JPH10510166A - Methods and compositions for reducing cholesterol absorption - Google Patents

Abstract

  (57) [Summary] Described are compositions derived from all or part of the carboxy-terminal region of human bile salt activated lipase (BAL). When they are taken orally, they compete for the binding of native BAL to the intestinal surface, thus suppressing the physiological role of BAL in mediating the transport of cholesterol to enterocytes, and consequently, blood from the intestine. Decreases cholesterol absorption into the stream. Useful derivatives of the carboxy-terminal region of BAL are derived from all or part of the region containing amino acid residues 539 to 722, and are mucin-like containing at least three repeating proline-rich units of 11 amino acid residues each. Having a structure.

Description

DETAILED DESCRIPTION OF THE INVENTION            Methods and compositions for reducing cholesterol absorption                                 Background of the Invention   The present invention relates to the field of dietetics and more particularly to cholesterol in the intestine. The use of bile salt activated lipase to reduce the uptake of cholesterol.   The U.S. Government has approved this invention under Research Grant No. HD-23472 by the National Institutes of Health. Has certain rights.             Cardiovascular and other diseases associated with high cholesterol   Hyperlipidemia, especially hypercholesterolemia and hyperlipoproteinemia, are atheromatous It is one of the most dangerous factors causing arteriosclerosis. Hyperlipoproteinosis also affects the pancreas Involved in the progress of the flame. According to a long established theory, cholesterol (usually , A form of low-density lipoprotein (LDL) containing cholesterol) The higher the bell, the more cholesterol penetrates the arterial wall, causing atherosclerosis. (Brown and Goldstein, "High lipoproteinosis and Harrison's Principles of Internal Medicine 1650-16 61 (Braunwald et al., 1987)).   Cardiovascular disease is the leading cause of death in women and middle-aged American men. You. In 1988, more than 41,000 U.S. residents were affected by cardiovascular disease by the age of 50. He died of illness. However, it is a known cause of cardiovascular disease and stroke. Atherosclerosis begins to develop at a much lower age. Fat Streaks are common in the arterial wall of children, and high rates of annular artery injury It has been found in young people who have died or died accidentally. High serum cholesterol levels Children and adolescents have children and adolescents with normal cholesterol levels You will probably have more parents with coronary heart disease than those in the early stages. In childhood High serum cholesterol levels can affect autopsy results in adolescents and adolescents. Associated with atherosclerosis of the vena cava, and in people aged 15 to 34 years Both arterial and coronary atherosclerosis have cholesterol levels at necropsy. (Klag et al., New Eng. J. Med. 328 (5): 313-318 (February 4, 1993). Day)).   Cholesterol is a steroid hormone produced by certain endocrine glands, and hepatocytes Used by the main part of the synthesis of bile acids of origin, and is an essential component of the cell membrane is there. It is found only in animals. Related sterols are raw in plants However, plant sterols are not absorbed from the gastrointestinal tract. Dietary cholesterol Is mostly found in egg yolk and animal fats.   Intestinal cholesterol is taken directly from the diet and Bile salts and bile acids, synthesized in the liver and secreted into the intestine through the bile duct Derived from free cholesterol. Bile and dietary cholesterol Stele is absorbed from the lumen of the small intestine by cells of the intestinal epithelial lining, It is taken up and enters the chylomicron, but a small amount is lipoprotein ( VLDL). Both of these are secreted into the lymph and ultimately To join. Chylomicron and VLDL have their own triacylglycerols and And some of its cholesterol to endothelial, muscle, and adipose tissue cells I do. The cholesterol-rich chylomicron remnants and VLDL are then Sterols are returned to hepatocytes and other cells on the vessel wall along their pathways (Ganong, Review of Medical Physiology 249-250 (Lange Medical Publications, 1985)) . Enterocyte and hepatocyte-derived VLDL are responsible for the release of these triacylglycerols. Thus, it can be converted to very low density lipoprotein (LDL). LDL is the total plasma Contains three quarters of sterols.   In hypercholesterolemia, increased blood cholesterol levels are Mainly related to increasing degrees. However, the specific causes of hypercholesterolemia are complex. Yes, and change. At least one type of hypercholesterolemia is mainly Mutation in the LDL receptor gene that removes cholesterol from blood in the liver More provoked. In general, hypercholesterolemia is High cholesterol uptake from the intestine into the circulating blood. Intrusion occurs.   Suppressing hypercholesterolemia delays the development of atherosclerosis And the progression of atherosclerosis is slowed, and therefore humans and other spirits The risk of coronary heart disease in primates is reduced. Specifically, animals, especially primates In, relatively complex plaques induced by hyperlipidemia regress, When hyperlipidemia is removed, the progression of atherosclerosis is terminated. Therefore, Prevent telomeres, interrupt progression, and possibly reduce risk factors Should promote regression of existing lesions (Blerman, Vascular Disorders: Atherosclerosis and Other Forms of Atherosclerosis Harrison's Principles of Internal Medicine 1014-1024 (Braunwald et al., 1987) )).   Some forms of hyperlipidemia, including hypercholesterolemia, are Some recovery may be possible using the current technology. However, the current technology is perfect Have not been successful, and often involve unwanted side effects and complications . Taking cholesterol-lowering drugs can reduce serum cholesterol by 20% . However, drugs are not always certain for hypercholesterolemia, for example , Lovastatin (Lovastatin), mevastatin (mevastatin), cholestyramine (Cholestyramine) (Questran), clofibrate, probuco (Probucol), and some hyperlipidemic drugs such as nicotinic acid Serious side effects, including increased mortality from complications, or, for example, constipation (cholestyrami May have less severe side effects such as skin flushing, muscle dysfunction, or Has the effect of lowering blood triacylglycerol, not blood cholesterol. I can do it. Diet is usually encouraged for all patients with hypercholesterolemia Is not always effective.   Thus, there is a need for effective methods and compositions that provide blood lipid levels ( Specifically, to lower cholesterol levels (especially Disease states that have no significant side effects) and are associated with high blood lipid levels (especially , Patients at high risk for heart disease or who have had a heart attack Is effective in treating   Therefore, an object of the present invention is to provide serum cholesterol in patients in need thereof. It is to provide a composition and a method for use in lowering the temperature.                                 Summary of the Invention   All or part of the carboxy-terminal region of human bile salt-activated lipase (BAL) Are described. When they are taken orally, they Compete for BAL binding to the intestinal surface and thus mediate the transport of cholesterol to enterocytes. Depresses the physiological role of BAL in blood transfusion, and consequently, Decreases cholesterol absorption. Useful derivatives of the carboxy-terminal region of BAL Is derived from all or part of the region comprising amino acid residues 539 to 722, and Has at least 3 repeating proline-rich units of 11 amino acid residues each It has a mucin-like structure. Preferred proline-rich units are Sussus sequence PVPPTGDSGAP (SEQ ID NO: 6).                              BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows C-tail O-glycosylated carbohydrate binding to lectin-like receptor , Cholesterol and cholesterol ester binding to BAL, Proposed binding of BAL to intestinal endothelial cells via hydrolysis of sterol esters Is shown.   FIG. 2 shows the elution of endothelial BAL lipolytic activity, and isotonic pressure from mouse intestinal mucosa. Phosphate buffer, fucose (0.1M), galactose (0.1M), heparin (10mg / m l) or NaCl (0.3 M) was measured at μmol / g / h.   