WO2001019970A2 - Chymotrypsin-free trypsin - Google Patents

Abstract

The present invention relates to recombinantly produced, essentially chymotrypsin-free trypsin and trypsinogen products. The present invention also includes reagents and processes for producing recombinant trypsinogen which can be subsequently activated to form essentially chymotrypsin-free trypsin.

Description

CHY OTRYPSIN-FREE TRYPSIN

This application claims priority of Provisional Application Serial No. 60/154,019 filed September 15, 1999.

The invention relates generally to recombinant DNA technology. More specifically, the present invention relates to recombinantly produced trypsin and trypsinogen, as well as to methods of making the same.

Trypsin is a widely used serine protease which cleaves the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine . In animals, trypsin plays a pivotal role among pancreatic enzymes in the activation of endopeptidases. These pancreatic enzymes are secreted through the pancreatic duct into the duodenum of the small intestine in response to a hormone signal generated when food passes from the stomach. They are not, however, synthesized in their final active form. Rather, they are made as slightly longer catalytically inactive molecules called zymogenε . The names given to some of these zymogens include trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase . These zymogens must themselves be cleaved proteolytically to yield active enzymes.

The first step of the activation cascade is the activation of trypsin from trypsinogen in the duodenum. Enteropeptidase (also known as enterokinase) is a protease produced by duodenal epithelial cells which activates pancreatic trypsinogen to trypsin by excising a hexapeptide leader sequence from the amino-terminus of trypsinogen. Trypsin in turn autocatalytically activates more trypsinogen to trypsin and also acts on other proenzymes, thus, for example, liberating the endopeptidases chymotrypsin and elastase as well as carboxypeptidases A and B. This battery of enzymes work together with pepsin produced in the stomach and other proteases secreted by the intestinal wall cells to digest most ingested proteins into free amino acids, which can be absorbed by the intestinal epithelium. The enzymes themselves are continually subjected to autodigestion and other degradative processes so that high levels of these enzymes never accumulate in the intestine. In the pancreas, several factors oppose trypsinogen autoactivation, whereas in the duodenum, all the conditions favor trypsinogen activation by enteropeptidase.

To activate trypsin from its inactive precursor, a leader sequence, which in the bovine enzyme consists of Valine-Aspartate-Aspartate-Aspartate-Aspartate-Lysine ( (Asp) ₄-Lys) , on the amino-terminus of trypsinogen is enzymatically removed. Trypsinogens of many different species have been cloned and characterized. The pattern of (Asp)^-Lys at the amino-terminus, however, is well conserved in all of these precursors. Mireille Rovery, Limi ted Proteolyses in Pancreatic Chymotrypsinogens and Trypsinogens, 70 Biochi ie 1131 (1988) .

Serine proteases such as trypsin have a variety of uses. They are useful for the characterization of other proteins as well as in the manufacturing process of other recombinant bioproducts . For example, small recombinant proteins are often expressed first as fusion proteins to facilitate their purification and enhance their stability. The fusion proteins can be engineered such that a leader sequence can be cleaved from the native protein sequence by trypsin. Any internal lysines or arginines that are not part of the leader sequence can be chemically protected from cleavage by trypsin.

Trypsinogens from various species have been isolated and characterized. Craik, C.S. et al . (1984) J. Biol . Chem. 259:14255-14264; Fletcher, T.S. et al . (1987) Biochemistry 26:3081-3086. However, bovine trypsin, isolated from bovine pancreas, is now largely used in research laboratories and is the trypsin of choice for protein processing in the pharmaceutical industry. Even after extensive purification of animal-derived trypsin, however, there are contaminating activities in most preparations that can have undesirable consequences for both experimental research and pharmaceutical therapeutic protein processing. For example, the emergence of diseases such as bovine spongifor encephalopathy (BSE) has raised concerns about the use of enzymes from animal original in industrial processes.

In addition, strict guidelines and regulations issued by the Food and Drug Administration as well as other national and international regulatory bodies has led to a need for pure trypsin of recombinant origin. The present invention addresses this need by providing recombinantly expressed trypsinogens which can be purified in large quantities. These trypsinogens can be produced stably in a variety of expression systems and subsequently activated to provide pure trypsin for use in both experimental research and industrial therapeutic protein processing.

Heretofore, the efficient manufacture of large quantities of recombinant trypsin useful in the manufacture of protein pharmaceuticals has been problematic. Problems have stemmed from such factors as the instability of mature trypsin in expression systems, the activation of trypsinogen during expression by endogenous host cell enzymes and subsequent damage to cell membranes, the low solubility of expressed protein in bacterial host cell systems, and improper folding of the protein in various host cell systems. Moreover, presently available commercial preparations all suffer from the presence of contaminating chymotryptic activity, which either necessitates the addition of inhibitors or results in illegitimate cleavage products . One type of bacterial expression system has been developed for rat ionic trypsin. Vasquez, J.R. et al . , An Expression System for Trypsin, J . of Cell. Biochem. , 39:265- 276 (1989). In this system the rat trypsin hexapeptide leader sequence is replaced with the phoA signal peptide which directs the secretion of trypsin to the periplasmic space of E. coli . The signal peptide is removed from the fusion protein during secretion into the periplasmic space. Thus, in the Vasquez process, active trypsin is a secreted active protein. This is problematic for the following reasons. First, as explained below, active trypsin is less stable than its inactive zymogen form because it autodigestε. Second, active trypsin can be toxic to host cells and/or cause damage to cell membranes during the secretion process. Finally, such a process is incapable of producing trypsin in a cost-effective manner because it requires a delicate "spheroplasting" technique that is difficult to implement on a commercial scale. Spheroplasting is a well-known technique that involves digesting the bacterial cell wall, thereby liberating the material trapped in the periplasmic space without disrupting the main cell membrane.

Trypsin contains three internal trypsin cleavage sites in addition to the cleavage site in the leader sequence, and trypsin has a strong affinity for itself. These features account for the consistent failure of others to develop an effective recombinant expression system. Because of these internal cleavages sites and this high self-affinity, the recombinant trypsinogen becomes activated to mature trypsin during expression and/or secretion. These activated trypsin molecules then cleave other recombinantly produced trypsin enzymes at internal cleavage sites and render these enzymes inactive. The resulting mixture of recombinant trypsin peptides contains only a small percentage of intact active trypsin. This self-cleavage contributes to low yields of recombinant trypsinogen or trypsin. Thus, there exists a need in the art for an efficient and inexpensive means to produce recombinant trypsin which can then be used to safely and consistently manufacture other protein therapeutics, without unwanted cleavage products. Accordingly, the present invention provides an efficient and relatively inexpensive process to manufacture recombinant trypsin that circumvents the disadvantages of the prior art processes.

First, the present invention provides trypsin intracellularly, as an inactive trypsinogen zymogen, improving stability, reducing toxicity to cells and avoiding the need for spheroplasting. Second, because trypsinogen is made in inclusion bodies, it is not subject to autodegradation. The invention provides a means to move away from animal-sourced trypsin and avoid the problems of degradation, contamination, instability, and damage to cell membranes which occurs during expression and/or secretion of recombinantly produced trypsinogen.

It is, therefore, an object of the invention to address one or more of the foregoing deficiencies in the art.

According to this object, the following embodiments of the invention are provided.

According to this object, the invention discloses preparations of trypsin that are essentially chymotrypsin- free. In one embodiment these trypsin preparations are essentially free from any contaminating mammalian or yeast materials, excluding the trypsin itself.

Also according to this object, the invention provides trypsinogen aggregates that contain trypsinogen and bacterial cellular debris.

In other embodiment, the invention provides liquid compositions containing trypsinogen that has an N-terminal methionine, a chaotrope and a reducing agent. In still another embodiment, the invention provides purified trypsinogen preparations that contain a trypsinogen moiety with two extra amino acids at the N-terminus, one being methionine.

The invention also provides according to this object a recombinant expression vector capable of intracellularly expressing a trypsinogen in a prokaryote. In one embodiment, the vector contains a nucleic acid encoding trypsinogen that is operably linked to a control sequence that is capable of directing intracellular trypsinogen expression in a prokaryotic organism.

Also according to this object, the invention provides essentially chymotrypsin-free trypsinogen, prepared by a method that entails contacting a trypsinogen-containing inclusion body with a solution containing chaotrope, thereby solubilizing said protein, contacting a refolding solution with more than one aliquot of the solubilized protein, such that the resulting concentration of protein in the refolding solution is from about 20 mg/1 to about 100 mg/1, and activating the refolded trypsinogen, thereby forming trypsin.

In another aspect, the invention provides a method of refolding a protein that involves contacting an inclusion body with a solution containing chaotrope, thereby solubilizing said protein, and transferring the solubilized protein into a refolding solution in more than one aliquot, such that the resulting concentration of protein in the refolding solution is from about 20 mg/1 to about 100 mg/1.

