WO2001051624A2 - Carboxypeptidase b free of animal products and contaminating enyzme activity - Google Patents

Abstract

The invention provides highly purified preparations of Carboxypeptidase B. These preparations are generally free of contaminating protease activity, like chymotrypsin or trypsin activity, free of animal products, bacterial cell components and other contaminants that result from conventional purification processes. The highly purified Carboxypeptidase B preparations are particularly suited for the manufacture of recombinant proteins that require delicate proteolytic processes in their manufacture. Compositions containing the present Carboxypeptidase B and such recombinant proteins are also disclosed. Finally, an efficient yeast-based purification system, with high yield downstream processing steps is provided with which the inventive preparations may be made.

Description

CARBOXYPEPTIDASE B FREE OF ANIMAL PRODUCTS AND CONTAMINATING

ENYZME ACTIVITY

This application claims the benefit of U.S. Provisional Application No. 60/175,781, filed January 12, 2000.

The invention relates generally to recombinant DNA technology. More specifically, the present invention relates to recombinantly produced carboxypeptidase B (CpB) , as well as methods of making the same.

The family of enzymes known as carboxypeptidases is well known in the art. As used in the -art, the term "carboxypeptidase B" generically refers to metallo- exopeptidases which preferentially cleave basic residues from the carboxy terminus of proteins. The amino acid sequences of rat, human and bovine tissue procarboxypeptidases are similar. See Eaton, D. L . , J. Biol . Chem. 266, No. 32, 21833-21838, 1991.

Carboxypeptidase B, also known as Peptidyl-L-lysine (-L- arginine) hydrolase (EC 3.4.17.2) is a zinc-containing pancreatic exopeptidase which specifically removes C-terminal Arginine, Lysine or Ornithine from peptides . Barrett and McDonald, MAMMALIAN PROTEASES, A GLOSSARY AND BIBLIOGRAPHY, Vol. 2 (Academic Press 1985); Coll et al . , EMBO J. 10: 1-9 (1991).

Naturally occurring carboxypeptidase B is produced as an inactive precursor protein, which goes through several maturation steps that result in the final active enzyme. For example, the rat enzyme is produced from a precursor protein, preprocarboxypeptidase B, containing a 108 amino acid long N- terminal fragment which includes the signal sequence (13 amino acids) and an activation peptide (95 amino acids) . Preprocarboxypeptidase B is enzymatically inactive. During transport of preprocarboxypeptidase B to the endoplasmatic reticulum, the signal peptide is cleaved off; the resulting enzymatically inactive procarboxypeptidase B precursor is secreted from the cell. The enzymatically active carboxypeptidase B is then formed by cleavage of the activation peptide by trypsin. Aviles et al . , Biochem. Biophys. Res. Comm. 130: 97-103 (1985).

Carboxypeptidase B is widely used for commercial and research purposes, such as in the production of insulin and other biologically active polypeptides, and in protein sequence analysis. Commercially available carboxypeptidase B, purified from porcine pancreas, however, is very expensive and is not totally free of other proteases. Moreover, CpB typically contains additional animal-derived products, which may include viruses, prions, or other deleterious agents. It is thus desirable to have a defined source of protein that is free of such risk and can be used to produce biopharmaceutical agents, like insulin.

The complete nucleotide sequence of a porcine CpB is known. U.S. Patent No. 5,672,496 (1997). The complete amino acid sequence of bovine carboxypeptidase B also has been published. Titani et al . , Proc . Nat'l Acad. Sci . 72: 1666-- 1670 (1975) . In addition, the complete nucleotide sequence of the rat gene and the human cDNA, for CpB, have been published. Clauser et al . , J . Biol. Chem. 263: 17837-17845 (1988); Yamamoto et al . , J. Biol. Chem. 267: 2575-2581 (1992).

Although recombinant expression systems for the human, rat and porcine enzymes are available, they all suffer from certain deficiencies. Yamamoto et al . , J . Biol. Chem. 267: 2575-2581 (1992); U.S. Patent No. 5,948,668 (1999); and U.S. Patent No. 5,672,496 (1997). The human system generates inactive protein. The rat system is based on an E. coli system that yields protein in inclusion bodies, which necessitates unfolding the protein with chaotropes reducing agents, then refolding it. Such a system typically generates an appreciable amount of misfolded and inactive protein. Finally, although the porcine system is yeast-based, it was not heretofore known that expression in yeast using a defined medium and specific purification steps could yield large- scale, low-cost CpB that meets unprecedented purity characteristics .

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.

Thus, according to this object, the invention provides Carboxypeptidase B (CpB) that has one or more of the following properties: (1) essentially free of contaminating protease. activity; (2) essentially free of bacterial cell components, urea, guanidium chloride, small molecule protease inhibitors and reducing agents; (3) essentially free of animal-derived products . The inventive CpB preferably has the same primary amino acid sequence as native, mature porcine CpB. Also according to this object of the invention, CpB compositions are provided that contain, in addition to the inventive CpB, a second protein that contains at least one non-native basic amino acid at the C-terminus. In one embodiment, the composition contains a second protein having the following configuration: A-X-B, wherein A corresponds to any protein, X corresponds independently to at least one basic amino acid, like arginine and lysine, and B is at least one amino acid. The second protein may be for example, insulin, an insulin analog or an insulin derivative. In the composition, CpB and the second protein typically are present in a weight ratio of from about 10:1 to about 1:10000. In another embodiment, the composition can contain a protease in addition to CpB, such as trypsin.

The invention .also provides further to this object, a commercial unit of CpB. In general a commercial unit contains at least about 50 milligrams of the inventive CpB. In other embodiments, a commercial unit can contain at least about 500 milligrams or at least about 1 kilogram of CpB.