Fig. 3 shows ethylene bis (oxyethylene nitrilo) tetravine to remove BAL. Radioactivity in the intestines of rats treated or untreated with acid (EGTA) Of cholesterol in the form of nmol / g / h of soluble cholesterol (oleate) ester Including   FIG. 4 shows 1 mM EGTA, 0.2 mg / ml C-tail and control in rat intestine. 4 shows a graph of cholesterol uptake rate (nmol / g / h) with respect to cholesterol.   FIG. 5 shows that with and without the C-tail,^ThreeH] -Cholesterol olee In rats fed rats^ThreeH] -Cholesterol level (nmol / mL) vs. hour It is a graph which shows a time (hour). Feeding C-tail isolated from BAL in human milk Blood in isolated rats^ThreeH] -Cholesterol levels are indicated by crosses. Feed C-tail In the blood of unfed rats [^ThreeH] -Cholesterol levels are indicated by squares. Blood sun Pull the [^ThreeAfter feeding H] -cholesterol oleate, collect at hourly intervals for up to 5 hours. I took it.   FIG. 6 shows the use of C-tail isolated from BAL in human milk (shaded) or Not (no diagonal lines)^ThreeBlood of rats fed H] -cholesterol oleate During[^ThreeMean (from 6 experiments) of H] -cholesterol level (nmol / mL) versus time ( hour). Blood samples were radiolabeled cholesterol olee Samples were collected at hourly intervals for up to 5 hours after feeding the first tube of the sheet. Data bar The horizontal line at the top of (bar) indicates the standard error (see Table XIV).   FIG. 7 shows that the intestine was 1) 6 mM in order to remove bound BAL prior to the uptake experiment. EGTA solution containing urocholate (column 1), 2) BAL bound is not removed Buffer without Taurocholate (column 2), 3) 6 mM Taurocore Buffer containing column (column 3), buffer containing 30 mM taurocholate (column 4) Of triolein uptake rate (μmol / g / h) in rats washed in It is rough. Data represent the average of the results of five experiments (see Table XV). All four groups of animals received milky radiolabeled triolein. Was.   FIG. 8 shows the intestine in 1) EGTA solution to remove bound BAL prior to uptake experiments. Liquid (column 1) (shaded), and 2) buffer so that bound BAL is not removed Rate of taurocholate uptake in rats washed with solution (hatched) (μ mol / g / h). Data represent the average of the results of five experiments (Table XVI See). The left and right column pairs are 1 mM and 6 mM radioactive labels, respectively. Shows the uptake rate by Taurocholate.                              Detailed description of the invention   All or part of the carboxy terminal region of bile salt activated lipase (BAL) Included compositions, or their functional equivalents, are described. They form on the intestinal surface. Competes with natural BAL in the intestine when combined with free cholesterol taken up in the bloodstream. Removal of cholesterol ester (eater) or free cholesterol in the form Decrease the function of endogenous BAL to mediate the Bile salt activated lipase   Warm-blooded animals can be found in mammary cells or in some digestive systems, including the pancreas, stomach and small intestine. Forms of Rippers with Different Structures and Activities Secreted by Different Cells Synthesize ze. Bile salts that are substantially inactive per se on physiological substrates Activated lipase (BAL) is activated in the intestine by bile salts. BAL, pancreas And by a few species of mammary glands, including humans, gorillas, cats and dogs. Is synthesized and secreted. The amino acid and cDNA sequences of BAL in human milk are It is the same as that of ruboxyl esterase, and lysophospholipase, Resterol esterase, sterol ester hydrolase, non-specific lipase , Lipase A, carboxylester lipase, and cholesterol ester Closely related to a lipase called terhydrolase, or The same, and depending on the species, mainly for the number of repeat units in the carboxy region (Wang and Hartsuck, Biochim. Biophys. Acta 1166: 1-19 (1993)). Pancreatic BAL is another type of non-bile salt such as pancreatic lipase and phospholipase It is clearly distinguished from activated lipase.   In the intestinal cavity, BAL is located on the intestinal surface, probably through a specific receptor Attaches to cell surface. BAL is stimulated by EGTA, galactose and fucose. Can be released from the surface, but may be heparin, isotonic buffer, or salt as shown below. It cannot be released by sodium chloride. BA is required when bile salts are required L to hydrolyze cholesterol esters to free cholesterol, Alternatively, it is essential for binding to free cholesterol in food. This only Since known pancreatic lipolytic enzymes can mediate cholesterol uptake, Both of these processes are necessary to enable cholesterol uptake. You. BAL is also a source of acylglycerol, phospholipids, and vitamin esters. Hydrolyze ruboxyl ester bonds to form fatty acids and glycerol, Act on immobilized, micellar or soluble substrates. Bile salts are BAL Causes a conformational change of the Lipophilicity and additional lipid binding capacity in forming the enzyme-substrate complex. It is believed to provide. In addition, bile salts are not fatty acids during BAL catalysis. It is believed to act as an acceptor.   The proposed mechanism of action of BAL is shown in FIG. BAL is C-tail O-grid Binds lectin-like receptors on the surface of intestinal epithelial cells via cosylated carbohydrates I do. Enzyme catalytic unit with heparin binding site and active site exposed And stays away from epithelial cells. Then, free cholesterol or kore Sterol esters bind to the active site, and If so, it is hydrolyzed to free cholesterol. Next, the catalyst unit is It binds to heparin on the vesicle surface and transfers cholesterol into cells.   Cholesterol, fatty acids and monoacyl derived from lipolis by intestinal BAL Glycerol is taken up by intestinal epithelial lining cells (mucosal cells) where it is taken up. These components are re-esterified to intracellular triacylglycerols. In cells Cholesterol can be reduced by these re-additions with the addition of apolipoproteins and phospholipids. Interacts with steerylated triacylglycerols, with kilomicrons and ultra-low Forming density lipoproteins. These kilo-micron and ultra-low density lipotan Parkin is secreted into small lymph vessels that ultimately join the vasculature for systemic circulation . Carboxy-terminal region of BAL molecule   Complete and mature human BAL contains 722 amino acid residues (SEQ ID NO: 1). The carboxy-terminal region of BAL refers to the region in the native BAL molecule that includes residues 539-722 . This carboxy-terminal region of BAL is compatible with derivatives of this region that retain intestinal binding activity. Both are referred to herein as "C-tails". The C-tail of the human BAL molecule is , Have many O-linked oligosaccharide units forming a mucin-like structure. Natural arbor The C-tail amino acid sequence consists of 16 repeats, each consisting of 11 amino acid residues. Contains a phosphorus-rich unit, most of which is PVPPTGDSGAP (SEQ ID NO: 6). It has a consensus sequence (Baba et al., Biochemistry 30: 500-510 (1991)). β-desorption Yes By doing so, the natural C-tail is mainly threonine and also one serine It is determined that O-glycosylation occurs to a lesser extent when it occurs at residues. Was. Serine residues with adjacent aspartic acids are preferred for O-glycosylation. (Elhammer et al., J. Biol. Chem. 268: 10029-10038 (1993)). Peptides prepared by cyanogen bromide digestion of the C-tail are carbohydrates of native BAL It was found to contain most of the products (Baba et al. (1991)).   As shown below, a C-tail bond referred to herein as "T-BAL". Truncated forms of BAL lacking the junction do not allow the enzyme to bind to the intestinal surface. It is not effective at transferring cholesterol into enterocytes. Similarly, C -Only the tail binds to the intestinal surface and may actually compete with native BAL for this binding However, the catalytic unit is neither functional nor present, I can't move it. C-tail protein   It is derived from all or part of the region containing amino acid residues 539 to 722, Whip containing at least three repetitive proline-rich units having amino acid residues Derivatives of the carboxy-terminal region of BAL having a C-like structure are referred to herein as C- Called the tail. As used herein, a proline-rich unit is Any of the 11 repeating amino acid groups present in the naturally occurring form of BAL, or When combined with two or more other proline-rich units, it results in intestinal epithelial cells. Those proteins that bind and / or become proteins that inhibit the binding of natural BAL Derivatives of carboxylic acids. A preferred proline-rich unit is the consensus sequence PVPPTGDSGA -P (SEQ ID NO: 6). As used herein, "C-tail tamper" "Quality" means any that contains three or more proline-rich units as defined above. This protein, which refers to the protein itself, binds to intestinal epithelial cells and / or Suppresses BAL binding. This is for example the mucin-like C-tail region of the native BAL. This can be achieved by using all or part BAL. Natural BAL is C -In the form of a tail protein.   