The invention further provides method of producing trypsin involving contacting a trypsinogen-containing aggregate with a denaturation solution renaturing said trypsinogen, and activating the renatured trypsinogen to form essentially chymotrypsin-free trypsin. In still another method of producing trypsin, a method is provided that entails expressing trypsinogen inside a bacterial cell by recombinant DNA methods, isolating said trypsinogen and processing said isolated trypsinogen thereby forming essentially chymotrypsin-free trypsin.

Figure 1A shows a schematic plasmap of pHKY603, showing the major structural features.

Figure IB shows the complete DNA and protein sequences of met-phe-bovine trypsinogen.

Figure 2 shows a sample chromatogram of a tryptic digest of glucagon.

The invention relates in general to essentially chymotrypsin-free preparations of trypsinogen and trypsin. These uniquely pure preparations are particularly suited for use in the manufacture of recombinant protein pharmaceuticals. Such biopharmaceuticals are, in many instances, produced as fusion proteins, which must be matured proteolytically to a final product by the action of a protease like trypsin.

Another aspect of the invention relates to methods of preparing such high purity trypsin products. These methods generally entail recombinantly producing trypsinogen inside a prokaryotic cell. The trypsinogen is then isolated and may be activated to form essentially chymotrypsin-free trypsin .

As noted above, commercial preparations of bovine trypsin, however, generally contain substantial amounts of chymotryptic activity, even the multiply crystallized trypsin. The presence of chymotrypsin produces illegitimate cleavage products in protein digests, yielding a heterogeneous final product. This is particularly problematic in obtaining marketing approval for pharmaceutical agents, which requires a certain uniformity of product and manufacturing process . Although chymotryptic activity may be inhibited by adding specific inhibitors, such addition merely complicates the purification process and yields an additional component to the purification scheme and adds another issue in obtaining marketing approval .

One product of the invention is a highly purified preparation of trypsin. (Trypsin is defined below.) This highly purified trypsin meets at least one of the following criteria of purity. Generally such preparations are greater than about 90 percent pure. More typically, however, these preparations are more than about 95 percent pure and preferably they are at least about 99 percent pure, and generally at least about 99.5, 99.9 or 99.99 percent pure. Some preparations have no detectable contaminants .

One method of estimating purity is sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining and densitometry . Percent purity is expressed as a ratio of the trypsin peak area to total peak area .

Purity may also be evaluated by reverse phase high performance liquid chromatography method as presented generally below in the Examples. In such a case, percent purity is evaluated by comparing the integration of the trypsin peak to all peaks. The preparations of the invention generally show no chymotrypsin by this method.

Purity may also be as measured by specific activity using the TAME assay, detailed below. Reference pure material should yield about 235 U/mg.

Importantly, preferred trypsin preparations are "essentially chymotrypsin-free." As used herein, "essentially chymotrypsin-free" denotes a preparation that has less than about 0.01% chymotrypsin (by weight, relative to trypsin) . More preferred essentially chymotrypsin-free preparations generally have less than about 0.005%, and even more preferred preparations have less than about 0.001% chymotrypsin and most preferred preparations have less than about 0.0005% chymotrypsin.

Some highly purified preparations meet the foregoing criteria of purity when measured using the ultra-sensitive glucagon-based high performance liquid chromatography (HPLC) method presented below in Example 4. In such an assay, preferred compositions show no detectable chymotryptic glucagon peaks .

The trypsin preparations of the invention generally are essentially mammalian protein-free. As used herein "essentially mammalian protein-free" compositions refer to any of the inventive compositions that are essentially free of all mammalian proteins, except, of course, for the recombinantly produced product. In general, this is achieved by avoiding the addition of mammalian proteins, like casein (added to bacterial cultures), and producing the proteins in a non-mammalian host. Excluding the recombinant protein, typical compositions have less than about 1% mammalian protein, but usually have less than about 0.5% mammalian protein or less than about 0.1% mammalian protein. Again, excluding the recombinant protein, more preferred compositions have less than about 0.01% mammalian protein and most preferred compositions have no detectable mammalian protein. The skilled artisan will be aware of numerous methods, such as enzyme-linked immunosorbent assays (ELISAs) for detecting contaminating mammalian proteins .

The trypsin preparations of the invention generally are also essentially free of any yeast-derived products. As used herein "essentially yeast product-free" compositions refer to any of the inventive compositions that are essentially free of all yeast-and/or fungal-derived components, like yeast proteins carbohydrates and lipids, aflatoxin and the like. In general, this is achieved by avoiding the addition of yeast products, like yeast extract (added to bacterial cultures) and producing the proteins in a non-yeast host. For example, typical compositions have less than about 1% yeast protein, but usually have less than about 0.5% yeast protein or less than about 0.1% yeast protein. More preferred compositions have less than about 0.01% yeast protein and most preferred compositions have no detectable yeast protein and/or no detectable yeast-derived components. The skilled artisan will be aware of numerous methods, such as enzyme-linked immunosorbent assays (ELISAs) for detecting contaminating mammalian proteins.

Conveniently, the essentially chymotrypsin-free trypsin of the invention may be assembled into "commercial units" that are suitable for sale. Each commercial unit generally comprises a bulk quantity of essentially chymotrypsin-free trypsin. A bulk quantity usually comprises at least about 10 mg of essentially chymotrypsin-free trypsin unit. More typically, however, larger quantities will be present, such as at least about 50 mg per unit, at least about 100 mg per unit, at least about 500 mg per unit or at least about 1 gram per unit. Even larger quantities, e . g. , a unit of at least about fifty grams, a unit of at least about a hundred grams unit, or a unit of at least about a kilogram(s), also are contemplated.

The term "commercial unit" also contemplates assemblages of smaller commercial units to form larger ones. For example a one-kilogram commercial unit of essentially chymotrypsin-free trypsin may be provided as a thousand one- gram commercial units. Generally, a commercial unit will contain essentially chymotrypsin-free trypsin as a liquid solution. It may, however, be present in a solid form, such as a freeze-dried (lyophilized) powder. Bulking agents and stabilizers (like calcium) are optionally included. A commercial unit also includes the packaging containing the essentially chymotrypsin-free trypsin, and optionally includes printed product specifications, an inventory control number and/or instructions for use.

Another product of the invention is an "aggregate" that contains trypsinogen protein and bacterial cell debris. In such aggregates, the trypsinogen is predominantly found in a non-native conformation and may have intermolecular and improper intramolecular disulfide bonds. Bacterial inclusion bodies are representative of such aggregates . As the artisan will appreciate, inclusion bodies often result when proteins are produced recombinantly in bacteria. They typically contain unfolded and mis-folded, thus mostly inactive, recombinantly produced protein, along with nucleic acids, lipids and other cellular debris, including bacterial proteins. While such aggregates are often considered a problem, they do offer certain benefits. In the present case, the aggregated trypsinogen is inaccessible to proteases, including trypsin, which could prematurely activate it and result in toxic effects on the host cell. Moreover, the isolated aggregates are storage stable and, thus, they represent a storage-stable source (they are stable for at least months at -20 to -70°C) that may readily be converted to highly purified trypsin.

Preferred aggregates comprise trypsinogen having an N- terminal methionine. Other preferred aggregates comprise trypsinogen having an N-terminal methionine and at least one additional amino acid prior to the native first-position amino acid (e.g., valine in bovine trypsinogen); phenylalanine is particularly preferred. Still other preferred aggregates contain a trypsin cleavage site, like that in native trypsinogen, by which the trypsinogen may be activated to trypsin. In bovine trypsinogen, for instance, the cleavage site follows the hexapeptide leader sequence Val (Asp) ₄Lys . The trypsinogen aggregates of the invention are also preferably essentially mammalian protein-free and/or essentially yeast product-free. The same general parameters regarding the inventive trypsin products, provided above, also apply to the trypsinogen aggregates.

Generally, the trypsinogen aggregates of the invention are provided in a large, bulk quantity. Such a bulk quantity is particularly suited for further processing to obtain a large quantity of highly purified trypsin. Again, a bulk quantity usually comprises at least about 10 mg of trypsinogen aggregate. More typically, however, a larger quantity will be present, such as at least about 50 mg, at least about 100 mg, at least about 500 mg or at least about 1 gram. An even larger quantity, e . g. , at least about fifty grams, a hundred grams, or kilogram(s), also is contemplated. An assemblage of such bulk quantities is contemplated. An assemblage can include a centrifuge rotor (flow-through or otherwise), a cold room, a refrigerator, a freezer, an ice bucket, a flask, a beaker, a filtration system and any other container capable of containing such bulk quantities .

Another product of the invention is a trypsinogen- containing solution. This solution generally also contains a chaotropic agent, like urea or guanidium chloride, and a reducing agent, like dithiothreitol, cysteine or β- mercaptoethanol . The concentrations of the chaotropic agent and the reducing agent will generally be sufficient to maintain the trypsinogen in an unfolded and reduced state. Most typically, the chaotrope will be at a concentration of at least about 5 molar and the reducing agent will be at least about 5 millimolar, but reducing agent generally will be at least about 20 millimolar. More preferred solutions have at least about 6 molar chaotrope and 50 millimolar reducing agent. Even more preferred solutions have about 7 molar chaotrope and about 100 millimolar reducing agent. Generally, the protein concentrations in these solutions will be from about 1 to about 5 g/1.