Also in furtherance of this object, the invention provides a method of protein purification. The method typically involves adding to a sample, containing a protein of interest and a protease, an effective amount of a protein- based protease inhibitor, and separating the resultant inhibitor-protease complex from the protein of interest. In one embodiment, the method is adapted to purifying CpB, prepared by the following method: (a) expressing a gene encoding pro-CpB in an eukaryotic organism, thereby forming a pro-CpB expression product, (b) activating the expression product by conversion of pro-CpB to CpB, (c) resolving the CpB on a hydrophobic medium, (d) contacting the resolved CpB with a protease inhibitor, and (e) resolving the contacted CpB of (d) on an ion exchange medium. The CpB product resulting from this method also is within the scope of the invention. Figure 1 shows a comparison of tryptic digest of native porcine carboxypeptidase B with recombinant carboxypeptidase B made according to the invention.

The invention relates to high-grade, purified carboxypeptidase B (CpB) . The inventive CpB is essentially free of contaminating proteases, like trypsin and chymotrypsin. The inventive CpB is useful in processing recombinant proteins to their mature forms and, thus, the invention also relates to compositions comprising the inventive CpB with recombinant proteins. The inventive CpB preferably is purified using a protein-based protease inhibitor. The addition of such an inhibitor during purification, when a contaminating protease is present, inactivates its cognate protease, by binding to it, and alters the protease chromatographic profile sufficiently to allow more efficient separation of the protease from the desired CpB material .

One product of the invention is a highly purified preparation of carboxypeptidase B. ("carboxypeptidase B" is used conventionally and defined below. ) This highly purified carboxypeptidase B meets at least one of the following criteria of purity. Any one or more of the following methods of determining purity can be used. 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, 99.99 or 99.999 percent pure. As used .herein, unless otherwise indicated, expressions of purity are by weight. Some preparations have no detectable contaminants . One method of determining purity by weight is sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS- PAGE) with silver staining and densitometry. Percent purity is expressed as a ratio of the carboxypeptidase B peak area to total peak area, which represents total protein content.

Purity by weight may also be evaluated by standard reverse phase high performance liquid chromatography method as presented generally below in the Examples. In such a case, percent purity of a CpB preparation is evaluated by comparing the integration of the carboxypeptidase B peak to all peaks when the protein is run on HPLC. The preparations of the invention generally show no trypsin, chymotrypsin or carboxypeptidase A activity by this HPLC method.

Purity by weight may also be as measured by specific activity using the hippuryl-L-arginine assay, detailed below and described by Folk et al . J. Biol. Chem. 235: 2272 (1960). Reference pure porcine material should yield at least about 200 U/mg in this assay.

Importantly, preferred carboxypeptidase B preparations are essentially free of contaminating, non-CpB proteases. As used herein, "essentially contaminating protease-free" denotes a preparation that has less than about 0.01% chymotrypsin, trypsin, and/or carboxypeptidase A. Again, all percentages are by weight, relative to carboxypeptidase B, unless otherwise indicated. More preferred essentially contaminating protease- free preparations generally have less than about 0.005% or less than about 0.001% chymotrypsin, trypsin, and/or carboxypeptidase A. In all cases, the measurements of contaminating enzymes are performed in the absence of protease inhibitors added to the assay (although there may be some trace amounts of protein-based protease inhibitors left over from purification) and, thus, these assay measure the absence of protease, not merely the absence of protease activity. Such protease inhibitors include phenylmethylsulfonyl fluoride (PMSF) , diisopropylfluorophosphate (DFP) , Antipain, AEBSF (4- (2-Aminoethyl) -benzenesulfonyl fluoride hydrochloride) , p- APMSF (4 (Amidinophenyl)methanesulfonyl fluoride) , Benzamidine, 3,4-DCI (3 , 4-Dichloroisocoumarin) , Leupeptin, TLCK (L-l-Chloro-3- [4-tosylamido] -7-amino-2-heptanone hydrochloride), chymostatin, and TPCK; (L-l-Chloro-3- [4- tosylamido] -4-phenyl-2-butanone) .

Some highly purified preparations of the invention 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 tryptic, chymotryptic or carboxypeptidase A glucagon peaks.

The carboxypeptidase B 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 nutrients added to yeast cultures, and producing the proteins in a non- mammalian host, like a yeast. 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%, 0.005% or 0.001% mammalian protein and most preferred compositions have no detectable mammalian protein. The skilled artisan will be aware of numerous methods, such as enzyme-linked immunosorbant assays (ELISAs) for detecting contaminating mammalian proteins. Moreover, these preparations are also generally free of animal-derived products, which may include viruses, prions, or other deleterious agents .

The carboxypeptidase B preparations of the invention generally are also essentially free of any bacteria-derived products. As u'sed herein "essentially bacterial product-free" compositions refer to any of the inventive compositions that are essentially free of all bacterially-derived components, like bacterial proteins, carbohydrates and lipids. In general, this is achieved by avoiding the use of bacterial hosts, maintaining aseptic conditions, and avoiding the addition of bacterial products. For example, typical compositions have less than about 1% bacterial protein, but usually have less than about 0.5% bacterial protein or less than about 0.1% bacterial protein. More preferred compositions have less than about 0.01%, 0.005% or 0.001% bacterial protein and most preferred compositions have no detectable bacterial protein and/or no detectable bacteria- derived components. The skilled artisan will be aware of numerous methods, such as enzyme-linked immunosorbant assays (ELISAs) for detecting contaminating bacterial proteins. In addition, since they are not produced in bacteria, and thus avoid inclusion bodies, the CpB preparations of the invention will be free of chaotropes, like urea and guanidiu chloride, and reducing agents, like b-mercaptoethanol and dithiothreitol. In general, the inventive carboxypeptidase B preparations are also free of certain protease inhibitors, like phenylmethylsulfonyl fluoride (PMSF) , diisopropylfluorophosphate (DFP) Antipain, AEBSF (4- (2- Aminoethyl) -benzenesulfonyl fluoride hydrochloride), p-APMSF (4 (Amidinophenyl)methanesulfonyl fluoride), Benzamidine, 3,4- DCI (3 , 4-Dichloroisocoumarin) , Leupeptin, TLCK (L-l-Chloro-3- [4-tosylamido] -7-amino-2-heptanone hydrochloride) , chymostatin, and TPCK; (L-l-Chloro-3- [4-tosylamido] -4-phenyl-2- butanone) . Again, the only way to ensure the absence of such products is to avoid adding them in the first place.