The C-tail protein has at least three amino acid residues each consisting of 11 amino acid residues. Should contain repeated proline-rich units. Has only four repeating units Rat pancreatic esterase C-tail also binds to the rat intestinal surface. Preferred C The tail protein has at least 10 and most preferably at least 16 prolines Has a rich unit. C-tay with fewer proline-rich units The protein is expected to bind to the intestinal surface with lower affinity. The binding affinity of any of the C-tail proteins differs from that of the consensus sequence (SEQ ID NO: 2). It can be enhanced by always closely matching proline rich units. C-tay Proteins are constructed by linking three or more proline-rich units. Can be achieved. Here, the proline unit is the proline rich from any BAL. Having the natural amino acid sequence of the human proline-rich unit The amino acid sequence or derivatives of these amino acid sequences, and as a result, Protein can bind to intestinal epithelial cells and / or inhibit the binding of native BAL Hold power. Amino acid sequence variant   C-tail proteins differ in the number of threonine and serine residues. Amino acids that are O-glycosylated to some extent and do not significantly interfere with binding to the intestinal surface. Loss, substitution or addition. To C-tail protein that does not change binding Substitutions, deletions, or additions are readily determined by screening assays. It is. Protein may bind to gut surface in screening assays Possible and then removed by washing with buffer while increasing the salt concentration. It is. One example of a BAL containing a deletion that does not affect C-tail binding is an amino acid residue. BAL lacking a heparin binding site, postulated to be between groups 56 and 62. (Baba et al. (1991)).   Amino acid sequence variants of the C-tail protein may include one or more of three classes: In the range. That is, it is a substitution variant, an insertion variant, or a deletion variant. . Insertions can be amino and / or carboxyl terminal fusions, and single or Contains an in-sequence insertion of multiple amino acid residues. The fusion is made with mature BAL, e.g., human B In the case of AL, homologous to BAL, a secretory leader from another secreted human protein And a hybrid with a C-tail protein having the polypeptide of Fusion is A polypeptide that is homologous to BAL and a host cell but not homologous to BAL. La And a polypeptide having a polypeptide that is heterologous to both the host cell and the BAL. Includes hybrids with tail proteins. Preferred fusions are prokaryotic, yeast, Prokaryotic peptides or signal peptides of bacterial or host cell signal sequences Amino-terminal fusion with any of the tides. The signal sequence is usually It is necessary to lack any residue mature sequence from the protein that induces ligation None, but preferred to avoid secretion of C-tail protein fusions.   The insertion may also be a copy of the proline-rich unit repeat region of the C-tail protein. Can be introduced into the dosing sequence. Such insertions may involve the addition of unrelated amino acids or It may include the insertion of one or more additional proline-rich units. Ami inserted In the context of amino acids, "unrelated" amino acids are the proline-rich amino acids of BAL. Refers to an amino acid sequence that is not related to the knit sequence. Proline rich unit In some cases, the inserted unit may be a consensus sequence (SEQ ID NO: 6) or individual human A heterologous unit from non-human BAL with additional repeats of the phosphorus-rich unit possible. However, in the case of insertion of an unrelated amino acid, the insertion is usually amino or Is smaller than a carboxyl-terminal fusion, or more than a proline-rich unit And small insertions on the order of 1 to 4 residues.   A variant of the inserted amino acid sequence (variant) of the C-tail protein is a target C-tail protein. One or more amino acid residues introduced at a predetermined site in a protein It is. Most commonly, insertional variants are those that convert a heterologous protein or polypeptide to C -Failed to the amino or carboxyl terminus of the tail protein You. Preferably, these heterologous polypeptides are proline present in human BAL. It is a heterogeneous form of rich units. Immunogenic C-tail protein derivatives are A polypeptide of sufficient size to confer epidemiogenicity can be crosslinked in vitro. Culture of recombinant cells transformed with the DNA encoding the This is done by fusing. Such an immunogenic polypeptide is trpLE And bacterial polypeptides such as β-galactosidase.   Deletions remove one or more amino acid residues from the C-tail protein sequence. And characterized by: Deletions include deletions of entire proline-rich units Is preferred. If individual amino acids in the proline-rich unit are deleted, At any site within the C-tail protein molecule, only about 2-6 residues are missing. Lost.   These variants are usually associated with a nucleoside in the DNA encoding the C-tail protein. DNA encoding the variant is prepared by site-directed mutagenesis of the Prepared by expressing the DNA in cell culture. However, about 100 A variant C-tail protein fragment having up to 150 residues is It can be easily prepared by in vitro synthesis. Variants are typically native Exhibit the same biological activity as the analog present in As will be described in more detail below, the variants also have the properties of C-tail proteins. Is selected to be modified.   The site for introducing the amino acid sequence variant is determined in advance, but the mutation itself is Need not be determined in advance. For example, to optimize mutation execution at a given position Random mutagenesis at the target colon or region to The modified C-tail protein is used for optimal combination of desired properties. Can be screened. At a predetermined site in DNA with a known sequence Techniques for making substitution mutations are well known, for example, M13 primer mutagenesis is there.   Amino acid substitutions are typically of single residues. Insertion is usually about 1-10 Of amino acid residues, or the entire proline-rich unit; About 1 to 30 residues, or over the entire proline-rich unit; deletion or insertion The entry is preferably made in neighboring pairs. That is, deletion of 2 residues or 2 Insertion of a residue. Substitution, deletion, insertion, or Any combination may be combined. Obviously, the variant BAL or C-tailer Mutations made in the DNA encoding the protein cause the sequence to be Must not be displaced out of the frame, and preferably produce secondary mRNA structures. It does not form complementary regions as obtained (EP 75,444A).   Substitution variants are those in which at least one residue in the C-tail protein has been removed. , And a different residue inserted in its place. Such substitutions are generally Finally, it is desirable to ultimately modify the characteristics of the C-tail protein or BAL If In accordance with Table I below.   By choosing less conservative substitutions than those in Table I, ie, (a) Polypeptide backbone structure in the replacement region (eg, sheet or helical configuration) ), (B) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. By choosing more significantly different residues in their effect on maintenance, Substantial changes in performance or immunological identity are created. BAL or C-tay Positions that are generally expected to produce the largest changes in protein properties. In other words, (a) a hydrophilic residue (eg, a seryl or threonyl residue) is Residues (e.g., leucyl, isoleucyl, phenylalanyl, (Or an alanyl residue). Substitution); (b) cysteine or proline replaced any other residue (C) having a positively charged side chain Residues (eg, lysyl, arginyl, or histidyl) Replaced charged residues (eg glutamyl, aspatil) Or (d) has a bulky side chain Residues (eg, phenylalanine) are substituted with residues without side chains (eg, glycine ) Is replaced by (or replaced by).   