These inventive solutions, however, do not contain any chromatographic marker dyes, like bromophenol blue. Of course one use of trypsinogen solutions is as an electrophoretic marker for trypsinogen (or other proteins) , whereby the addition of those dyes is beneficial, but such dye-containing solutions are not within the scope of the invention. Yet another use of the solutions of the invention is in the preparation of the highly purified trypsin of the invention.

Preferred solutions comprise trypsinogen having an N- terminal methionine. Other preferred solutions comprise trypsinogen having an N-terminal methionine and at least one additional amino acid prior to the native first-position amino acid (e.g., valine in bovine trypsinogen); phenylalanine is particularly preferred. Still other preferred solutions contain a trypsin cleavage site, like that in native trypsinogen, by which the trypsinogen may be activated to trypsin. In bovine trypsinogen, for instance, the cleavage site follows the hexapeptide leader sequence Val (Asp) _<Lys .

The trypsinogen solutions of the invention are also preferably essentially mammalian protein-free and/or essentially yeast product-free. The same general parameters regarding the inventive trypsin products, provided above, also apply to the trypsinogen solutions.

Like the other products of the invention, trypsinogen solutions are usually provided in a large, bulk quantity. Such a bulk quantity is particularly suited for further processing to obtain a large quantity of highly purified trypsin. Again, a bulk quantity is usually comprised of at least about 10 mg of trypsinogen in solution. More typically, however, a larger quantity will be present, such as at least about 50 mg, at least about 100 mg, at least about 500 mg or at least about 1 gram. Even larger quantities, e.g., at least about fifty grams, a hundred grams, or kilogram(s), also are contemplated. In general, while they may be in smaller volumes, the bulk quantities typically will consist of larger volumes, like at least about 100 ml, at least about 1 liter, at least about 100 liters and at least about 1000 liters. An assemblage of such bulk quantities is contemplated. An assemblage includes a centrifuge (flow-through or otherwise), a cold room, a refrigerator, a freezer, an ice bucket, a flask, a beaker, a tank, a filtration system and any other container capable of containing such bulk quantities.

Still another product of the invention is purified trypsinogen. This highly purified trypsinogen meets at least one of the following criteria of purity. Generally such preparations are greater than about 90 percent pure. More typically, however, these preparations are more than about 95 percent pure and preferably they are at least about 99 percent pure. Some preparations have no detectable contaminants .

One method of estimating purity is sodium dodecyl sulfate - polyacrylamide gel electrophoresiε (SDS-PAGE) with silver staining and densitometry . Percent purity is expressed as a ratio of the trypsin peak area to total peak area .

Purity may also be evaluated by reverse phase high performance liquid chromatography method as presented generally below in the Examples. In such a case, percent purity is evaluated by comparing the integration of the trypsin peak to all peaks.

In general, the purified trypsinogen of the invention contains amino acid modifications that result from the optimization of the coding sequence for expression in a prokaryote. The most evident modification is an N-terminal methionine codon. Preferred embodiments also have an N- terminal methionine and at least one additional amino acid prior to the native first-position amino acid (e.g., valine in bovine trypsinogen) ; phenylalanine is particularly preferred. A particularly preferred embodiment contains the sequence shown in Figure IB.

The purified trypsinogen of the invention are also preferably essentially mammalian protein-free and/or essentially yeast product-free. The same general parameters regarding the inventive trypsin products, provided above, also apply to the purified trypsinogen.

The purified trypsinogen of the invention may be assembled into commercial units exactly as described above for the essentially chymotrypsin-free trypsin, including the exact same quantities of material.

Recombinant protein expression is well known in the art, and important features are set out below. The present invention also contemplates a recombinant expression system for the intracellular expression of trypsinogen in a prokaryotic cell. Thus, the system does not include signal sequence (s) that direct secretion of the trypsinogen or matured trypsin. In fact, the trypsinogen made by this system generally accumulates inside the cell as insoluble inclusion bodies.

In one aspect, the expression system comprises a trypsinogen gene, for example a bovine trypsinogen gene. Preferably this trypsinogen gene has been at least partially codon optimized for use in E . coli . Codon optimization is a well known process of re-engineering a gene to conserve the amino acid sequence, yet utilize the most prevalent codons for a particular organism. Figure IB shows a preferred example that has been partially optimized (rare codons have been eliminated) . Guidance relating to codon optimization and rare codons is found, for example, in Itakura et al , Science 198: 1056 (1977), Goeddel et al . , Proc . Nat ' 1. Acad. Sci. 76: 106 (1979), Itakura et al , U.S. Patent No. 4,356,270 (1982), Edge et al , Nature 292: 756 (1981) and Feretti et al . , Proc. Nat ' 1 Acad. Sci. 83: 599 (1986).

In a preferred embodiment, the trypsinogen gene encodes the following amino acid sequence: Methionine-X-trypsinogen, wherein X is any amino acid, but is preferably phenylalanine . The "trypsinogen" component of the expression vector encodes a trypsinogen, as defined herein.

The methods of the invention generally involve the intracellular recombinant production of trypsinogen in a prokaryotic cell . General methods useful in recombinant protein production in prokaryotes are known, and described briefly below.

The basic steps in the recombinant production of desired proteins are:

a) construction of a synthetic or semi-synthetic DNA encoding the trypsinogen,- b) integrating said DNA into a prokaryotic expression vector in a manner suitable for the expression of the trypsinogen, either alone or as a fusion protein; c) transforming an appropriate prokaryotic host cell with said expression vector, d) culturing the transformed host cell in a manner to express the trypsinogen; and e) recovering and purifying the recombinantly produced trypsinogen . As indicated, the methods of the invention are characterized generally by the (a) intracellular production of (b) an inactive zymogen form of trypsin that (c) may be activated by the action of trypsin (or other protease) to form a mature enzyme. The intracellular production is beneficially accomplished in a prokaryotic organism like E. coli , which produces the protein in insoluble inclusion bodies. The inclusion bodies ensure that the trypsinogen is not prematurely activated to trypsin, alleviating adverse effects on the host and on the ultimate purity and integrity of the final product.

Moreover, to achieve the mammalian protein-free compositions of the invention, the host organism is typically grown in a defined synthetic medium, like the one presented below in the Examples, in the absence of casein or any other supplemental mammalian proteins. This assures that the resultant product is mammalian protein-free, except of course for the trypsinogen that is produced by the host. In order to obtain the essentially yeast product-free compositions, likewise, the addition of yeast extract or other yeast products is avoided by using a defined synthetic medium, as discussed below.

Following cell lysis by well known mechanical or chemical means, the inclusion bodies may be isolated by a combination of centrifugation and washing with water or a suitable buffer to remove excess cell debris. Ethylenediamine tetraacetic acid (EDTA) at 5 to 10 mM is also beneficial in the washing step. The isolated inclusion bodies are stable for extended periods of time at -10 to - 70°C.

In the preparation of trypsin, the isolated inclusion bodies are solubilized, which results in the unfolding of the trypsinogen protein, including the reduction of any disulfide bridges, and disaggregation of the debris in the inclusion body. This is accomplished with the aid of a chaotropic solution, like urea and/or guanidine hydrochloride; arginine may also be used. Generally, to completely unfold the protein, the chaotrope should be present in an amount of at least about 6 molar, but at least about 7 molar is more effective. In addition, the disulfide bridges are reduced by the action of a reducing agent (generally about 5 to 100 mM is suitable) , such as dithiothreitol , dithioerythreitol, beta-mercaptoethanol , cysteine, and glutathione. In the case of cysteine and glutathione, it is sometimes beneficial to use preparations that are about 10% to about 33% in their reduced forms

(cystine and glutathione disulfide, respectively) . It is also beneficial to use an elevated (above neutral) pH, generally above pH 8, but more beneficially above about pH 9, e . g. , around pH 9.5. EDTA at about 10 to 50 mM is also useful .

The solubilized inclusion bodies may then be refolded according to standard methods or a preferred modified batch procedure. In this procedure, the trypsinogen is refolded by sequential pulsing of solubilized material into a single aliquot of refolding solution, described below. The process of denaturation is essentially reversed by lowering the concentration of chaotrope and reducing agent, as a result of dilution, thereby allowing the protein to refold and re- oxidize. In order for refolding and oxidation to occur, the amount of chaotrope and reducing agent must be lowered below a certain threshold. Thus, the ratio of the aliquot of solubilized material added to the refolding solution should be such that the concentration of chaotrope (contributed by the added solubilized material) is kept low enough to allow the trypsinogen refold properly, i.e., to assume a conformation capable of being processed to form trypsin. Typically, the concentration of chaotrope that allows refolding is between about 3 and about 4.5 molar. Trypsinogen is believed to undergo a conformational change at about 3.5 molar.

