein.
The present invention also encompasses host-vector systems for expressing enzymatically active rFC. The host cells in these embodiments of the invention are insect cells, preferably leptidopteran cells. The vectors in these embodiments support replication of inserted DNA in insect cells and expression of heterologous DNA in insect cells. The vectors are preferably baculovirus or plasmid vectors. The heterologous DNA is sufficient to encode a Factor C enzyme of a horseshoe crab, preferably of the genus Carcinoscorpius, Tachypleus or Limulus. The recombinant Factor C will preferably at least have the LPS binding activity of Factor C and more preferably will have both LPS binding activity and serine protease activity. Preferred heterologous DN
Ding Jeak Ling
Ho Bow
Hines Ja'Na
National University of Singapore
Smith Lynette R. F.
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