Conveniently, the essentially contaminating protease-free carboxypeptidase B of the invention may be assembled into "commercial units" that are suitable for sale. Each commercial unit generally comprises a bulk quantity of essentially contaminating enzyme -free carboxypeptidase B. A bulk quantity usually comprises at least about 10 mg of essentially contaminating enzyme -free carboxypeptidase B 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 a 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 contaminating enzyme-free carboxypeptidase B may be provided as a thousand one-gram commercial units. Generally, a commercial unit will contain essentially contaminating enzyme -free carboxypeptidase B as a liquid solution. It may, however, be present in a solid form, such as a freeze-dried (lyophylized) powder. Bulking agents and stabilizers (like calcium) are optionally included. A commercial unit also includes the packaging containing the essentially contaminating protease- free carboxypeptidase B, and optionally includes printed product specifications, an inventory control number and/or instructions for use.

The invention further contemplates certain compositions containing CpB. The compositions are useful in preparing proteins which, by their nature, are in need of proteolytic processing. For example, the inventive compositions are useful in preparing recombinantly produced proteins, especially those produced as pro-proteins or fusion proteins .

The inventive compositions have as their essential ingredients CpB, as described above, plus a target protein (a.k.a. a protein of interest) . This target protein is preferably recombinantly produced and in need of some form of proteolytic processing in order to form the "mature" protein. For example, recombinantly produced insulin, insulin analogs and insulin derivatives are typically produced as a single chain that must be processed to an active "mature" form by the action of proteases. This generally is done by the combined action of trypsin and carboxypeptidase B. Accordingly, some inventive compositions consist essentially of a foregoing inventive CpB preparation and insulin, insulin derivatives, or insulin analogs, including the corresponding pre- and prepro- forms of insulin, insulin derivatives, and insulin analogs. Such insulin compositions optionally may contain an additional protease that is helpful in processing the recombinant protein to a final active form. Additional proteases include trypsin. Target proteins that are in need of proteolytic processing include fusion proteins (which may also be pro- proteins) , wherein the immature protein, as it exists prior to proteolytic cleavage, contains at least one non-native amino acid at its C-terminal end. This C-terminal amino acid is a basic amino acid, such as lysine or arginine, which can be cleaved off by CpB to form "mature" protein. In this context "mature protein" does not necessarily mean the protein as it is found in nature. Rather, it means the final, active or optimal form of the protein. The "mature protein" very well could diverge significantly from the sequence as found in nature .

Target fusion target proteins comprise at least one, but preferably two, basic amino acids immediately C-terminal to the "mature" protein sequence, but generally they contain additional amino acids beyond the two basic amino acids . The C-terminal sequence beyond the basic amino acid(s), if present, can be of any variety. For example, this extra C- terminal sequence may confer specific binding characteristics that aid in purification. The two basic amino acids in the target protein render the target protein susceptible to cleavage by trypsin. Hence, when processing such a target, trypsin typically will be added to the target protein preparation to cleave the target . The resultant cleavage product may then be added to the above described CpB, with or without removing the trypsin first, thereby forming an inventive composition. The CpB will then "polish" the C- terminal end of the target protein by removing the extra basic amino acid(s) .

Thus, in preferred compositions, the target protein has the following configuration: A-X-B wherein A corresponds to any protein, X corresponds independently to at least one amino acid selected from basic amino acids, like arginine and lysine, and B is at least one amino acid situated C-terminal of A-X, and may be, for example, a protein domain that aids in purifying the target protein, including His-Tag and the like.

Usually the ratio of CpB to target protein in the inventive compositions is from about 10 : 1 (w/w) to about 1/10,000 (w/w). Preferably, however, the ratio of CpB to target protein in the inventive compositions is from about 1/10 (w/w) to about 1/2000 (w/w) .

The invention further comprehends methods of protein purification that are useful in making the inventive CpB and CpB-containing compositions, but the inventive methods can be used in many other contexts . The inventive methods may be used, for example, to purify any protein where it is desirable to avoid proteolytic degradation. These methods are based in part on the surprising observation that adding a protein to a protein purification protocol facilitates the purification, rather than hinders it. It is axiomatic in protein purification that the goal is to remove contaminating proteins from target protein, and it is generally not advantageous to add non-target protein to the target protein preparation. Contrary to conventional wisdom, however, the present inventors discovered that the addition of a protein-based protease inhibitor actually facilitates purification of target proteins .

The inventive purification methods avoid entirely small molecule protease inhibitors, like phenylmethylsulfonyl fluoride (PMSF) , diisopropylfluorophosphate (DFP) , and the like. Rather, they employ at least one protein-based inhibitor. Surprisingly, the addition of such an inhibitor alters the chromatographic profile of the cognate protease, thereby facilitating its removal. Thus, utilizing the present methods results not only in a preparation free of protease activity, but also free of protease (active and inactive forms) . Moreover, these methods result in much lower levels of protease activity than conventional methods. This is demonstrated using the HPLC-based glucagon digestion assay, presented in Example 4.