In a preferred method that is applicable to refolding inclusion body-localized proteins generally (not just trypsinogen) , each pulse (aliquot) typically increases the protein concentration such that aggregation does not substantially occur - proper folding is preferred, for example, in the refolding solution by about 10 mg/1 to a typical total maximum of about 60 mg/1, resulting in a dilution of about 100- to about 1000-fold. Generally about 2 to about 10 pulses are used, with about 3 to about 8 being more preferred, and about 4 to about 7 being even more preferred. In some instances, the refolding solution will contain from about 20 mg/liter to about 100 mg/liter of protein. In a preferred embodiment, the refolding solution will contain from about 30 mg/1 to about 80 mg/1.

When adding the solubilized material to the refolding solution, smaller pulses (i.e, resulting in lower final concentrations) are quite acceptable, as are slightly larger pulses. In general, final concentrations per pulse of about 1 mg/1 to about 12 mg/1 are useful. Such a concentration assures that the local concentration of unfolded material is kept at a low enough concentration to prevent unacceptable levels of aggregation (a symptom of improper folding) , thereby reducing the yield of properly folded protein. Thus, if substantial aggregation occurs, smaller pulses can be employed.

Another mechanism of lowering the local protein concentration and thus increasing the yield of properly folded protein is to increase the rate at which the solubilized material is dispersed in the refolding solution. This can be accomplished by using devices for achieving higher rates of mixing, dispersion devices, and the like, or combinations. Such devices include jet nozzles for transferring the sample to the refolding solution, high speed mixers, ultrasound, high-flow recirculating loops, turbine agitators and the like. For example, whereas normal methods result in sample application to the refolding solution at a rate of about 1 m/sec, using a spray nozzle to propel the sample increases this by 1-2 orders of magnitude, to about 10 to 50 m/sec and generally to at least about 5 m/sec. Similarly, the rate of mixing can be increased using a high speed mixer from the typical about 1-10 m/sec at the propeller tip to about 10 to about 100 or more m/sec at the propeller tip.

In this preferred method, between each pulse, the solution is allowed to equilibrate. This equilibration is carried out for a time sufficient to allow the refolding of each aliquot to proceed to near completion prior to the addition of the next aliquot, thereby reducing the likelihood of illegitimate inter- and intra-molecular interaction and favoring the proper intramolecular interactions. The pulse/equilibration rationale is based on the inventor's insight that, while higher concentrations promote aggregation (thereby reducing yield of properly- folded material), properly folded protein is less susceptible to aggregation. Thus, if each pulse is allowed to refold prior to the addition of the next pulse, higher concentrations of protein can be achieved in the refolding reaction .

Refolding can be monitored by reverse phase HPLC, as described below. Refolding is usually complete (equilibrium obtained) in about thirty to forty minutes. This technique has the advantage of allowing higher protein concentrations than conventional methods, thereby substantially reducing the volume of the refolding reaction. It also avoids having to empty the reaction chamber as often because it results in a higher final concentration of protein than conventional methods .

The refolding solution is generally comprised of a buffer that maintains an elevated pH, for example pH 9, and optionally some calcium ion (e.g. CaCl₂) which helps to stabilize the enzyme. One suitable buffer contains 5 mM Tris, 3 mM cysteine, 1 mM cystine and 50 mM CaCl₂ at a pH of approximately 9. Some standard conditions suitable in general for refolding inclusion bodies, which also may be employed in the present methods, may be found in the literature, like Rudolph et al . , U.S. Patent No. 5,077,392 (1991). The refolding reaction is beneficially accomplished below room temperature and usually from about 4 °C to about 15 °C . The reaction is quenched by reducing the pH to below neutral, ideally to between about pH 3 to about pH 5.

The quenching usually results in the formation of some aggregate which can be removed by centrifugation or another suitable method. The optionally clarified trypsinogen solution is then subjected to classical protein purification steps, as detailed below. Although in some instances it is beneficial to purify, via chromatography or other suitable means, the trypsinogen in its unfolded state (in the presence of high concentrations of chaotrope and/or reducing agent), in most instances, the trypsinogen will be refolded prior to any chromatographic or other purification steps.

The terms and abbreviations used in this document have their normal meanings unless otherwise designated. For example, "°C" refers to degrees Celsius; "mmol" refers to millimole or millimoles; "mg" refers to milligrams; "ml" refers to milliliters; "μg" refers to micrograms; and "μl" refers to microliters.

All nucleic acid sequences, unless otherwise designated, are written in the direction from the 5' end to the 3' end, frequently referred to as "5' to 3'".

All amino acid or protein sequences, unless otherwise designated, are written commencing with the amino terminus ( "N-terminus " ) and concluding with the carboxy terminus ( "C- terminus" ) .

The term "recombinant DNA expression vector" as used herein refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated. The term "expression vector system" as used herein refers to a recombinant DNA expression vector in combination with one or more trans-acting factors that specifically influence transcription, stability, or replication of the recombinant DNA expression vector. The trans-acting factor may be expressed from a co-transfected plasmid, virus, or other extrachromosomal element, or may be expressed from a gene integrated within the chromosome.

The term "promoter" refers to a DNA sequence which directs transcription of DNA to RNA.

The term "processed polypeptide" refers to a polypeptide or protein wherein the N-terminal leader sequence has been removed to yield the desired polypeptide of interest.

The term "trypsinogen" refers to an inactive serine protease zymogen which can be converted to trypsin (defined below) by removal of a leader sequence. Trypsinogen variants are also included. The native bovine enzyme, for example, has a leader comprising the sequence Val (Asp) ₄Lys . In the context of the invention, any native leader may be eliminated and replaced with another leader that is removable by alternative means. The important feature is that the protein with the leader has no trypsin activity, whereas the converted protein does . Preferred trypsinogens also have a methionine codon at the N-terminus, and may have an additional amino acid preceding the native valine (or corresponding amino acid in a non-native leader) . "Trypsinogen" encompasses both trypsinogen proteins and non- native analogs. Examples of native trypsinogens include human, bovine, porcine, ovine, rodent (e.g., rat or mouse) and the like. See, for example, Walsh (1970) Trypsinogens and trypsins of various species . Methods Enzvmol . 19, 41-63.

As used in this application, the term "met-X- trypsinogen" refers to trypsinogen, as defined above, with an additional methionine and one other amino acid at the N- terminus . Thus, "met-phe-trypsinogen" refers to trypsinogen with an additional methionine and an additional phenylalanine at the N-terminus. The bovine "met-phe- trypsinogen, " for example, has the N-terminal sequence MetPheVal (Asp) ₄Lyε- .

The term "trypsin" or "trypsin-li e enzymes" are used interchangeably to refer to proteases which have the ability to cleave a peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine, in a polypeptide. Trypsin proteins are generally those classified as EC 3.4.21.4 (formerly EC 3.4.4.4). Both native and non-native trypsins are included. Trypsin proteins have been isolated and characterized from numerous species including bovine, rat, and humans. Le Huerou et al . (1990) Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen . Structural identi ty wi thin the trypsin family, Eur . J. Biochem. 193, 767-773; Craik, C.S. et al . (1984) J. Biol . Chem. 259:14255-14264; Fletcher, T.S. et al . (1987) Biochemistry 26:3081-3086.

Detailed mechanisms for the catalytic hydrolysis of peptide and ester substrates by serine proteases have been established and trypsin is a well-researched member of this class of enzymes. A large number of trypsin mutants have been made in order to elucidate the catalytic mechanism leading to proteolysiε. Knights, R.J. et al . (1976) J . Biol. Chem. 251:222-228. In addition, many Biochemistry text books use trypsin aε an example when discussing general enzyme structure and function. Mathews van Holde, Biochemistry 355 (1990) . Thus, a protein having trypsin activity includes a large group of enzymes which are well- conserved between specieε and which function by cleaving the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine. Accordingly, the artisan will be familiar with a wide array of trypsin (and thus trypsinogen) analogs useful in the invention. The term "enterokinase" or "enteropeptidase" refer to proteases generally produced in epithelial cells that activate trypsinogen to trypsin by cleaving off the trypsinogen leader sequence from the amino terminus of the protein.

The term "trypsinogen analogs" includes any protein having a non-native sequence which may be activated to form trypsin, typically via a proteolytic or chemical cleavage. Such analogs specifically include trypsinogen which has been mutated εuch that it cannot be converted to active trypsin by the action of trypsin or trypsin-like enzy eε, but rather muεt be converted via εome alternative cleavage mechanism. For example, different proteolytic enzymes and their cleavage sites may be employed, as may chemicals mean, like cyanogen bromide cleavage of methionine residues . The artisan will be familiar with a multitude of such schemes. Trypsinogen analogs also include trypsinogen that has a non- native amino acid sequence.

The term "trypsinogen variants" generally includes sequences related to the native trypsinogen sequences that retain the prescribed functional characteristics and share greater than about 55 percent sequence identity with the native sequences at the DNA level . Still other analogs share greater than about 65 percent identity or greater than about 70 percent identity. Yet others share greater than about 75 percent identity or greater than about 80 percent identity. On the protein level preferred analogs have greater than about 75 percent identity or greater than about 80 percent identity. More preferred analogs will have at the protein level greater than about 85 percent identity or greater than about 90 percent identity. Such analogs may be prepared with reference to the sequences in Figure IB.

