Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-08-31
2002-07-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069700, C435S023000, C435S174000, C435S252300, C435S320100, C424S094640, C536S023200, C536S023400
Reexamination Certificate
active
06420157
ABSTRACT:
BACKGROUND OF THE INVENTION
Members of the trypsin/chymotrypsin-like (S1) serine protease family play pivotal roles in a multitude of diverse physiological processes, including digestive processes and regulatory amplification cascades through the proteolytic activation of inactive zymogen precursors. In many instances protease substrates within these cascades are themselves the inactive form, or zymogen, of a “downstream” serine protease. Well-known examples of serine protease-mediated regulation include blood coagulation, (Davie, et al (1991).
Biochemistry
30:10363-70), kinin formation (Proud and Kaplan (1988).
Ann Rev Immunol
6:49-83) and the complement system (Reid and Porter (1981).
Ann Rev Biochemistry
50:433-464). Although these proteolytic pathways have been known for sometime, it is likely that the discovery of novel serine protease genes and their products will enhance our understanding of regulation within these existing cascades, and lead to the elucidation of entirely novel protease networks.
The S1 family of serine proteases is the largest family of peptidases (Rawlings and Barrett (1994).
Methods Enzymol
244:19-61). As described above, members of this diverse family perform diverse functions including food digestion, blood coagulation and fibrinolysis, complement activation as well as other immune or inflammatory responses. It is likely that these functions in both normal physiology and during diseased states, currently under investigation by numerous laboratories, will become better understood in the near future. The discovery of novel S1 serine protease cDNAs will enhance our understanding of the complex pathways controlled by these enzymes. These functions will undoubtedly be aided by the ability to express large amounts of the active protease, which is then amenable to biochemical analyses.
In the vast majority of cases, maturation of an S1 serine protease zymogen into an active form by proteolytic cleavage, results in transformation into a protease of enhanced catalytic efficiency. Zymogenicity (Tachias and Madison (1996).
J Biol Chem
271:28749-28752), the degree of enhanced catalytic efficiency, varies widely among individual members of the serine protease family. Proteolytic cleavage of the conserved amino terminus zymogen activation sequence results in an aliphatic amino acid, most frequently isoleucine (Ile-16 chymotrypsin numbering), becoming protonated and thus, positively charged. The event that accompanies zymogen activation is the creation of a rigid substrate specificity pocket generated by a salt bridge between the aliphatic amino acid and a highly conserved residue aspartic acid (Asp-194 chymotrypsin numbering) one amino acid upstream from the active-site serine (Ser-195 chymotrypsin numbering) within the catalytic domain (Huber and Bode (1978).
Acc Chem Res
11:114-22).
Proteases are used in non-natural environments for various commercial purposes including laundry detergents, food processing, fabric processing and skin care products. In laundry detergents, the protease is employed to break down organic, poorly soluble compounds to more soluble forms that can be more easily dissolved in detergent and water. In this capacity the protease acts as a “stain remover.” Examples of food processing include tenderizing meats and producing cheese. Proteases are used in fabric processing, for example, to treat wool in order prevent fabric shrinkage. Proteases may be included in skin care products to remove scales on the skin surface that build up due to an imbalance in the rate of desquamation. Common proteases used in some of these applications are derived from prokaryotic or eukaryotic cells that are easily grown for industrial manufacture of their enzymes, for example a common species used is Bacillus as described in U.S. Pat. No. 5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serine proteases isolated from a fungus,
Tritirachium album
, for use in laundry detergent compositions. Unfortunately use of some proteases is limited by their potential to cause allergic reactions in sensitive individuals or by reduced efficiency when used in a non-natural environment. It is anticipated that protease proteins derived from non-human sources would be more likely to induce an immune response in a sensitive individual. Because of these limitations, there is a need for alternative proteases that are less immunogenic to sensitive individuals and/or provides efficient proteolytic activity in a non-natural environment. The advent of recombinant technology allows expression of any species' proteins in a host suitable for industrial manufacture.
A major drawback in the expression of full-length serine protease cDNAs has been overwhelming potential for the production of inactive zymogen. These zymogen precursors often have little or no proteolytic activity and thus must be activated by either one of two methods currently available. One method relies on autoactivation (Little, et al. (1997).
J Biol Chem
272:25135-25142), which may occur in homogeneous purified protease preparations, that often requires high protein concentrations, and must be rigorously evaluated on a protease specific basis. The second method uses a surrogate protease, such as trypsin, to cleave the desired serine protease. The surrogate protease must then be either inactivated (Takayama, et al. (1997).
J Biol Chem
272:21582-21588) or physically removed from the desired activated protease. (Hansson, et al. (1994).
J Biol Chem
269:19420-6). In both methods, the exact conditions must be established empirically and activating reactions monitored carefully, since inadequate activation or over-digestion would result in a heterogeneous population of active and inactive zymogen protein. Some investigators studying particular members of the S1 serine protease family have exploited the use of restriction proteinases on the activation of zymogens expressed in either bacterium (Wang, et al. (1995).
Biol Chem
376:681-4) or mammalian cells (Yamashiro, et al. (1997).
Biochim Biophys Acta
1350:11-14). In one report, the authors successfully engineered the secretion of proteolytically processed and activated murine granzyme B by taking advantage of the endogenous yeast KEX2 signal peptidase in a
Pichia pastoris
expression system (Phain et al. (1998).
J. Biol. Chem
. 273:1629-1633). U.S. Pat. No. 5,326,700 shows modification of the tissue plasminogen activator (t-PA) molecule such that the polypeptide is cleaved by the expression host cell to yield mature protein upon secretion from the cell. This example of a specific modification, while simple, suffers from the requirement that the associated protease is expressed within the host cell at such levels as to cleave the t-PA, which would be expressed in large quantities relative to other host proteins. Similarly, U.S. Pat. Nos. 5,270,178 and 5,196,322 describe modification of the protein C cleavage site such that it becomes a more efficient substrate of the protease thrombin. These examples of activating recombinant zymogens clearly have the added value to permit expression and activation of several serine proteases, however there remains unmet needs in the field. The example of Pham et al clearly limits the expression system available for use due to the nature of the signal peptide. The other examples describe enzyme specific engineered constructs that do not easily predict a generic method to which other serine proteases may be applied.
Introduction of proteolytic cleavage sites into fusion proteins is well known in the art. However, it is the present invention, for the first time, that creates a fusion protein designed for the generic activation of S1 serine proteases by the introduction of a propeptide region with a predefined, easily processed, cleavage site. Inclusion of the catalytic domain of a serine protease into the fusion gene allows the specific enzyme's activity to be preserved without the requirement of a specific activating enzyme. Because the protein is proteolytically processed using commercially available en
Andrade-Grodon Patricia
Darrow Andrew
Qi Jenson
Achutamurthy Ponnathapu
Moore William W.
Ortho-McNeil Pharmaceutical , Inc.
Wallen III John W.
LandOfFree
Zymogen activation system does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Zymogen activation system, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Zymogen activation system will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2863044