An exemplary method entails providing a sample, which contains a target protein; adding to that sample an effective amount of a protein-based protease inhibitor, thereby forming an inhibitor-protease complex; and separating the complex from the protein of interest. The sample usually represents a stage in a conventional protein purification process. The "effective amount" of inhibitor is an amount sufficient to essentially completely inactivate the cognate protease. Inactivation can be measured using the assay set out in Example 4. While stoichiometric amounts would be optimal, an excess generally will be required to fully inactivate the protease. In gauging an "effective amount," the artisan will appreciate that it is usually advantageous not to add a large excess of inhibitor, however, since at some level it could begin to complicate the purification process. The protease is inhibited by binding to the inhibitor, and this complex surprisingly confers dramatically altered chromatographic properties on the contaminating protease, which aids in purification. Thus, once formed, the complex may be isolated from the target protein by any suitable means of purification, like chromatography. Conventional protein purification methods are well known and can be found in any suitable reference. See, for example, Deutscher, METHODS IN ENZYMOLOGY, vol. 182 (Academic Press, Inc. 1990) .

Protein-based protease inhibitors useful in the present methods include any conventional protein that inhibits the enzymatic activity of a protease by binding specifically to it. Examples include trypsin inhibitors and chymotrypsin inhibitors. A preferred protease inhibitor is soybean trypsin inhibitor. This preference is based on the fact that trypsin is an integral part of the manufacturing process for rCpB (it is used to activate proCpB into mature CpB, as set out below in the examples) , and trypsin soybean inhibitor is an inexpensive, non-toxic, and' very effective inhibitor of trypsin.

As stated above, the inventive purification methods are applicable to the purification of CpB. Generally, these methods entail providing a CpB-containing sample; adding to that sample an effective amount of a protein-based protease inhibitor, thereby forming an inhibitor-protease complex; and separating the complex from the CpB. One preferred method includes expressing a gene encoding pro-CpB in an eukaryotic organism, thereby forming a pro-CpB expression product; activating the expression product of by conversion of pro-CpB to CpB; resolving the resultant CpB on a hydrophobic medium; contacting the resolved CpB with a protein-based protease inhibitor, thereby forming an inhibitor-protease complex; and resolving the CpB from the complex using an ion exchange medium. Standard protein purification protocols, using hydrophobic and ion exchange media, that are supportive of this method can be found in Deutscher, supra . The terms and abbreviations used in this document have their normal meanings unless otherwise designated. For example, ^*Λ°C" refers to degrees Celsius; " mol" refers to millimole or millimoles; "mg" refers to milligrams; "ml" refers to illiliters; "μg" refers to icrograms; and "μl" refers to microliters.

The term "procarboxypeptidase B" refers to an inactive pro-enzyme form of carboxypeptidase B.

The terms "carboxypeptidase B" or "carboxypeptidase B- like enzymes" are used interchangeably to refer to proteases which have the ability to remove C-terminal arginine, lysine and ornithine residues from proteins. These enzymes may have native or altered sequences, relative to known enzymes. These terms also encompass generically "carboxypeptidase B variants," defined below.

The term "carboxypeptidase B variants" generally includes sequences related to the native carboxypeptidase B 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 SEQ ID NO.: 1. The term "protein-based protease inhibitors" refers to proteins that have the ability specifically to inhibit the enzymatic activity of at least one protease, typically by binding to the protease. Examples include •2-Antiplasmin, Aprotinin; bovine pancreatic trypsin inhibitor (BPTI) , Chicken egg white ovomucoid, Lima bean trypsin inhibitor, «2- Macroglobulin , »l-Proteinase inhibitor; »l-antitrypsin, Turkey egg white trypsin inhibitor, trypsin soybean inhibitor. Protein-based protease inhibitors do not include small molecule inhibitors, like PMSF, DFP, Antipain, AEBSF (4- (2- Aminoethyl) -benzenesulfonyl fluoride hydrochloride) , p-APMSF (4 (Amidinophenyl)methanesulfonyl fluoride), Benzamidine, 3,4- DCI (3 , 4-Dichloroisocoumarin) , Leupeptin, TLCK (L-l-Chloro-3- [4-tosylamido] -7-amino-2-heptanone hydrochloride) , chymostatin, and TPCK; (L-l-Chloro-3- [4-tosylamido] -4-phenyl-2- butanone) , and the like.

The term " insulin analogue " is used in its conventional sense to refer to insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including non-coding amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids. Examples of conventional insulin analogs can be found in Norup et al . , U.S. Pat. No. 5,866,538 (1999).

The term "insulin derivative" refers to insulin or an insulin analogue in which at least one organic substituent is bound to one or more of the amino acids . Examples of conventional insulin derivatives can be found in Norup et al . , U.S. Pat. No. 5,866,538 (1999). This section sets forth useful, non-exclusive methodologies for practicing the invention. The CpB of the invention is prepared in an eukaryotic cell. General methods useful in recombinant protein production in eukaryotes are known, and described briefly below.

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

a) Isolation or construction of a DNA encoding CpB; b) integrating said DNA into an eukaryotic expression vector in a manner suitable for the expression of the

CpB, either alone or as a fusion protein; c) transforming an appropriate host cell with said expression vector, d) culturing the transformed host cell in a manner to express the CpB; and e) recovering and purifying the recombinantly produced CpB.

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 CpB that is produced by the host. In order to obtain the essentially bacterial product-free compositions, likewise, the use of a bacterial host and the addition of bacterial products is avoided by using a defined synthetic medium, as discussed below.