As used herein, "percent identity" is used with reference to the Blast 2 algorithm, which is available at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST), using default parameterε . References pertaining to this algorithm include: thoεe found at http : / /www.ncbi .nlm.nih.gov/BLAST/blast_references .html ; Altεchul, S.F., Giεh, W. , Miller, W. , Myerε, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol . Biol. 215:403-410; Gish, W. & Stateε, D.J. (1993) "Identification of protein coding regionε by database similarity search . " Nature Genet . 3:266-272; Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzvmol . 266:131-141; Altεchul, S.F., Madden, T.L., Schaffer, A.A. , Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein databaεe εearch program . " Nucleic Acids Res. 25:3389-3402; and Zhang, J. & Madden, T.L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analyεis and annotation." Genome Res . 7:649-656.

The term "trypsin analogs" refers to the mature formε of the trypsinogen analogs of the invention.

The term "autoactivation" or "autocatalytic" refers to the ability of trypsin to activate trypsinogen by cleaving the leader sequence to produce more active trypsin.

This section sets forth useful, non-exclusive methodologies for practicing the invention.

Wild-type trypsinogen genes can be obtained by a plurality of recombinant DNA techniqueε including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis . { See e . g. , T. Maniatis et al . , Molecular Cloning: A Laboratory Manual, (2d ed. 1989) . Sourceε of trypsinogen genes can be identified by searching GenBank at http://www.ncbi.nlm.nih.gov/ and by conducting Blast searcheε of, for example, bovine trypεinogen (pancreatic cationic pretrypεinogen is Accession No. D38507 and anionic trypsinogen is Accession No. X54703), at http://www.ncbi.nlm.nih.gov/blast/blast.cgi. The isolated gene can then be modified or mutated to encode any one of a variety of trypεinogen analogs.

The isolated nucleic acids of the present invention can be prepared by direct chemical syntheεiε by methods such as the phosphotriester method of Narang, et al . , Meth. Enzymol . 68:90-99 (1979); the phoεphodiester method of Brown, et al . , Meth. Enzvmol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al . , Tetra . Letts. 22:1859-1862 (1981) ; the solid phase phosphoramidite trieεter method deεcribed by Beaucage and Caruthers, Tetra. Letts. 22 (20) : 1859-1862 (1981), e.g., using an automated syntheεizer, e.g., as deεcribed in Needham-VanDevanter , et al., Nucleic Acids Res. 12:6159-6168 (1984); and the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis generally produces a single-εtranded oligonucleotide, which may be converted into double-εtranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA poly erase using the single strand as a template.

The trypsinogen cDNA can be isolated from a library constructed from any tissue in which said gene iε expreεsed. Methods for constructing cDNA libraries in a suitable vector such as a plaεmid or phage for propagation in prokaryotic or eukaryotic cells are well known to those skilled in the art. (See e.g., MANIATIS ET AL., supra) . Suitable cloning vectors are well known and are widely available.

In one method, mRNA is isolated from a suitable tissue, and first strand cDNA syntheεiε iε carried out. A second round of DNA synthesis can be carried out for the production of the second strand. If desired, the double-stranded cDNA can be cloned into any suitable vector, for example, a plasmid, thereby forming a cDNA library. In addition, a variety of different cDNA libraries can be purchased commercially (Clontech Laboratories Inc., Palo Alto, California) .

Oligonucleotide primers targeted to any suitable region of the trypεinogen gene can be uεed for PCR amplification. See e . g. PCR PROTOCOLS: A GUIDE TO METHOD AND APPLICATION (M. Inniε et al . eds . , 1990). The PCR amplification compriseε template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetuε, Norwalk, CT) . A positive reεult is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis .

The present invention alεo relateε to vectors that include isolated nucleic acid molecules of the present invention, host cellε that are genetically engineered with the recombinant vectorε, and the production of trypεinogen polypeptides or fragments thereof by recombinant techniques .

The nucleotides encoding trypεinogenε can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced into a host by transfection, electroporation or other conventional methods. Bacterial viral vectors can be packaged in vitro using packaging cell extracts commercially available and then transfected into host bacterial cells.

In the case of expresεion vectors, the trypsinogen will be operably linked to a control sequence that directs intracellular expression. Such a control sequence generally comprises a promoter, a ribosome binding site and an ATG start codon (where one is not present in the native sequence) . Promoters and ribosome binding sites suitable for use in icroorganiεmε are well-known in the art. Since the preεent vectors are adapted for making only intracellular proteins, secretion signals are not included. In general, the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, to name a few. Other suitable promoterε will be known to the εkilled artiεan. The expreεεion conεtructε will further contain εiteε for tranεcription initiation, termination and, in the tranεcribed region, a riboεome binding εite for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated.

Expression vectors will preferably include at least one selectable marker. Such markers include tetracycline, ampicillin, kanamycin, or chloramphenicol resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli , Streptomyces and Salmonella typhimurium cells. Appropriate culture mediums and conditionε for the above-described host cells are known in the art. Vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectorε, Phageεcript vectors, Bluescript vectorε, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Other εuitable vectors will be readily apparent to the skilled artisan.

Introduction of a vector construct into a host cell can be effected by calcium phosphate transformation, DEAE- dextran mediated transformation, cationic lipid-mediated transformation, electroporation, transduction, infection or other methodε . Such methodε are deεcribed in many εtandard laboratory manualε, εuch aε Sambrook, εupra, Chapterε 1-4 and 16-18; Auεubel , εupra, Chapters 1, 9, 13, 15, 16. Trypsinogen of the present invention can be expressed in a modified form, such as a fusion protein, and can include additional heterologous functional regions. For instance, a region of additional amino acids can be added to the N-terminus of an analog to improve εtability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to facilitate purification. Such regions can be removed prior to final preparation of an active enzyme. Such methods are described in many standard laboratory manuals, such as Sambrook, εupra, Chapterε 17.29-17.42 and 18.1-18.74; Auεubel, supra, Chapters 16, 17 and 18.

Using trypsinogen nucleic acids, one may expresε the encoded protein in a recombinantly engineered prokaryotic cell, such as a bacterium. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time) , because they have been genetically altered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in the numerous expreεεion systems available for expresεion of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes will be made.

In summary, the expresεion of iεolated nucleic acids encoding a trypsinogen protein will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible) , followed by incorporation into an expresεion vector. The vectors can be suitable for replication and integration in prokaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences and promoterε useful for regulation of the expresεion of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectorε which contain, at the minimum, a εtrong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator .

One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expresεion, incorporation of the targeting molecule into a fusion protein, or purification of the protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to facilitate purification of the protein or other cleavages to create conveniently located restriction sites or termination codons .

Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli ; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lacta ase (penicillinase) and lactose (lac) promoter systems (Chang, et al . , Nature 198:1056 (1977)), the tryptophan (trp) promoter εyεtem (Goeddel, et al . , Nucleic Acids Res. 8:4057 (1980)), the bacteriaphage T7 promoter and RNA polymerase, and the bacteriaphage lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al . , Nature 292:128

(1981)). The incluεion of εelection markers in DNA vectors transfected in E. coli iε also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol. The vector is selected to allow introduction into the appropriate hoεt cell. Bacterial vectorε are typically of plaεmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plaεmid vector iε used, the bacterial cells are transformed with the plasmid vector DNA. Expreεεion εyεtems for expresεing a protein of the preεent invention are available using Bacillus sp . and Salmonella (Palva, et al., Gene 22:229-235 (1983); Mosbach, et al . , Nature 302:543-545 (1983)).

Once an expreεsion vector carrying the trypsinogen gene is transformed into a suitable hoεt cell uεing standard methodε, cells that contain the vector are propagated under conditions suitable for expression of the recombinant trypsinogen protein. For example, if the recombinant gene has been placed under the control of an inducible promoter, suitable growth conditions would incorporate the appropriate inducer. The recombinantly-produced protein may be purified from cellular extracts of transformed cells by any suitable means .

Most favorably trypεinogens are expressed in a manner that resultε in the substantial majority of recombinant protein localizing within insoluble inclusion bodies. In large part, this will occur without any intervention by the artiεan. However, certain measures may be taken to increase the fraction of inclusion body-localized trypsinogen. For instance, growth conditions are known to have a profound effect, which may be determined empirically. Important variables include temperature (e.g., 25°C to 42°C for E. coli ) , medium (minimal verεus rich) , dissolved oxygen, pH, and the like. Moreover, a variety of strains may be tried.

Inclusion bodies consist in large part of unfolded and/or misfolded recombinant protein that iε generally inactive. The advantages of inclusion bodieε include increased resistance to proteaseε and ease of purification, since they generally may be separated from the majority of cellular proteins by centrifugation and washing steps. Moreover, the increased resistance to proteaseε preventε the premature maturation of trypεinogen to trypεin, would likely would have toxic effectε . Thuε, inclusion bodies allow for production of essentially native trypsinogen in an inactive form that can be subεequently activated in a controlled fashion, described below, to yield highly purified trypεin. The iεolation of incluεion bodies is known, as detailed in Rudolph et al . , U.S. Patent No. 5,077,392 (1991), which iε hereby incorporated by reference in itε entirety.