Using substitution or deletion mutagenesis, N-linked glycosylation sites or O-linked A lycosylation site is added (eg, asparaginyl residue in the Asn-X-Thr glycosylation site). Eliminated or by BAL or C-tail protein Or alter the half-life of the protein. Or, Non-glycosylated BAL or C-tail protein is produced in recombinant prokaryotic cell culture. Can be achieved. Such non-glycosylated forms have lost intestinal binding activity or Or is expected to decrease. Of cysteine or other unstable residues Deletions may also be desirable, for example, in improving oxidative stability or in BAL It may be desirable in choosing a disulfide bond configuration. Potential tongue Deletions or substitutions of proteolytic sites (eg, Arg) can be made, for example, at one of the basic residues. By two deletions or substitution by glutaminyl or histidyl residues, Achieved.   In order to competitively inhibit cholesterol binding, an inactivated catalytic site A full-length BAL may be used. Since the catalytic site has been deactivated, BAL Cholesterol uptake cannot be performed easily. Catalytic site, the activity of recombinant BAL Deletion or substitution of an amino acid at a site can result in inactivation. catalyst The site may also be a chemical modification of BAL at or near the active site, for example, Catalytic site of irreversible enzyme inhibitor by proteolysis or other enzymatic reactions Or by means of destroying the three-dimensional arrangement of the BAL catalytic units ( It is inactivated because the binding of the C-tail to the gut is independent of its configuration). Breaking Examples of breaking means include surfactants and heat.   Useful for not hydrolyzing cholesterol esters or binding to heparin Various BAL derivatives are polypeptides that may or may not be substantially homologous to BAL. Including peptides. These BAL derivatives are used for recombinant or organic synthesis of BAL fragments. By preparation or by introducing amino acid sequence variants into the complete BAL Cholesterol ester hydrolysis and / or heparin binding as defined above Produced by not displaying activity.   Does not hydrolyze cholesterol esters as described above or binds to heparin Incompatible BAL derivatives are useful as immunogens for raising antibodies against active BAL It is. Such BAL derivatives, called "BAL protein antagonists," It can be used to neutralize cholesterol uptake-mediated activity or BAL. This Some BAL protein antagonists bind to the intestinal lining, thereby binding native BAL. Can block the match. BAL protein antagonists use various cholesterol Abnormalities, such as hypercholesterolemia, especially due to dietary cholesterol intake It is useful for treating hypercholesterolemia that has worsened.   BAL, BAL derivatives, and C-tail protein molecules are also covalently modified I can do it. Such modifications can be targeted by recovered or synthesized proteins. An organic derivatizing agent (organic derivative) capable of reacting a amino acid residue with a selected side chain or terminal residue.  derivatizing agent). Or selected Proteins may be Modified Using Post-Translational Modifications in Selected Recombinant Host Cells . As is known to those skilled in the art, the resulting covalent derivative can be used as an immunogen, Or to identify residues important for biological activity, as well as half-life and binding It is useful for altering the pharmacological properties of the molecule, such as affinity.   Certain post-translational inductions may involve the production of recombinant host cells against the expressed polypeptide. Results. Glutaminyl and asparaginyl residues are often translated. Later, it was deamidated and the corresponding glutamyl residue and aspara It becomes a chill residue. Alternatively, these residues are deamidated under mildly acidic conditions. It is. Either form of these residues may be used.   Other post-translational modifications include hydroxylation of proline and lysine, Phosphorylation of lysine or trionyl residue hydroxyl group, lysine, arginine , And methylation of the O-amino group of the histidine side chain (Creighton, Proteins: Struc Nature and Molecular Properties, pp. 79-86, (W.H.Freeman & Co., San Francisc o, 1983)), acetylation of the N-terminal amine, and optionally Amidation of the thiol. Production of C tail protein   Several methods exist for the production of C tail proteins. For example, our business One can obtain natural or recombinant BAL and use a protease (eg, Syn, chymotrypsin, pepsin and others) or Met-X binding Can be used to cleave the natural C tail using chemical methods such as cleaving with cyanogen bromide . Alternatively, one of skill in the art will appreciate that in eukaryotic hosts, the C tail region of BAL or A gene or cDNA encoding a sequence containing any C tail protein of It can be expressed either on its own or as a fusion with other non-BAL DNA sequences. So The non-BAL DNA sequence of either expresses or purifies the recombinant C tail protein. Can be easier. Recombinant BAL or C tail protein fusions will Can be subjected to cleavage and purification to obtain.   BAL is used, for example, in human and certain species of milk or intestinal or pancreatic juice of animals. Can be purified from such natural sources. But this is impractical on a large scale . BAL is preferably prepared using standard recombinant DNA techniques and eukaryotic host cells (eg, , Yeast or cultured mammals or insect cells) (In which case the C tail can be properly glycosylated) or in humans Not produced in the milk of transgenic animals.   Methods for isolating BAL from milk are described in Wang and Johnson, Anal. Biochem. 133: 457 (1 983) and is incorporated herein by reference. As described in this specification Used to describe the status of the BAL or C tail protein produced. `` Essentially free '' or `` essentially pure '' means, for example, that BAL In vivo, when obtained by extraction and purification from fluids and / or tissues Contain proteins or other substances that normally bind to BAL in the physiological environment of Means that Other substances are, for example, causative factors of acquired insufficiency syndrome (AIDS) And infectious organisms such as BAL and CTE produced by the method of the present invention Il protein is more than 95% pure.   To synthesize a recombinant BAL or C tail protein, one of skill in the art CDNA sequence (SEQ ID NO: 3) encoding sulphate activated lipase (SEQ ID NO: 2) (Ta ng and Wang, U.S. Patent No. 5,200,183; Baba et al. (1991); or Nilsson et al., Eur. . J. Biochem. 192: 543-550 (1990), all of which are incorporated herein by reference. Can be used. This sequence corresponds to the C Deletion, addition, or substitution of the sequence encoding Such C tail proteins can be readily adapted for expression.   DNA isolates may be chemically modified with or without 3 ′ and / or 5 ′ flanking regions. It is understood to mean chemically synthesized DNA, cDNA, or genomic DNA. Code BAL DNA derived from a) liver, breast, pancreas, or other tissues that contain BAL mRNA from a particular animal Obtaining the original cDNA library, b) in the cDNA library containing the homologous sequence To detect clones, human BAL or a fragment thereof (usually less than 100 bp) Performing hybridization analysis using labeled DNA encoding And c) analyze clones by restriction enzyme analysis and nucleic acid sequencing Can be obtained from other sources besides humans. Full length claw If the fragment is not present in the library, the appropriate fragment is opened here. Using the indicated nucleic acid sequences to recover from various clones, Can be assembled at a common restriction site to encode a full-length clone encoding BAL. You.   “Transformation” is when DNA is transferred as an extrachromosomal element or by chromosomal integration. Refers to the introduction of DNA into an organism so that it can be replicated. Not specifically indicated As long as the method of transformation of a host cell used herein is described by Graham and van de r Eb, Virology 52: 456-457 (1973). However, the bite by nucleic acid injection Or other methods of introducing DNA into cells, such as by fusion or protoplast fusion Can also be used. Used by prokaryotic cells or cells containing rigid cell wall structures If so, suitable transfection methods are described in Cohen et al., Proc. Natl. Acad . Sci. USA 69: 2110 (1972). Reason.   