Wild-type carboxypeptidase B genes can be obtained by a plurality of recombinant DNA techniques 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). Sources of carboxypeptidase B genes can be identified by searching GenBank at http://www.ncbi.nlm.nih.gov/ and by conducting Blast searches, for example, of porcine carboxypeptidase B at http://www.ncbi.nlm.nih.gov/blast/blast.cgi. The porcine sequence can be found at Accession number AJ133775 (or U.S. Patent No. 5,672,496) and the human at either AJ224886 or NM_001871. The rat sequence is set out in its entirety in U.S. Patent No. 5,948,668; see also Accession Nos . M23953, M23959 and M23950.

The isolated carboxypeptidase B nucleic acids can be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al . , Meth. Enzymol .

68:90-99 (1979); the phosphodiester method of Brown, et al . , Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage, et al . , Tetra. Letts. 22:1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22 (20) : 1859-1862 (1981), e . g. , using an automated synthesizer, e.g., as described 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- stranded oligonucleotide, which may be converted into double- stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.

The carboxypeptidase B cDNA can be isolated from a library constructed from any tissue in which said gene is expressed. Methods for constructing cDNA libraries in a suitable vector such as a plasmid 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 synthesis is 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 carboxypeptidase B gene can be used for PCR amplification. See e . g. PCR PROTOCOLS: A GUIDE TO METHOD AND

APPLICATION (M. Innis et al . eds . , 1990). The PCR amplification comprises template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) . A positive result is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis .

The present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of carboxypeptidase B polypeptides or fragments thereof by recombinant techniques .

The nucleotides encoding carboxypeptidase B 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 .

In the case of expression vectors, the carboxypeptidase B will be operably linked to a control sequence that directs 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 microorganisms are well-known in the art.

In general, the DNA insert should be operatively linked to an appropriate promoter. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site 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 RNA to be translated.

Expression vectors will preferably include at least one selectable marker for propagation. Such markers include tetracycline, ampicillin, kanamycin, or chloramphenicol resistance genes for culturing in E. coli and other bacteria. Moreover, since preferred methods of expression are carried out in yeast, the vector preferably will have at least one yeast selectable marker, like HIS4 and the like. Such markers are well known.

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 methods . Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

Carboxypeptidase B 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 stability 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, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Using carboxypeptidase B nucleic acids, one may express the encoded protein in a recombinantly engineered eukaryotic cell, such as a yeast cell. 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 expression systems available for expression 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 expression of isolated nucleic acids encoding a carboxypeptidase B 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 expression vector. The vectors can be suitable for replication in prokaryotes, and subsequent propagation in eukaryotic, preferably yeast, hosts. Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression 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 vectors which contain, at the minimum, a strong 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, expression, 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 .

Once an expression vector carrying the carboxypeptidase B gene is transformed into a suitable host cell using standard methods, cells that contain the vector are propagated under conditions suitable for expression of the recombinant carboxypeptidase B 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 .

Methods of protein purification are known to the artisan. Preferred methods employ at least one chroma ographic separation, like hydrophobic interaction chromatography and ion exchange chromatography. Particularly preferred methods employ a protein-based protease inhibitor, like soybean trypsin inhibitor, in order to remove proteases.

Generally, CpB is produced as a pro-enzyme, and needs to be activated by the action of trypsin, or another suitable protease. In a preferred purification method, trypsin (preferably yeast-derived recombinantly-produced trypsin, in order to avoid introduction of undesirable mammalian and bacterial cellular components) is employed, and, following activation, the trypsin is inhibited by the action of a protein-based protease inhibitor. The inhibitor complexes with trypsin, which alters the chromatographic profile of trypsin, making it easier to purify away from CpB. While CpB may be activated at any step in purification, it is usually done prior to any chromatography steps. In a preferred method, however, the protein-based protease inhibitor is added after at least one chromatography step.

A particularly preferred method employs a hydrophobic interaction column, followed by addition of the protein-based protease inhibitor, and then an ion exchange column, as detailed in Example 2.

The following examples more fully describe the workings of the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1; RECOMBINANT PRODUCTION OF CPB

The host-expression vector system uses the SMD1163 strain of Pichia pastoris (his4 pep4 prBl) and the pLGD43 plasmid (See U.S. Pat. No. 5,672,496 (1997)), a shuttle vector between E. coli and Pichia pastoris . This vector allows a single copy of the recombinant porcine proCpB gene to be stably integrated in the host genome at a known location.

PLGD43 contains some E. coli portions, derived from pBR322 (ColEl replicon region and the ampicillin resistance gene used as a selectable marker) . The Pichia pastoris regions include two expression cassettes, the HIS4 gene encoding the histidinyl dehydrogenase, used as a marker to complement the histidine auxotrophy of the host and a 650 base pair fragment of the 3' end of the AOX1 gene (3¹ AOXl-B) necessary for site directed integration of the recombinant gene in the Pichia pastoris genome.

Each expression cassette consists of the 5' untranslated region from the Pichia pastoris alcohol oxidase 1 (5' UTR A0X1) gene, a promoter, a signal peptide, the recombinant porcine pro-CpB gene and the 3 ' untranslated region from the Pichia pastoris alcohol oxidase 1 (3' UTR A0X1) gene as a terminator. The promoter used here is that for the gene encoding glyceraldehyde 3-phosphate 'dehydrogenase (pGAPDH) in Pichia pastoris . Though this gene is part of the glycolysis pathway, its expression is unexpectedly regulated by the presence of glucose: it is only active when the glucose concentration drops below a certain level . The signal peptide used here is the human serum albumin signal peptide (preHSA) .