One problem preεented by incluεion bodies, however, iε the ultimate recovery of properly folded activated protein. In order to obtain active protein, the inclusion bodies must be solubilized, generally under denaturing and reducing conditions which resultε in unfolded protein. Thiε iε generally facilitated by chaotrope like guanidine hydrochloride or urea, in combination with reducing agents like cysteine, beta-mercaptoethanol, 1, 4-dithioerythritol or dithiothreitol . Refolding is achieved by the controlled removal of the denaturing conditions. Oxidation of disulfide bridges is done by removing the reducing agent and with the optional use of oxidizers like copper sulfate, a combination of reduced glutathione/glutathione disulfide or a combination of cysteine/cystine.

Trypεinogens of the present invention can be purified either in their unfolded state or following refolding. It has been observed, however, that refolding prior to purification is beneficial. Well-known methods of protein purification may be employed, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, reverεed-phaεe chromatography, hydroxylapatite chromatography, and lectin chromatography. Moεt preferably, depth filtration iε employed coupled with cation exchange chromatography. Exampleε of trypεinogen purification are deεcribed below.

Additionally, the trypsinogen may be fused at its N- terminal end to several histidine residues. This "histidine tag" enables a single-step protein purification method referred to as "immobilized metal ion affinity chromatography" (IMAC), essentially as described in U.S. Patent 4,569,794. The IMAC method enables rapid isolation of εubstantially pure protein starting from a crude extract of cells that express a recombinant protein, as described above .

Following the isolation of trypsinogen, it may be activated to form trypsin. Methods of activating trypsinogen are well known. Esεentially, it entailε cleaving the zymogen to liberate active trypsin. In native trypsinogen, it can be accomplished, for example, with trypsin itself or with enteropeptidase (enterokinase) . Alternatively, heterologouε proteolytic sites may be utilized between the leader sequence and the trypsin moiety. For example, an aminopeptidaεe site could replace the trypsin cleavage site and aminopeptidase could then be uεed to activate the enzyme. The trypεin εo generated may then be subject to additional purification steps.

The following examples more fully describe the workings of the present invention. Thoεe εkilled in the art will recognize that the particular reagentε, equipment, and procedureε deεcribed are merely illuεtrative and are not intended to limit the present invention in any manner. EXAMPLES EXAMPLE 1 : USING A TRYPSINOGEN PRODUCING EXPRESSION SYSTEM

This example illustrates the use of a trypsinogen- producing expreεεion vector in a fermentation process.

The host-expreεεion vector system used for the production of r-trypsin in this example consists of a derivative of E. coli K12 , RV308, as the host, and a derivative of the plasmid pBR322, called pHKY603, which codes for the production of Methionyl-phenylanalyl-bovine trypsinogen (biosynthetic Met-Phe-bovine trypsinogen) as the expression vector. The resulting strain RV308/pHKY603 is called ELTRP-1.

RV308 is lambda-, F^~ , streptomycin resistant (sm^R), gal

308^", and moderately thiamine- (ATCC 31608) . This strain was first described in the literature in 1980 (J. Mol .

Biol. 1980; 139:147-161). It derives from a long line of laboratory strains propagated almost continuously εince the original K12 εtrain was isolated. RV308 grows well in minimal media, synthesizing all necessary macromolecules from ammonium, phosphate and magnesium saltε, trace metalε and glucoεe . It is a facultative anaerobe, but is usually fermented under aerobic conditions. It is stable at room temperature and below, but is rapidly inactivated at higher temperatures, and readily killed by steam sterilization. It is stable in a pH range of 5.5-8.0, and inactivated at pH extremes .

pHKY603 is a derivative of the pBR322 plasmid. It contains the origin of replication and the rop copy number control element of pBR322, aε well aε a tetracycline resistance gene used aε a selectable marker.

The expresεion of recombinant trypsinogen iε directed by a modified version (p548) of the leftward RNA tranεcription promoter pL of the E. coli bacteriophage lambda. p548 containε a copy of the E. coli integration host factor (IHF) upstream of the pL promoter, which stimulates transcription from the promoter. The promoter iε repressed at low temperature (<34^SC) by a thermolabile mutant repressor coded by the bacteriophage lambda el⁸⁵⁷ gene contained on the plasmid. Inactivation of the repreεεor protein at temperatureε higher than 35 ^SC allowε full expreεsion of the gene product after activation of the promoter .

The gene coding for Met-Phe bovine trypsinogen is fully εynthetic and waε obtained using published sequences for bovine trypsinogen and human trypsinogen. The portion of pHKY603 containing the promoter-operator sequence and the Met-Phe bovine trypsinogen gene was sequenced after cloning to rule out any sequence alteration

pHKY603 is maintained as a free plasmid in RV308. Recipient RV308 cells were made competent by a CaCl₂ treatment in ice, followed by a brief heat-shock at 32⁹C in the presence of pHKY603. Following a short incubation (1 to 2 hours) at 34²C in TY media, the cells were plated on nutrient agar containing tetracycline (10 μg/ml) . Since the tetracycline resistance is borne on the plasmid, only transformants can grow under selective pressure. These transformants were used to establish a master cell bank consisting of cells grown under εelective preεεure (tetracycline 10 μg/ml) and εtored frozen in 1.5 ml aliquotε at -190²C (vapor phase of liquid nitrogen) in 10% glycerol from beef or pork tallow. The quality of this cell bank was confirmed using several techniques, including restriction analysiε and sequencing of the plaεmid, and phenotype checking of the hoεt .

In order to remove all animal derived raw materialε from the r-trypεin proceεs (bacto-peptone from the culture media and glycerol), one ampoule of the cell bank was thawed and plated on YES broth. From thiε culture, one colony waε εelected and streaked on YES broth again. Several isolated colonieε from this culture were spotted onto selective media to confirm host and plasmid markers (lac-, Sm^R and Tc^R) . A phenotypically correct colony was inoculated into YES-broth containing tetracycline (10 μg/ml) and incubated for 12 hours on a rotary shaker at 30²C. The resulting culture waε supplemented with 10% synthetic glycerol and aεeptically transferred into sterile 1.5 ml cryovials which were stored at -190²C (vapor phase of liquid nitrogen) , resulting in an animal product-free cell bank. The quality of this cell bank was confirmed using several techniques, including restriction analysis and sequencing of the plaεmid, and phenotype checking of the host.

The procesε waε initiated by removing one ampoule of the animal product-free cell bank (RV308/pHKY603 ) from itε liquid nitrogen storage. The content of the ampoule was aseptically streaked on YES-agar and incubated at 30^SC overnight, to obtain a supply of well isolated clones, which were in turn spotted onto εelective media to confirm hoεt and plaεmid markerε (lac-, Sm^R and Tc^R) . A phenotypically correct clone waε inoculated into YES-broth containing tetracycline (10 μg/ml) and incubated 6-8 hourε on a rotary εhaker. The broth waε εupplemented with 10% synthetic glycerol and aseptically tranεferred into 0.3 ml sterile cryogenic strawε, which were stored in the vapor phase of liquid nitrogen (-190²C) . These straws constitute a working cell bank.

One straw from the working cell bank is thawed at 30²C and used to inoculate 50 ml of YES-broth containing tetracycline (10 mg/ml) . After incubation for 7 hourε at 30°C, the broth iε aεeptically diεpensed (approximately 1.1 ml) into pre-εterilized cryogenic vialε, and frozen in the vapor phase of liquid nitrogen (-190°C) . To start a fermentation, a single cryogenic vial is removed, thawed at not more than 30°C, and used to inoculate a continuation YES-broth culture containing tetracycline (lOμg/ml) . The culture iε incubated at 30²C on a rotary εhaker for 4 hourε, and iε used to inoculate several 2 liter shake flaskε containing 500 ml of defined medium (modified M-9) with glucose, salts and tetracycline (10 μg/ml) . The cultures are incubated on a rotary εhaker at 30²C for 8 to 10 hours, and pooled into a sterile transfer vessel .

A seed vessel containing a defined medium εupplemented with glucoεe, salts, thiamine and tetracycline iε inoculated with the contents of the transfer vessel. This culture is allowed to grow at 30²C under controlled conditions. The contents of the seed vessel then is used to inoculate the 6000 liter production fermentor containing a defined medium supplemented with glucose, salts, thiamine and tetracycline. In the production vessel, a growth phase at 30²C (6 to 8 hours) is followed by a rapid temperature increase to 36²C where the production phase is initiated. An a ino-acid feed (containing L-Serine at 2%, DL-Methionine at 0.6%, Potassium phosphate dibasic at 0.62% and Potassium phosphate monobasic at 2.1%) is added at induction time and throughout the remainder of the process. The total fermentation time is about 18 hours. At the end of the fermentation, the temperature in the fermentor is optionally increased to approximately 60^SC for approximately 15 minutes.