Standard construction of appropriate vectors containing the desired coding and control sequences Various coupling techniques are used. Isolate plasmids or DNA fragments , Tailored, and the desired form to form the required plasmid Is reconnected.   Since glycosylation of the C tail is essential, prokaryotic cells bind to the receptor. It is not useful as a compatible C tail expression host. However, in general, prokaryotes Used for the cloning of DNA sequences in the construction of useful vectors for expression. You. For example, E. coli W3110 (F, prototrophic, ATCC No. 27325), Bacillus subtil bacilli like us, and Salmonella typhimurium or Serratia marcescans Other Enterobacteriaceae, such as various Pseudomonas species, may be used.   Generally, it contains promoter and control sequences from a species compatible with the host cell. Plasmid vectors are used with these hosts. Vectors are usually , A replication site and one or more marker sequences. The marker sequence is transformed It may provide for phenotypic selection of cells. For example, E. coli is E. coli. Plasmid from E. coli species Is typically transformed with a derivative of pBR322 (Boliver et al., Gene 2). : 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance And thus provide a simple means of identifying transformed cells. pBR322P Rasmid or other bacterial plasmids are also commonly used in recombinant DNA construction Contains or contains promoters and other regulatory elements It has been modified as follows.   Suitable promoters for use in prokaryotic hosts include β-lactamase and lactamase. The ketose promoter system (Chang et al., Nature 275: 615 (1978); and Goeddel et al. , Nature 281: 544 (1979)), alkaline phosphatase, tryptophan (trp). Promoter systems (Goeddel et al., Nucleic Acids Res. 8: 4057 (1980) and European Patent Application No. 36,776) and hybrid promoters such as the tac promoter (De Boer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)). You. However, other functional bacterial promoters are also suitable. These nucleotides The sequences are generally known, and the skilled artisan will provide any necessary restriction sites. Do In order to use these nucleotide sequences in the BAL Alternatively, it can be operably linked to DNA encoding the C-tail protein (Siebenlis t et al., Cell 20: 269 (1980)). Promoters for use in bacterial systems also include BAL Or Shine-Dane operably linked to DNA encoding the C-tail protein Contains the Lugarno (S.D.) sequence.   For analysis to confirm the correct sequence in the constructed plasmid, the ligation mixture was E. E. coli K12 strain 294 (ATCC 31446) Transformants, if appropriate, due to ampicillin or tetracycline resistance Selected. Plasmids from transformants were prepared, analyzed by restriction, and And / or Messing et al., Nucleic Acids Res. 9: 309 (1981) Alternatively, the sequence was determined by the method of Maxam et al., Methods in Enzymology 65: 499 (1980). It is.   The host cell can be transformed with the expression vector and induce the promoter, A conventional nutrient culture modified as appropriate for selection of transformants or gene amplification It can be cultured in the ground. Culture conditions (eg, temperature and pH) are selected for expression. Conditions previously used in selected host cells, and will be apparent to those skilled in the art.   "Transfection" refers to the uptake of an expression vector by a host cell, This is regardless of whether any coding sequences are actually expressed. Many tigers Transfection methods are known to those skilled in the art (eg, CaPO_FourAnd electro Poration). Successful transfection generally involves the production of this vector. Recognized when any sign of movement occurs within the host cell.   To facilitate understanding of the following examples, certain frequent methods and / or Describe the term.   “Plasmids” are capitalized with a lower case p preceded and / or followed by capital letters And / or represented by numbers. The starting plasmids herein are commercially available. More available, publicly available without restriction, or published Can be constructed from available plasmids according to the procedures described. In addition, plus Plasmids equivalent to mid are known in the art and will be apparent to those of skill in the art. Is.   "Digestion" of DNA is the catalysis of DNA by restriction enzymes that act only on specific sequences of DNA Say the target disconnection. The various restriction enzymes used herein are commercially available. And their reaction conditions, cofactors, and other requirements are known to those of skill in the art. Was used. For analytical purposes, typically 1 μg of plasmid or DNA plasmid is used. The fragment is used with about 2 units of enzyme in about 20 μl of buffer. For the purpose of isolating DNA fragments for plasmid construction, typically , 5 to 50 μg of DNA are digested in large volumes by 20 to 250 units of enzyme. specific Appropriate buffers and amounts of substrate for the restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 ° C are usually used, but should be Therefore it can change. After digestion, the reaction is electrophoresed on a polyacrylamide gel. Apply directly and isolate the desired fragment.   The "recovery" or "isolation" of a given DNA fragment from a restriction digest is Separation of digests on polyacrylamide or agarose gels by electrophoresis, Transfer of marker DNA fragments with known fragment mobility and molecular weight Identification of desired fragment by comparison with mobility, gel containing desired fragment Refers to excision of compartments and separation of gel from DNA. This procedure is generally known (Lawn et al., Nucleic Acids Res. 9: 6103-6114 (1981), and Goeddel et al. , (1980)).   "Dephosphorylation" is the result of treatment with bacterial alkaline phosphatase (BAP). Refers to the removal of the terminal 5 'phosphate. This procedure involves two restriction digestions of the DNA fragment. Prevents the formation of end "cyclization" or ring closure. "Cyclization" or formation of a ring closure Prevent insertion of another DNA fragment at the restriction site. Procedures for dephosphorylation And reagents are conventional (Maniatis et al., Molecular Cloning, 133-134 (Cold  Spring Harbor, 1982)). The reaction using BAP is performed at 68 ° C. in 50 mM Tris, Inhibits any exonuclease activity that may be present in the enzyme preparation. reaction Is performed for one hour. After the reaction, the DNA fragment is gel purified.   "Ligation" refers to the formation of a phosphodiester bond between two double-stranded nucleic acid fragments Say the process (Maniatis et al., Ibid., 146). Unless otherwise stated, consolidation is public. Using known buffers and conditions, an approximately equimolar amount of 0.5 μg ligated DNA flag This can be done with 10 units of T4 DNA ligase ("ligase") per ment.   "Filling" or "blunting" refers to single-stranded and restriction digested sticky ends. Refers to a procedure by converting nucleic acids to double stranded. This cuts out the sticky ends and To form blunt ends. This process involves the use of one or a few other restriction enzymes. Restriction ends that may be sticky with the ends created by Converts endogenous endonuclease compatible ends or other filled sticky ends It is a versatile means to Typically, blunting is performed with 2-15 μg of target DNA. 10mM MgCl_Two, 1 mM dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5) buffer In about 37 ° C., 8 units of Klenow fragment of DNA polymerase I and Incubate in the presence of 250 μM of each of the four deoxynucleoside triphosphates. This is done by doing Incubations generally involve phenol and The reaction is terminated after 30 minutes by extraction with chloroform and ethanol precipitation.   Many of the above procedures for mutating, manipulating, and recombining nucleic acid molecules are also , Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, 1989) can be found in standard laboratory manuals.   