Commercial production of proteases that is high yielding, economical and provides ease in manufacture and processing would provide considerable advantages to many industries. Proteases are used in a variety of commercial applications, including pharmaceutical uses, medical processes, lab processes, in sequencing amino acids, among others. Many proteases used in such commercial applications are obtained from sources that are difficult and costly to maintain, that are not high yielding, and include undesirable contaminants.
Among the problems encountered are that either animal organs or bacteria are the common sources for proteases. For example, pepsin is obtained from gastric mucosa, carboxypeptidase A and B are obtained from the pancreas of animals, and leucine aminopeptidase from the kidney and intestinal mucosa. Contamination by undesirable components produced by the animal cells can impact the final product. Bacterial sources typically cannot produce the protease in reliable or sufficient quantity to be useful for commercial purposes. An example of proteases obtained from bacteria include subtilisin and themolysin, obtained from strains of Bacillus. As noted by James Wells and David Estell, “Most enzymes are expressed in minute amounts, and no generic solution is available for the expression of large amounts of an active enzyme from its cloned gene.” Wells, J. and Estell, D. “Subtilisin: An Enzyme Designed to Be Engineered” Trends in Biochemical Sciences (a review) vol. 13 pp.291–297.
By way of example, in the digestive process, a hormone action is triggered that releases digestive juice made by the pancreas. This juice contains several precursors or zymogens, including trypsinogen, and chymotrypsinogen, among others. Trypsinogen is a protein which is the precursor or zymogen molecule of trypsin. By action of enterokinase, which removes a hexapeptide from the NH2-terminal end of the trypsinogen molecule, trypsin is formed. Trypsin is a protease which hydrolyzes the peptide bonds of the oligopeptides in the intestine by cleaving on the carboxyl side of lysine and arginine residues.
Trypsin also activates chymotrypsinogen to chymotrypsin. Chymotrypsin hydrolyzes the peptide bonds involving phenylalanine, tyrosine and tryptophan.
Trypsin has a number of uses in the biological sciences and in the medical field An example is the use of trypsin in identifying the sequence of amino acids. It is useful in many processes where its' selective cleaving can be employed. For example, because its' cleavage is specific to select amino acids, it can be used to break down a polypeptide into fragments of known number. Importantly, this substrate specificity is also useful in converting biosynthetically produced molecules to preferred molecules. It is used in this manner to convert proinsulin to insulin by removal of the connecting peptide. Thus, trypsin has many commercially valuable uses.
The current source of trypsin is the organs of animals, with bovine and porcine pancreas the primary common source of the enzyme. There are numerous difficulties associated with obtaining trypsinogen or trypsin from these sources. One is that there is considerable contamination by other proteases. Chymotrypsin is one of the additional proteases in the contaminants that may cleave the product in an undesired manner.
Further, there are obvious expenses and handling concerns when shipping and using animal pancreas. They must be fresh, kept sterile, shipped in a manner to maintain freshness of the organ, and require special storage space of sufficient size to accommodate the animal organs. It also requires the care, feeding, and slaughtering of the animals that are the source. Additionally, some users of the end product have concerns about use of enzymes prepared from animal sources as components in human products.
Prior attempts to avoid these problems have included expressing trypsinogen in bacteria. A European patent application describes introducing trypsin and trypsinogen into E. coli, and selecting for transformed bacteria by use of an antibiotic resistance marker. See Greaney, EP 0 587 681. They used a variety of E. coli host cells in the construction of vectors and expression systems. There, the inventors reported that while some bacterial strains expressed the protein, others would not.
The present invention overcomes these obstacles by providing for a plant having a heterologous DNA sequence that expresses a protease. In one preferred embodiment, the protease is trypsin or trypsinogen. The inventors have been able to achieve expression at commercially acceptable levels of production and provide for a homogeneous product which does not contain the contaminants associated with animal sources. Since the source is plant, other undesirable proteases produced in the host are not a problem. The inventors have also found there are advantages to expressing proteases in seed. The seed is rich in protease inhibitors, thus enhancing stability. This is an even further advantage when the protease is not the inactive zymogen, (such as trypsinogen) but the active protease (such as trypsin). The storage, shipping and expense associated with expressing the enzyme in plants is vastly superior to the animal or bacterial sources.
Thus, it is an object of the invention to provide for plants and plant cells having DNA comprising heterologous nucleotide sequences encoding a protease.
It is a further object of this invention to provide for expression of proteases at commercially acceptable levels.
Another object of the invention is to provide for production of proteases which is not contaminated with animal proteases and other contaminants.
Yet another object of the invention is to provide for production of proteases in seed.
A still further object of the invention is to provide for a method of production of proteases in a biomass of plants.
Another object of the invention is to provide for production of proteases which is economical and provides for ease in production and handling.
A further object of the invention is to provide for production of trypsin or trypsinogen in plants at commercially acceptable levels and without contamination from animal proteases.
These and the other objectives will become apparent by the description below.
All references cited herein are incorporated herein by reference.