A sample of broth is removed from the tank prior to the heat treatment and streaked on L-agar for iεolated colonieε. Theεe colonies are spotted onto selective media to confirm the maintenance of the proper phenotype markers . The εame sample is may also be asεayed for contamination and incluεion bodieε formation (microεcopic examination) aε well as product accumulation, for example, by HPLC .

Table 2 shows the composition of a typical fermentation medium. - 31

EXAMPLE 2 ; ISOLATION OF TRYPSINOGEN AND MATURATION TO TRYPSIN

Thiε example illustrates how to use an expression system to make the trypsinogen aggregate products, the trypsinogen solutions and the essentially chymotrypsin-free trypsin products of the invention.

Fermentation broth, prepared aε detailed above, is first concentrated by centrifugation (solid-liquid separation) . The concentrate containing the cells is processed over a cell disrupter (homogenizer or equivalent) until sufficient cell breakage is detected by microscopic evaluation. After dilution of the disrupted cells in water, a solid-εolid separation is performed by differential centrifugation in two independent passes to purify the incluεion bodies away from the cell debris resulting from the cell lysis. EDTA (approximately 5 mM) may also be added to facilitate the separation. The concentrated inclusion bodies can be εtored at 2 to 8°C for a few days or frozen at -10 to -20°C for several months before being forward procesεed; they repreεent a trypsinogen aggregate product of the invention. Recombinant trypsinogen contains 12 cysteinyl residues generating 6 disulfide bridges. In the inclusion bodieε, the molecule iε in an heterogeneouε εtate of partial unfolding, hence itε inεolubility . To achieve the refolding of thiε molecule, the incluεion bodieε are firεt εolubilized in approximately 7 M urea/100 mM cyεteine/10 mM EDTA at a pH of approximately 9.5 to reach a concentration of r- trypεinogen of no more than 4 g/1. The reεultant solution is an inventive trypεinogen εolution.

For further processing, the foregoing trypsinogen solution is then added in successive aliquots of equal volume to a refolding solution containing approximately 5 mM Tris, 3 mM cysteine, 1 mM cyεtine and 50 mM CaCl₂ at a pH of approximately 9 to reach a r-trypsinogen concentration of approximately 10 mg/1 for each addition. After each addition, the reaction is allowed to proceed for approximately 40 minuteε at 4 to 15 °C . After six aliquotε of εolubilized granules have been added to the refolding tank, the reaction (now at 60 mg/1) is quenched by adjusting the pH to 3 to 5. The concentration of properly folded r- trypsinogen is determined by reversed-phase HPLC .

Exemplary conditions for HPLC measurement of trypsinogen are as followε:

Mobile Phaεe A (10% Acetonitrile, 0.1% Trifluoroacetic acid)

Mobile Phaεe B (90% Acetonitrile, 0.1% Trifluoroacetic acid)

Column: Vydac C4 4.6mm X 50mm (EN: 214TP5405).

UV detection: 214nm.

Column oven: 50°C.

Gradient :

A large amount of improperly folded proteinε and other impurities precipitates upon acidification of the fold reaction. This precipitate iε removed by εolid-liquid separation on a continuous centrifuge and dead-end filtration (approximately 1 micron cut-off) . Alternatively, this precipitate can be removed using a microfiltration syεtem with membranes ranging from 0.1 to 0.65 microns cutoff.

The clarified folded r-trypsinogen iε captured by cation-exchange chromatography uεing SP550-C resin (Toso-

Haaε) equilibrated in an approximately 50 mM acetic acid/100 mM NaCl pH 3.5 buffer system. The charge is applied downflow at approximately 300 to 500 cm/hr, and the column is washed using the equilibration buffer. The product is eluted by applying a step or a linear gradient using an approximately 50 mM acetic acid/2 M NaCl pH 3.5 buffer system. The resulting fractions are assayed by reversed- phase HPLC and pooled into a mainstream. Thiε step iε performed at 2-8°C.

The activation of the captured r-trypsinogen into r- trypεin in vi tro can be achieved in two ways: by autoactivation at a neutral pH or by seeding with a small amount of r-trypsin at a neutral pH. The mainstream pool from the capture step is diluted to a r-trypsinogen concentration lower than 2 g/1, and the pH of the solution is raised to approximately 8. Tris (approximately 70 mM) and CaCl₂ (approximately 50 mM) are added to the reaction. In the caεe of autoactivation, the reaction iε allowed to react at 2 to 8°C for up to 24 hourε. In the case of a seeded activation, a small amount of r-trypsin

(approximately 1% w/w) is added to the solution, and the reaction is allowed to proceed at 2 to 8°C for approximately 6 hours. In both cases, the advancement of the reaction is monitored by reversed-phase HPLC. Upon completion, the reaction iε quenched by adjusting the pH to approximately 3. The activated r-trypsin iε further purified by affinity chromatography uεing benzamidine εepharoεe resin (Pharmacia) . The resin is packed in a chromatography column and equilibrated with a buffer containing approximately 70 mM Tris/50 mM CaCl₂/100 mM NaCl at pH approximately 8. The activation reaction is applied to the column after a pH adjustment to approximately 8, to allow the r-trypsin to bind to the benzamidine groups chemically linked onto the sepharose backbone. The resin is then washed with the equilibration buffer, and the r-trypsin is eluted by applying a buffer containing approximately 50 mM acetic acid/100 mM NaCl/50 mM CaCl₂ at a pH of approximately 3.5. The resulting fractions are assayed by reversed-phase HPLC and pooled appropriately.

The benzamidine mainstream is concentrated to a final concentration of approximately 5 g/1 in a tangential flow filtration unit using a membrane with a 10,000 dalton nominal molecular weight cut-off, and may be diafiltered with a 50mM acetic acid buffer to lower the salt concentration in the final preparation. The concentrated material is filtered on a 0.22 micron filtration unit and stored in polypropylene containers with polypropylene lids at approximately -70°C.

EXAMPLE 3 : PRODUCT CHARACTERIZATION

This assay setε out a method uεeful in assessing the quality of the inventive products, especially the esεentially chymotrypεin-free products. The data presented below are from proteins prepared according to the methods preεented above.

The purity of r-trypεin iε meaεured by SDS-Page under non-reducing conditionε, followed by densitometric analysis of the bands. The potential impurities that can be measured are small fragments of trypsin or E. coli polypeptides . The method is deεigned to meaεure contamination of the r-trypsin by E. coli proteins from the fermentation. The method also detects trypsin, two chain trypsin, and two other proteolysiε productε of trypεin. Theεe variantε of trypsin are counted as product in the calculation of purity. All bands included as trypsin product have been identified as bovine trypsin sequences as measured by direct sequence analysis of bands from control sample of bovine trypsin eluted from gels. Bands found at all other molecular weights are considered impurities. Out of nine preparations, each showed a purity > 99%, as meaεured by SDS-PAGE under non- reducing conditions.

The activity of trypεin can be measured by following the degradation of a synthetic substrate (TAME or Tosyl-Arg- Methyl-Ester) as measured by a change in absorbance at 247 nm over time. This method is essentially as described by Hummel, Can. J. Biochem. Phvsiol . 37: 1393 (1959). The specific activity (expressed in Units/mg) iε calculated aε the ratio between the activity (expressed in Units per ml) and the protein concentration (expressed in mg/ml) . Generally, high quality trypsin will have a specific activity of >190U/mg. Preferably, the specific activity should exceed 210U/mg, with superior preparations exceeding 220 U/mg. The average specific activity for nine runε was 239.8 U/mg with a standard deviation of 13.7 U/mg.

The specificity of a lot of r-trypsin is determined by performing a complete tryptic digest of a reference standard of glandular glucagon at room temperature using a ratio of 1 mg of trypεin per 100 mg of εubεtrate over a period of 2 hourε, aε described below. Thiε exposure-ratio combination is equivalent to the reaction conditions in most processes using trypsin. The reaction is monitored by reversed-phase HPLC, aε described below. The main tryptic fragments of glucagon are εhown in Figure 2. The reεultε are reported as the ratio of the sum of the area for the tryptic fragments obtained with a sample versus that obtained with a standard (% specificity) . This asεay was designed to measure any degradation of the substrate resulting from contaminating proteases (particularly chymotrypsin) which could have co- purified with r-trypsin throughout the purification procesε.

Glucagon contains several chymotryptic sites and the asεay can detect very low levelε of chymotryptic contamination of trypεin (>0.01% by comparing the area of chymotryptic peaks to the area of tryptic peaks) . None of nine preparations, made as described above, showed any evidence of contaminating chymotrypsin enzymatic activity and gave an average specificity of 99.5% with a standard deviation of 1.08% (n=9), although >95% specificity in some circumstances may be acceptable. Details of this assay are presented below.

EXAMPLE 4 ; HPLC METHOD FOR DETECTING TRYPSIN VERSUS CHYMOTRYPSIN

Thiε aεsay is useful in measuring the purity of trypsin and simultaneously measuring chymotryptic activity.

Equipment

1) A suitable HPLC system capable of gradient elution, equipped with a UV detector and column heater, and chilled autosampler capable of keeping samples between 4 and 9°C.