Many eukaryotic expression systems, exons, are correctly cleaved, resulting in a human BAL cDNA and Both human BAL genomic DNA and recombinant human BAL It can be used to direct the synthesis of proteins. Human BAL gene expresses enzyme Is expected to be included in its untranslated region sequence. These regulatory sequences Can be used directly even in the expression of transgenic animals.   Recombinant BAL protein can be derived from human BAL cDNA or It can be produced from offspring. These include, for example, E. coli, Bacillus, yeast, fungi, insects BA in hosts such as cells, mammalian cells, and transgenic animals L expression. T-BAL (short form of BAL without C-tail) in E. coli Expression is described in Downs et al., Biochemistry 33: 7980-7985 (1994). Prokaryotic Protokaryotes can not remove mammalian introns from mRNA In biological systems, it is preferable to make appropriate modifications to express cDNA rather than gene. New However, because prokaryotes cannot glycosylate BAL, It is preferable to use a eukaryotic system. When using eukaryotic cells as hosts, Either the human BAL gene or the cDNA can be used to direct the synthesis of the enzyme. To direct the newly synthesized BAL to the interior of the rough endoplasmic reticulum If "" or "signal" sequences are present, glycosylation of BAL may also be present. You. This is because the intestinal surface interaction cleaves the polysaccharide at the C-tail, Reducing sterol uptake is important for the C-tail.   In all cases, the human BAL cDNA or gene contains an expression control element ( For example, transcription start signal, translation start signal, start codon, stop codon, translation Into appropriate expression vectors, including termination signals, polyadenylation signals, etc. Can be done. Suitable vectors are commercially available from various companies. BAL cDNA or After transfecting the recombinant vector containing the gene into host cells, these It can remain as extrachromosomal DNA or integrate into the host genome. Which Again, they may direct the synthesis of recombinant BAL in the host cell. Generating heterologous genes Some examples of the present are found in Methods in Enzymology, Volume 153, Chapters 23-34 (edited). Wu and Grossman, Academic Press, 1987). BAL synthesis of host cells Large-scale cultivation and enzyme purification are cost-effective commercial sources of BAL or C-tail production. Business means. Methods for large-scale production of enzymes are well known to those skilled in the art.   Some examples of useful expression systems for the C-tail of glycosylated BAL are It is shown below: (1) Using yeast and fungi as hosts: the source of expression of recombinant BAL in yeast The theory is similar to E. coli expression. Examples are described in Bitter et al., Methods in Enzymology 15 3: 516-544 (1987). Like E. coli, yeast host cells Either in the sol or, in this case, preferably, as a secreted protein Thus, a foreign gene can be expressed. Unlike E.coli expression, secretory expression in yeast Can be glycosylated. Many vectors available for yeast expression of secreted proteins There are promoters, promoters, and leaders. A good example is the methylotro Control by alcohol oxidation promoter in pick yeast Pichia pastoris Based system. Vectors pHIL-S1 and pPIC9 are commercially available (Invitrogen) And produce large volumes of secreted eukaryotic recombinant proteins (1-4 mg / L Human serum albumin, Barr et al., Pharmaceutical Engineering 12: 48-51 (1992); 12 mg / L tetanus toxic fragment C, Clare et al., Bio / Technology 9: 455-460 (19 91).   For expression in Saccharomyces, for example, plasmid YRp7 (Stinchcomb Et al., Nature 282: 39 (1979)); Kingsman et al., Gene 7: 141 (1979); Techemper et al., Gene.  10: 157 (1980)) is commonly used. This plasmid is already in tryptophan A mutant strain of yeast lacking the ability to grow on (eg, ATCC Accession No. 44076 or Provides a selectable marker for PEP4-1 (Jones, Genetics 85:12 (1977)) l Includes gene. Next, the existence of trpl lesions as a characterization of the yeast host cell genome Provides an effective means of selection by growing in the absence of tryptophan .   Suitable promoter sequences for using yeast hosts include 3-phosphoglycerate kinase ( 3-phosphoglycerata kinase (eg, enolase, glyceraldehyde-3-li Acid dehydrogenase, hexokinase, pyruvate decarboxylase, phos Hofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate Tomidase, pyruvate kinase, triose phosphate isomerase, phos Hoglucose isomerase, and glucokinase) (Hitzeman et al., J. Adv. Enzy me Reg. 7: 149 (1968); and Holland, Biochemistry 17: 4900 (1978)). Including   With an inducible promoter that has the added advantage of transcription controlled by growth conditions Certain other yeast promoters include alcohol dehydrogenase 2, isocytochrome C , Acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glycein Lualdehyde-3-phosphate dehydrogenase, and maltose and galactose This is the promoter region of the enzyme responsible for the utilization of the enzyme. Suitable for use in yeast expression Novel vectors and promoters are described in Hitzeman et al., European Patent Publication No. 73,657A. It is further described. Yeast enhancers are also available using yeast promoters. Used for profit.   Few fungal expression vectors have been successfully used to express heterologous genes Exists. Existing fungal expression vectors use the host genome after transfection. (Cullen et al., Molecular Cloning Vectors for Aspergil lus and Neurospora '' in A Survey of Molecular Cloning Vectors and their U ses (Butterworth Publishers, Stoneham, MA 1986). Tamper where leader is expressed If present before the codon codon, the secreted recombinant protein will be glycosylated. Can be done. Some examples of successful expression are bovine chymosin (Cullen et al., Bio / Te chnology 5: 369-378 (1987)) and acid proteases from different fungi (Gray et al., G ene 48: 41-53 (1987)).   (2) Insect cells as hosts: Ba for the synthesis of foreign genes in insect cells culovirus expression vectors contain many mammalian proteins and viral proteins. Has been successfully used to express quality as well as for rat pancreatic BAL (DiPersio et al., Protein Expr. Purif. 3: 114-120 (1992)). This system Can be glycosylated and also express recombinant proteins at high levels You. The use of this system has been reviewed in some detail (Luckow and Summers, "T. rends in the Development of Baculovirus Expression Vectors '' Bio / Technolo gy, September 11, 1987). Many vectors derived from baculovirus expression systems are commercially available. (Eg, pBlueBacHis A, B, C and pEBVHis A, B, C) and insect host cells Available from Invitrogen.   (3) Mammalian cells as hosts: many heterologous genes in mammalian cells Expression has been successfully accomplished for commercial purposes. Recombinant human tissue plasmino One example is a commercial product of Genactivator. Most of these expression vectors , Either a mammalian promoter or a viral promoter, polyadenyl Signal and appropriate regulatory elements for cloning into E. coli (anti- Biomaterial resistance gene). BAL or C-tail downstream of promoter After inserting the encoding DNA, the vector can be first cloned into E. coli and isolated. And can be transfected into mammalian cells. Neomycin or Any of the similar resistance selectable markers are co-transfected into mammalian cells obtain. Either neomycin or a similar resistance selection marker can be Or they can be co-transfected in the same vector. High level expression Therefore, a gene amplification system is advantageous. Secretes BAL or C-tail protein Transformed clones can be identified by enzyme assays or Western blots. You. Successful approaches to this approach using other mammalian proteins include glycosyls Recombinant protein (Poorman et al., Proteins 1: 139-145 (1986)) and human Interferon (Scahill et al., Proc. Natl. Acad. Sci., USA 80: 4654-4658 (1983)). Synthesis.   Preferred protocols for controlling transcription from a vector in a mammalian host cell Motors come from a variety of sources: for example, polyomas, simian wheels Virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most Preferably from a viral genome, such as a cytomegalovirus, or a heterologous mammal It can be obtained from an animal promoter, such as the β-actin promoter. SV40 ui The Russ early and late promoters surround the SV40 viral origin of replication. (See Fiers et al., Nature 273: 113 (1. 