The gene coding for porcine proCpB was obtained from a cDNA library made from total porcine pancreatic RNA, using the Polymerase Chain Reaction technique (PCR) and 5' and 3' primers designed based on the basis of published amino-acid sequences for porcine CpB and pro-CpB pro sequence . After sequencing of the gene and confirmation of the amino-acid sequence of the encoded protein, the gene was subcloned from its E. coli cloning vector (pCRlOOO, Invitrogen, USA) into the secretion vector pLGD43.

A seed culture of pLGD43 was used to inoculate a 6000 liter production fermentor containing a defined medium (M9) supplemented with glucose, salts, and trace minerals including biotin. In the production vessel, a growth phase at 30²C (14- 24 hours) was followed by a production phase signaled by the depletion of the initial glucose in the production vessel. The dissolved oxygen concentration (D02) was controlled by starting a supplemental feed of 80% liquid dextrose (QA127R) at a rate to maintain the D02 at the specified set point. Additional feeds of phosphate and trace minerals were added to the vessel at an age of approximately 48 hours. The fermentation was terminated at 72 hours, and cells were harvested live to recovery. EXAMPLE 2; PURIFICATION OF THE RECOMBINANT CPB

The culture prepared according to Example 1 was purified as follows. The sample was centrifuged at <10 °C in a Westfalia SC35. In a first step, cells and debris were removed at pH 6. The pH of the first pass centrate was raised to 7, salt allowed to precipitate, and a second pass of centrifugation was made.

The centrate was concentrated using a Millipore 10K PES spiral cartridges (UFl) at <10 °C to approximately 650 liters. The concentrate was diafiltered to remove salts and pigments and to add zinc to the process. Tris buffer and zinc were introduced into the process at this point via the diafiltration buffer, which contains 0.1 M Tris/0.1 mM zinc acetate at pH 7.0-7.3.

Activation of the diafiltered CpB was carried out at <10 °C . The pH was raised to 7.5 and recombinant trypsin was added at a ratio of 1/2000 to the mass of r-CpB (estimated from an HLA activity assay, as set out in Example 5). The solution was stirred for 14-18 hours. This step resulted in the maturation of the pro-r-CpB to mature r-CpB.

Next the mature r-CpB preparation was subjected to preparative hydrophobic interaction chromatography (HIC) at <10 °C. The mature r-CpB preparation was brought to a final buffer composition of 1 M ammonium sulfate/0.1 M Tris/0.1 mM zinc acetate/pH 7.2-7.5 and clarified by centrifugation using the SC35, followed by filtration using a Seitz 50S depth filter.

The HIC step was run at 4-8 °C. A TosoHaas HW40C guard column with a volume of approximately 30 liters and a Pharmacia Phenyl Sepharose Fast Flow high substitution capture column with a volume of approximately 80 liters were hooked up in series . The mature r-CpB preparation was loaded through the columns at 75 cm/hr, and the columns were washed at 75 cm/hr with 0.5 Phenyl column volume of Buffer A (1 M ammonium sulfate/0.1 M Tris/0.1 mM zinc acetate/pH 7.2-7.5) to flush the guard column. The guard column was removed, and the Phenyl column was washed with 1 column volume of Buffer A and eluted with a ten column-volume linear gradient of Buffer A to Buffer B (0.1 M Tris/0.1 mM zinc acetate/pH 7.2-7.5).

Fractions of 15% of column volume were collected during elution, and pooled according to their RP-HPLC purity, as set out in Example 4. Trypsin Soybean Inhibitor was added at twice the amount (w/w) of r-trypsin used for activation.

The major HIC fractions containing r-CpB were pooled and the pool was concentrated to 50-100 liters at 4-8 °C on 0.5 m² Pellicon 2 cartridges. The concentrate was diafiltered with 0.01 M Tris pH 7.8-8.2 buffer until the conductivity was <2 mS.

A Pharmacia Q Sepharose Fast Flow column with a volume of approximately 20 liters was used at 4-8 °C for the anion exchange chromatography. The column was charged with the diafiltered CpB pool at 75 cm/hr and washed with one column volume of Buffer A (0.02 M Tris/pH 7.9-8.1) at 75 cm/hr. A ten column-volume linear gradient from Buffer A to Buffer B (0.02 M Tris/0.25 M sodium chloride/pH 7.9-8.1) is run at 50 cm/hr. Fractions of 10% column volume were collected and pooled by RP-HPLC purity.

At 4-8 °C, Zinc acetate was added to 0.1 mM, and the solution was diluted to 4.5-5.5 mg/ml r-CpB with 0.02 M Tris/0.1 M sodium chloride/0.1 mM zinc acetate/pH 7.8-8.2. The final r-CpB solution was filtered and frozen at -20 °C .

EXAMPLE 3; CHARACTERIZATION OF THE RECOMBINANT CPB

N-terminal sequencing of material prepared according to Example 2 gave an identical N-terminal sequence to the porcine CpB standard. However, the CpB made according to the invention showed fewer and different proteolytic fragments.

rCpB

The primary sequence observed corresponds to the predicted N-terminus of Carboxypeptidase B. Two possible CpB- related tertiary sequences were observed at very low level (<5%) : the theoretical residues 138-147 of CpB and the theoretical residues 234-243 of CpB. These sequences do not appear to be the result of enzymatic degradation due to either trypsin or chymotrypsin, since the sequences do not correspond to sites for those proteases and, as described below in Example 6 , the inventive preparations lack these proteases .