2) Beckman DU70 Spectrophotometer or equivalent

3) Zorbax SB-C18, 0.46X15 cm, 80A, 5 micron packing (cat. # 883975-902)

4) Pipets able to dispenεe accurately from 10 to 5000μL

5) Stir plate

6) pH Meter

7 ) Vortex 8) Analytical Balance

9) Polypropylene microfuge tubeε 10) Polypropylene 15 mL tubeε or equivalent 11) 25°C water bath 12)0.45 micron mobile phase filtration assembly Materials

1) Glandular Glucagon Reference Standard 3 mg

2) Sigma Bovine Trypsin (bTrp) (Cat. No. T 8003) 3) 0.001 M HC1

4) 0.05 M HOAc

5) 50 mM Borate Buffer, pH 8.5 (see materialε 12 and 13)

6) 1 M CaCl₂ 7) 5 N HC1

8) Needle Waεh Solution - 50% ACN in Milli-Q water

9) Triflouroacetic acid 10) Acetonitrile

11) Phoεphoric Acid 12) Boric Acid

13 ) Concentrated NaOH

14) Ice

15) Milli-Q water or equivalent

Sample Preparation Glucagon Substrate Preparation : One vial of glucagon reference εtandard iε needed to assay two trypsin samples in triplicate. To each vial of glucagon reference standard, add 0.5 mL of 0.001 M HCl . Mix gently, and transfer the solution to a 15 mL polypropylene tube. Add 5 mL of the 50 mM borate buffer. Add 28 μL of the 1 M CaCl₂ stock solution and mix. Measure the pH and adjust to 8.0 +/- 0.1 with IN NaOH if necessary. Hold the εolution on ice until needed. The same substrate solution muεt be uεed for analysis of both the bovine trypsin (bTrp) control and the recombinant trypsin (rTrp) sampleε.

Glucagon Standard Preparation : Aliquot 250 μL of the glucagon εolution prepared in εtep a) into a 1.5 mL microfuge tube. Add 750 μL of the 50 mM borate buffer and mix. Add 50 μL of the 5 N HCl and mix. Hold on ice until needed. bTrp Standard Preparation : Weigh out approximately 1 mg of the Sigma bovine trypεin. Diεsolve in 1 mL of 0.05 M HOAc . Determine the concentration of the bTrp solution by measuring the A280 on the Spectrophotometer . See Data Analyεis section for calculations. Dilute the solution to 0.5 mg/mL with 0.05 M HOAc. Hold on ice until needed.

rTrp Sample Preparation : Determine the concentration of the rTrp sample by measuring the A280 on the Spectrophotometer. See Data Analysis for calculations. Dilute the εolution to 0.5 mg/mL with 0.05 M HOAc. Hold on ice until needed.

Enzyme reaction : For each sample, transfer a 1 mL aliquot of the glucagon reference standard substrate solution into a 1.5 mL eppendorf tube. Add 10 μL of the 0.5 mg/mL bTrp standard εolution or rTrp sample solution and vortex. Place each tube in a 25°C water bath and incubate for 2 hours. After 2 hours, quench the reaction by adding 50 μL of 5 N HCl. Samples must be held between 4 and 9°C prior to analysis or precipitation will occur. Samples should be analyzed within 12 hours after quenching. The bTrp standard must be analyzed uεing the same glucagon substrate solution and in the same HPLC sequence as the rTrp samples .

HPLC Conditions 1) Mobile Phase A - 0.1 % Phosphoric acid and 0.025% TFA

2) Mobile Phase B - 0.1 % phosphoric Acid and 0.025% TFA in ACN

3) Column: Zorbax C18, 5 μm, 80A, 15 cm x 0.46 cm 4) Injection Volume: 20 μL

5) Flow Rate: 1 mL/min

6) Detector: UV at 214 nm

7 ) Autosampler Temperature : 8°C 8) Column Oven Temperature: 60°C +/- 2°C 9) Gradient set aε followε:

10) Needle Wash: 1000 μL after each injection. See

Materials #8, above, for wash solution makeup.

11) Integrate all peaks from 250 to 1150 secondε A sample chromatogram is depicted in Figure 2. A typical HPLC run of trypsin-digested material yields 4 tryptic peaks at retention time of about 6.1 (3), 9.1 (2),

17.4 (1) and 18.7 (4) minutes (parenthetical indicate the ranking of the peaks in order of area, largest to smallest) . The same protocol used with chymotrypsin yielded 5 peaks having retention time of about 8.0 (3), 8.1 (2), 8.7 (5) ,

13.5 (1) and 13.6 (4) minutes (parenthetical indicate the ranking of the peaks in order of area, largest to smallest) . Thus, all chymotryptic and tryptic peaks resulting from glucagon digestion are clearly resolvable .

Standard trypεin εampleε were εpiked with 1%, 0.1% and 0.01% (by weight) of chymotrypεin (Sigma) and the reaction resolved by the same HPLC method. Peak resolution was obtained at all spiking levels, demonstrating that the detection limit of the assay is well below 0.01% by weight. No samples of inventive trypsin preparation that were tested showed any chymotryptic peaks, indicating that chymotrypsin was present in an amount lesε than 0.01%. By injecting more of the reaction mixture, the εenεitivity may be enhanced even f rther. The inherent limit on sensitivity is interference with the chymotryptic peaks by the tryptic peaks. Senεitivity may be increased, it is believed, to levels of lesε than 0.005%, 0.001% and even 0.0005%, merely by increasing the amount of material injected into the HPLC εyεtem.

Claims

WE CLAIM :

1. Trypsin that is esεentially chymotrypsin-free.

2. A trypsin according to Claim 1 that has less than about 0.01% chymotrypsin.

3. A trypsin according to Claim 2 that has leεε than about 0.005% chymotrypsin.

4. A trypsin according to Claim 3 that has less than about 0.001% chymotrypsin.

5. A trypsin according to Claim 1 that is bovine trypsin.

6. A trypsin according to Claim 5 that haε the amino acid sequence of Figure IB.

7. An isolated aggregate comprising trypsinogen and bacterial cellular debris.

8. A liquid composition, comprising a trypsinogen protein that has an N-terminal methionine residue, a chaotrope and a reducing agent .

9. Purified met-X-trypεinogen.

10. The purified met-X-trypsinogen according to Claim 9 that is met-phe-trypsinogen.

11. The purified met-X-trypsinogen according to Claim 9 that is bovine met-phe-trypsinogen .

12. A commercial unit, comprising at least about 50 milligrams of esεentially chymotrypsin-free trypsin.

13. A commercial unit according to Claim 12, comprising at least about 500 milligramε of essentially chymotrypsin-free trypsin.

14. A commercial unit according to Claim 13, comprising at least about 1 kilogram of essentially chymotrypsin-free trypsin.

15. A commercial unit, comprising at least about 50 milligrams of purified met-X-trypsinogen.

16. A commercial unit according to Claim 15, comprising at least about 500 grams of purified met-X- trypεinogen.

17. A commercial unit according to Claim 16, comprising at least about 1 kilogram of purified met-X- trypεinogen.

18. A recombinant expression vector, comprising a nucleic acid encoding trypsinogen that is operably linked to a control sequence that is capable of directing intracellular trypsinogen expreεεion in a prokaryotic organism.

19. A recombinant expression vector according to Claim 18 that encodes a trypsinogen gene that encodes no rare codons relative to E. coli codon usage.

20. A recombinant expression vector according to Claim 18 that encodes a gene shown in Figure IB.

21. Essentially chymotrypsin-free trypsinogen, prepared by the following method: (a) contacting a trypsinogen-containing inclusion body with a solution containing chaotrope, thereby solubilizing said protein,

(b) contacting a refolding solution with more than one aliquot of the εolubilized protein, such that the resulting concentration of protein in the refolding solution is from about 20 mg/1 to about 100 mg/1, and

(c) activating the trypsinogen resulting from (b) , thereby forming trypsin.

22. A method of refolding a protein, comprising:

(a) contacting an inclusion body with a solution containing chaotrope, thereby solubilizing εaid protein, and

(b) transferring the solubilized protein into a refolding solution in more than one aliquot, such that the reεulting concentration of protein in the refolding εolution iε from about 20 mg/1 to about 100 mg/1.

23. A method according to Claim 22, wherein the solubilized protein is contacted with refolding solution under conditions that facilitate rapid diεpersion of the solubilized protein in the refolding solution.

24. A method according to Claim 21, wherein the protein is bovine met-phe-trypsinogen .

25. A method of producing trypsin, comprising:

(a) contacting a trypsinogen-containing aggregate with a denaturation solution,

(b) renaturing said trypsinogen, and (c) activating the renatured trypsinogen to form essentially chymotrypsin-free trypsin.

26. A method of producing trypsin, comprising:

(a) expressing trypsinogen inside a bacterial cell by recombinant DNA methods ,

(b) isolating said trypsinogen, and

(c) procesεing said isolated trypsinogen thereby forming esεentially chymotrypsin-free trypsin.