978)). The immediate early promoter of the human cytomegalovirus is a HindIII E restriction fragment. It is easily obtained as a fragment (Greenaway et al., Gene 18: 355-360 (1982)). Mochi Of course, promoters from host cells or related species are also useful herein. is there.   Transcription of DNA encoding BAL or C-tail protein by higher eukaryotes Is increased by inserting an enhancer sequence into the vector. Enhancer Is a cis-acting element of DNA, usually about 10 to 300 bp, To increase its translation initiation ability. Enhancers are relatively directional and Position independent and 5 'of the transcription unit (Laimins et al., Proc. Natl. Acad. Sci. USA 78: 993 (1981)) and 3 '(Lusky et al., Mol. Cell. Bio. 3: 1108 (1983)), in the intron (B anerji et al., Cell 33: 729 (1983)), and within the coding sequence itself (Osborne et al., Mol Cell Bio. 4: 1293 (1984)). Mammalian genes (globulins, Lastase, albumin, α-fetoprotein, and insulin) Many enhancer sequences are known. However, typically, eukaryotic virus A conventional enhancer is used. The example shows the SV40 enhancer behind the replication origin. (Nucleotides 100-270), cytomegalovirus early promoter enhancer , A polyoma enhancer behind the origin of replication, and an adenovirus enhancer Including   Eukaryotic host cell (yeast, fungus, insect, plant, animal, human, or nucleated cell) The expression vector used in is also used to terminate transcription that can affect mRNA expression. It may also contain the sequences necessary for These regions encode the BAL or C-tail Transcribed as a polyadenylation segment in the untranslated portion of the mRNA. This 3 The 'untranslated region also includes a transcription termination site.   An expression vector can include a selection gene (also referred to as a selectable marker). Feeding Examples of suitable selectable markers for animal cells include dihydrofolate reductase (DHFR ), Thymidine kinase, or neomycin. Such a selection marker Upon successful transfer into a mammalian host cell, the transformed mammalian host cell Vesicles can survive even under selective pressure. Two widely used choice regimens There are different categories. The first category is cell metabolism and recruitment Based on the use of a mutant cell line that lacks the ability to grow independently of the prepared medium . Two examples are CHO DHFR cells and mouse LTK cells. These cells are Lack of ability to grow without the addition of nutrients such as thymidine or hypoxanthine I have. These cells have specific genes required for a complete nucleotide synthesis pathway These cells lack lost nucleotides provided in supplemented media. Unless you can survive. An alternative to media supplementation is the intact DHFR gene Or introducing the TK gene into cells lacking their respective genes, thus Is to change the proliferation requirement. Transformed with DHFR or TK gene Individual cells that did not survive in unsupplemented media.   The second category is dominant choice. It is used in any cell type And does not require the use of a mutant cell line. Typically These schemes use drugs that stop the growth of host cells. New genetics The offspring express the protein that confers drug resistance and produce from selection. Extend. An example of such a dominant selection is neomycin (Southern and Berg, Mo. lec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan and Berg, S. science 209: 1422 (1980)) or hygromycin (Sugden et al., Mol. Cell. Bi. ol. 5: 410-413 (1985)). The three examples above are eukaryotic G418 or neomycin (genome) were used, respectively, with bacterial genes under sex control. Neticin), Xgpt (mycophenolic acid), or hygromycin This leads to severe drug resistance.   "Amplification" refers to the expansion or replication of an isolated region within the chromosomal DNA of a cell. Amplification uses a selective agent (eg, methotrexate (MTX) to inactivate DHFR) Is achieved. Amplification of the DHFR gene or generation of continuous copies requires more MT Generates more DHFR, which is produced against X. Amplification pressure depends on medium By adding a higher amount of MTX, it can be applied despite the presence of endogenous DHFR. You. Amplification of the desired gene causes the mammalian host cell to encode the desired protein. Plasmids containing DNA and DHFR genes or enhancements to allow for simultaneous integration This can be achieved by co-transfecting with a width gene. Even higher By selecting only cells that can grow in the presence of MTX concentration, DHFR is required (this need is more Is done). The gene encoding the desired heterologous protein is co- As long as it is integrated, the replication of this gene is Bring in duplicates. As a result, the gene encoding the desired heterologous protein The increased number of copies (ie, the amplified gene) is Express the protein.   Disclosure vector encoding BAL or C-tail protein in higher eukaryotes Suitable suitable host cells for expressing the promoter include the following: Transformed monkey kidney CVI strain (COS-7, ATCC CRL 1651); human embryonic kidney strain (293, Graham J. et al. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cell-DHFR (CHO, Urlaub and Chasin, Proc . Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mathe r, Biol. Reprod. 23: 243-251 (1980)), monkey kidney cells (CVI, ATCC CCL 70); Licamid monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA , ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34); falo rat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75) ); Human hepatocytes (hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL 51) And TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68). (1982)).   (4) Expression of C-tail protein or BAL in transgenic animals : Transfer the human BAL gene into the genome of another animal for tissue-specific expression Techniques already exist (Jaenisch, Science 240: 1468-1474 (1988); Westp hal, FASEB J. 3: 117-120 (1989)). By using transgenic technology Several studies to alter milk composition are already ongoing (reviewed). For a discussion of Bremel et al. Dairy Sci. 72: 2826-2833 (1989)). Up The general approach, summarized in the three reviews listed in Sec. Promoters for proteins (eg, casein or milk lysozyme), BAL or Or the DNA encoding the C-tail protein, and the appropriate complementary element Is to construct a vector. The cloned vector was then newly fertilized Microinject into a bovine or ovine egg and transfer the egg to the fetus Populate the "nanny" for birth and birth. Transgenic offspring, Saza For gene transfer by immunoblotting and BAL or C-tail in milk Analyze for protein production. Cows and sheep contain BA in their milk. It is important to note that it does not produce L. In order to produce a high yielding strain, Transgenic animals can be bred. C-tail tamper secreted into milk The protein is completely glycosylated. Cows and sheep do not make milk BAL Thus, the recombinant product can be easily distinguished from the host milk protein.   Compounds containing sugars or sugar analogs that mimic the C-tail structure can also be chemically reacted Can be synthesized by These bind to the intestinal surface and are the same as the C-tail itself Can be used to compete with endogenous BAL in a fashion. The following example demonstrates galacto And fucose can elute endogenous bound BAL from rat intestinal surface This demonstrates that synthetic mimics of C-tails containing these sugars or Indicate that its structural analogs can be used to affect binding to the intestinal surface. And Since the C-tail contains repetitive sequences and multiple glycosylation sites, Synthetic mimetics can include multiple sugar-containing sites. The chemical bond of the sugar is Can be modeled on the basis of glycose structures or the structure of these oligosaccharide structures Analogs may include essential properties for effective binding to intestinal BAL receptor . Sugars can be chemically attached to polymers to create repeating units. Examples of suitable polymers Are polypeptides, polyethylene glycols, dextran-like sugar polymers, and Includes other synthetic polymers with appropriate functional groups for chemical attachment to sugars You. Pharmaceutical applications and compositions   As described in the examples below, BAL is administered to the intestinal surface via its C-tail oligosaccharide. It was found to bind to surface lectin-like receptors. This bond is This is an essential step in mediating cholesterol uptake. further, The isolated C-tail of BAL competes with BAL for binding to intestinal surface receptors. It was found to gain. In addition, the C tail can reduce intestinal BAL binding. Showed that it could competitively inhibit intestinal cholesterol uptake . The C-tail reduction of cholesterol uptake is specific. Because BAL Is the only enzyme in the gut that can mediate cholesterol uptake. BAL also hydrolyzes other fatty acid esters, thus allowing uptake of other fats. Can be easier. However, the C-tail of intestinal uptake of non-cholesterol fats The reduction is not specific. Because other lipases in the gut are also non-cholesterol This is because it is possible to digest dairy fat. Therefore, as defined above, the C tailor Protein can be a specific reduction in cholesterol uptake and / or other fat Administered as a therapeutic to an individual in need of a more general reduction in uptake, Thus, hyperlipoproteinosis, hypercholesterolemia, and atherosclerosis Treat diseases associated with keratosis.   