The primary sequence observed corresponds to the predicted N-terminus of Carboxypeptidase B. A significant amount of amino acids sequences (>10% each) other than the primary sequence were observed in this sample. The following is a list of possible sequences that were detected in this sample: residues 48-52 of CpB (a chymotryptic-like cut); residues 278-282 of CpB (a chymotryptic-like cut) ; residues

3-7 of CpB (a diamino peptidase-like cut) ; residues 5-9 of CpB (a loss of 4 AA from the N-terminus) ; residues 128-132 of CpB and (tryptic-like cut) . Upon reduction, alkylation and tryptic digest, the 3 lots of r-CpB and the pancreatic porcine CpB control generated the same HPLC pattern. See Figure 1. Further analysis by ESI-MS (Electro-spray ionization mass spectrometry) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization- Time Of Flight Mass Spectrometry) of the digestion products confirmed the similarity between the observed HPLC fragments, with only differences in mass peak intensities being noticeable.

EXAMPLE 4 ; HPLC ASSAY FOR CPB PURITY This example provides another assay useful to assess purity of CpB, for example to analyze fractions for pooling the Phenyl Sepharose and Fast Flow Q Sepharose CpB peaks in the purification set out above, and to assess the performance of processing steps by analysis of pools.

Briefly, HPLC was routinely accomplished using a

Phenomenex Jupiter, 5 •m C5, 4.6 X 150 mm column (part # OOF- 4052-EO), using a gradient of Mobile Phase A (9:1:0.001 v/v/v water/acetonitrile/trifluoroacetic acid) and Mobile Phase B (1:9:0.001 v/v/v water/ acetonitrile/trifluoroacetic acid) at 60 °C, with a flow rate of 1 ml/min and detection at 214 nm. The injection volume was 20 μl and the gradient was run as follows :

A standard curve was run with each sample using the following dilutions of 3.3 mg/ml CpB stock solution: 30 μg/ml; 100 μg/ml; 300 μg/ml. A microfuge was used to clarify samples where precipitates were visible. Where needed to bring the concentration within the standard curve, samples were diluted with Milli-Q water. CpB eluted between 510 and 570 seconds. Standard and sample chromatograms were integrated and used calculate CpB concentrations and total CpB obtained in purification.

EXAMPLE 5; ACTIVITY ASSAY (ΗLA ASSAY") FOR CPB

This example provides an assay useful to measure the enzymatic activity of Carboxypeptidase B (CpB) . The activity is measured using the small synthetic substrate Hippuryl-L- arginine (HLA) ; the hippuric acid released by the CpB is quantitated by HPLC, and CpB standards are used to relate the hippuric acid levels to units of CpB activity. One unit of activity will hydrolyze 1 μmole of HLA per minute at 25°C at pH 7.65.

Briefly, samples and standards are diluted with a phosphate buffer to a concentration range of 40 to 600 micrograms/ml. ProCpB samples are incubated with trypsin at a ratio of approximately 1/1 to 1/20 (w/w) for at least 5 minutes at 25°C. HLA is then added to CpB or activated proCpB samples to achieve a final substrate concentration of approximately 5 mM, and the reaction is allowed to proceed for 5 minutes at 25 °C . The reaction is quenched by the addition of 0.4M phosphoric acid to achieve a final concentration of 0.1M. Samples (25 μl) of the final quenched reaction were resolved on HPLC using an Alltech Adsorbospere UHS C18 5U column (cat. # 288117) with an Alltech Adsorbospere HS C18 5U guard column (cat. # 96079) . The system was maintained at room temperature with a flow rate of 1 ml/min and isocratic elution with 0.1 M phosphate pH 2.5/15% acetonitrile and detection at 229 nm.

EXAMPLE 6; HPLC METHOD FOR DETECTING CONTAMINATING PRQTKASES

The following glucagon-based assay was designed to measure any degradation of the substrate resulting from contaminating proteases which could have co-purified with r- CpB throughout the purification process. While glucagon is not digested by CpB, most proteases that might co-purify with CpB, such as chymotrypsin, trypsin, and the like, can digest glucagon.

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) Beck an DU70 Spectrophotometer

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

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

5) Stir plate

6) pH Meter

7 ) Vortex 8) Analytical Balance

9) Polypropylene microfuge tubes 10) Polypropylene 15 mL tubes

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) and Chymotrypsin (bChymo, Cat. No. C3142)

3) 0.001 M HC1

4) 0.05 M HOAc 5) 50 mM Borate Buffer, pH 8.5 (see materials 12 and 13)

6) 1 M CaCl₂

7) 5 N HC1

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

9) Triflouroacetic acid 10) Acetonitrile

11) Phosphoric 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 standard was used to assay two CpB samples in triplicate. To each vial of glucagon reference standard, 0.5 mL of 0.001 M HCl were added. After gentle mixing, the solution was transferred to a 15 mL polypropylene tube. 5 mL of the 50 mM borate buffer was added, followed by 28 μL of the 1 M CaCl₂ stock solution and mixing. The pH was adjust to 8.0 +/- 0.1 with IN NaOH, where necessary and the solution was held on ice until needed. Glucagon Standard Preparation : The glucagon solution prepared in step a) was divided into 250 μL aliquots into 1.5 L microfuge tubes. 750 μL of the 50 mM borate buffer was added, and followed by mixing. 50 μL of the 5 N HCl were added, and the sample again was mix and held old on ice until needed.

Trypsin/ chymotrypsin Standard Preparation : Approximately 1 mg of the Sigma bovine trypsin/chymotrypsin was dissolved in 1 mL of 0.05 M HOAc . The concentration of the bTrp solution was determined by measuring the A280 on the Spectrophotometer and using a trypsin extinction coefficient of 1.41 ml/mg/cm, and the bChymo solution by measuring the A280 and using an extinction coefficient of 2 ml/mg/cm. The solution was diluted to 0.5 mg/mL with 0.05 M HOAc and held on ice until needed.

rCpB Sample Preparation : The concentration of the rCpB sample was determined by measuring the A280 on the Spectrophotometer and using an extinction coefficient for rCpB = 2.13 ml/mg/cm. The solution was diluted to 0.5 mg/mL with 0.05 M HOAc and held on ice until needed.

Enzyme reaction : For each sample, a 1 mL aliquot of the glucagon reference standard substrate solution was transferred into a 1.5 mL Eppendorf tube, followed by addition of 10 μL of the 0.5 mg/mL protease standard solution or CpB sample solution and vortexing. Each tube was placed in a 25°C water bath and incubated for 2 hours. After 2 hours, the reaction was quenched by adding 50 μL of 5 N HCl and the samples were analyzed within 12 hours . 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 μ , 80A, 15 cm x 0.46 cm

4) Injection Volume: 20 μL

5) Flow Rate: 1 mL/min

6) Detector: UN at 214 nm

7) Autosampler Temperature: 8°C

8) Column Oven Temperature: 60°C +/- 2°C

9) Gradient set as follows:

10) Needle Wash: 1000 μL after each injection. See Materials #8, above, for wash solution makeup.

11) Integrate all peaks from 250 to 1150 seconds

A typical HPLC run of trypsin-digested glucagon yielded 4 tryptic peaks at retention time of about 6.1 (3), 9.1 (2),

17.4 (1) and 18.7 (4) minutes (parentheticals 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 (parentheticals 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 samples were spiked with 1%, 0.1% and 0.01% (by weight) of trypsin and chymotrypsin (Sigma) and the reaction resolved by the same HPLC methodPeak 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 r-CpB that were tested showed any tryptic or any chymotryptic peaks, indicating that trypsin and chymotrypsin were each present in an amount less than 0.01%. Furthermore, no shift in retention time was observed for the products of chymotrypsin digest of glucagon when inventive CpB was added to the reaction mix, indicating the absence of CpA activity in the inventive CpB preparation.

The detection limit using the experimental protocol presented above is about 0.01% for trypsin and chymotrypsin. By injecting more of the reaction mixture onto the column, however, the sensitivity may be enhanced even further. The inherent limit on sensitivity is interference of the proteolytic peaks with the intact glucagon peaks by the proteolytic peaks. Sensitivity may be increased, it is believed, to levels of less than 0.005%, 0.001% and even 0.0005%, merely by increasing the amount of material injected into the HPLC system.

Claims

WE CLAIM :

1. Carboxypeptidase B (CpB) that is essentially free of contaminating protease activity.

2. A CpB according to claim 1 that is free of bacterial cell components, urea, guanidium chloride, small molecule protease inhibitors and reducing agents.

3. A CpB according to claim 1 that is free of animal- derived products .

4. A CpB according to claim 1 that has the amino acid sequence of SEQ ID NO. : 1.

5. A composition consisting essentially of a CpB according to claim 1 and a second protein, wherein said second protein contains at least one basic amino acid at the C-terminus, wherein said basic amino acid is not found in the mature form of said second protein.

6. A composition according to claim 5, wherein said second protein contains at least two non-native basic amino acids.

7. A composition consisting essentially of a CpB according to claim 1 and a second protein, wherein said second protein has the following configuration:

A-X-B wherein A corresponds to any protein, X corresponds to at least one amino acid independently selected from the group consisting of arginine and lysine, and B is at least one amino acid.

8. A composition according to claim 7 , wherein said composition contains a protease in addition to CpB.

9. A composition consisting essentially of a CpB according to claim 1 and a second protein selected from the group consisting of insulin, insulin analogs, insulin derivatives, and pre- and prepro- forms of insulin, insulin analogs, and insulin derivatives.

10. A composition according to claim 6, wherein said Carboxypeptidase B and said second protein are present in a weight ratio of from about 10:1 to about 1:10000.

11. A commercial unit, comprising at least about 50 milligrams of CpB according to claim 1.

12. A commercial unit according to claim 11, comprising at least about 500 milligrams of CpB.

13. A commercial unit according to claim 12, comprising at least about 1 kilogram of CpB.

14. Carboxypeptidase B (CpB), prepared by the following method

(a) expressing a gene encoding pro-CpB in an eukaryotic organism, thereby forming a pro-CpB expression product,

(b) activating the expression product of (a) with a protease by conversion of pro-CpB to CpB,

(c) resolving the CpB of (b) on a hydrophobic medium,

(d) contacting the resolved CpB of (c) with a protein- based protease inhibitor, and

(e) resolving the contacted CpB of (d) on an ion exchange medium.

15. A method according to claim 14, wherein said protease is trypsin and said protein-based protease inhibitor is soybean trypsin inhibitor.

16. A method of producing Carboxypeptidase B, comprising

(a) expressing a gene encoding pro-CpB in an eukaryotic organism, thereby forming a pro-CpB expression product,

(b) activating the expression product of (a) with a protease by conversion of pro-CpB to CpB,

(c) resolving the CpB of (b) on a hydrophobic medium,

(d) contacting the resolved CpB of (c) with a protein- based protease inhibitor, and

(e) resolving the contacted CpB of (d) on an ion exchange medium.

17. A method according to claim 16, wherein said protease is trypsin and said protein-based protease inhibitor is soybean trypsin inhibitor.

18. A method of protein purification, comprising:

(a) providing a sample, which contains a protein of interest and a protease;

(b) adding to said sample an effective amount of a protein-based protease inhibitor, thereby forming an inhibitor-protease complex; and

(c) separating said complex from the protein of interest .

19. A method according to claim 17, wherein said protease is trypsin and said protein-based protease inhibitor is soybean trypsin inhibitor.