In a preferred embodiment, the C-tail protein is cholesterol in blood Uptake of cholesterol from food as measured by reduced levels Orally in an amount effective to reduce Dosage depends on formulation, excretion rate, intestinal surface Variability, such as the number of receptors for cholesterol, levels of cholesterol to be reduced, and And the frequency of administration, as well as other factors that are usually optimized by a physician.   Intestinal cholesterol uptake from food is about 200 mg per person per day is there. The body synthesizes about 500 mg of cholesterol per person per day. pancreas Secretes about 500 mg of cholesterol per person per day, which is Will be collected. Therefore, the C-tail composition on the intestinal surface is about 700 mg per person per day. Must be effective in reducing cholesterol levels from resterol uptake Must. When the decrease is below 500 mg a day, the body is Reduce emissions.   Administered to patients in an amount effective to reduce intestinal cholesterol uptake Enhances the pharmacological activity of C and C-tail proteins, thereby Pharmaceutical sets containing C-tail proteins designed to reduce roll levels The composition may contain a suitable pharmaceutical stabilizing compound, delivery vehicle, carrier, inert diluent, And / or other suitable for enteral (oral) administration by methods well known in the art. It can be prepared in combination with additives. The formulation is usually released into the stomach or intestines . C-tail proteins include lipids, pastes, suspensions, gels, powders, tablets, capsules , Food additives, or other standard forms. Pharmaceutically compatible knots Combination and / or adjuvant materials can be included as part of the composition. That Examples include binding of microcrystalline cellulose, gum tragacanth, or gelatin Excipients such as starch or lactose; alginic acid, Primogen^TMOr Disintegrants such as corn starch; magnesium stearate or lubricants such as rotes); aglidants such as colloidal silicon dioxide Sweeteners such as sucrose or saccharin; and / or peppermint, salici Odorants such as methyl luate or orange flavoring are included. Dosage unit form If a capsule, it may contain, in addition to materials of the above type, a liquid carrier. other Dosage unit form further comprises a sugar, shellac coating, or other enteric agent. I can see. C-tail protein, elixirs, suspensions, beverages, liquid food supplements Or substitutes, or fluids such as syrups; or cachets or candies Can be administered as a solid component such as C-tail protein, other blood fat Other active ingredients which do not impair the desired action, such as pharmaceutical compositions which reduce the quality, or It can be mixed with materials that supplement the desired effect.   In one preferred embodiment, the C-tail protein is a protein, poly Microparticles prepared from synthetic or natural polymers such as hydroxy acids or polysaccharides , Encapsulated in a carrier that is released into the small intestine, such as microcapsules or microspheres You. Suitable systems are known to those skilled in the art. Some microsphere preparations are used for oral drugs It has been proposed as a means of delivery. These preparations generally contain the encapsulated compound And helps to deliver this compound to the bloodstream. Enteric formulations are administered orally Has been widely used for many years to protect and delay release of drugs . Others designed to deliver compounds to the bloodstream and protect the encapsulated drug Preparations include zein as described in PCT / US90 / 06430 and PCT / US90 / 06433. Hydrophobic proteins; "Proteins" described in Steiner U.S. Pat. No. 4,976,968. Inoids (proteinoids); or UAB Research Foundation and Southern Rese Synthesis described in European Patent Application No. 0 333 523 by arch Institute Formed of polymer. EPA 0 333 523 is an anti-inflammatory drug used for oral administration of vaccines. It describes fine particles less than 10 microns in diameter, including the raw material. Encapsulated To prevent the uptake of C-tail protein into the blood and lymphatic system, Larger sizes are preferred for applications described in the textbook.   Fine particles are exposed to carboxyl groups when the smooth surface is corroded Poly [lactide-co-glycolide], polyanhydrides, and polyorthoesters. Which rapid bioerodible polymers; zein, modified zein, casein, gelatin, Proteins such as ruten, serum albumin or collagen, and cellulose , Dextran, polyhyaluronic acid, acrylic acid ester and methacrylic acid Natural polymers such as polysaccharides, such as polymers of steal and alginic acid; Hofazine (polyphosphazines), poly (vinyl alcohol), polyamide, poly Carbonate, polyalkylene, polyacrylamide, polyalkylene glycol , Polyalkylene oxide, polyalkylene terephthalate, polyvinyl acetate Ter, polyvinyl ester, polyvinyl halide, polyvinylpyrrolidone, Synthesis of Liglycolide, Polysiloxane, Polyurethane and its copolymer Polymers; and alkylcellulose, hydroxyalkylcellulose, cells Synthesizing such as rose ether, cellulose ester, and nitrocellulose And modified natural polymers. Typical polymers include methyl Lulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypro Pilme Chill cellulose, hydroxybutyl methyl cellulose, cellulose acetate, propyl cellulose Cellulose onion, cellulose acetate butyrate, cellulose acetate phthalate, carboxy Methyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, Poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl) Methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate) Acrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate) G), poly (phenyl methacrylate), poly (methyl acrylate), poly ( Isopropyl acrylate), poly (isobutyl acrylate), poly (octa Decyl acrylate), polyethylene, polypropylene, poly (ethylene glycol ), Poly (ethylene oxide), poly (ethylene terephthalate), poly ( Vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone, and Polyvinyl phenyl. Particularly typical bioerodible polymers include poly Actide, polyglycolide and its copolymer, poly (ethylene terephthalate) G), poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolact) Tonnes), poly [lactide-coglycolide], polyanhydrides, polyorthoesters, Formulations, as well as copolymers thereof.   These polymers are available from Sigma Chemical Co., St. Louis, MO., Polysciences, Warrenton, PA, Aldrich, Milwaukee, WI, Fluka, Ronkonkoma, NY, and BioR ad, Richmond, CA or other sources or use standard techniques. It can be synthesized from monomers obtained from these suppliers.   Pharmaceutical compositions containing C-tail proteins are stable under the conditions of manufacture and storage. List of compounds that must be approved for use by the Food and Drug Administration Use antioxidants such as vitamin E and ethoxyquin and bacteriostatics listed in And can be protected from contamination by microorganisms such as bacteria and fungi.   As described above, the inactivation and elimination rates of C-tail Other factors known to the human affect the amount of drug administered to the patient. The decisive criterion is , Whether the amount is effective in reducing cholesterol in the blood. In the blood Cholesterol levels are assayed by standard techniques used in clinical laboratories. It is done.   C-tail protein reduces intestinal uptake of cholesterol, Hypercholesterolemia, hypertriglyceridemia and atherosclerosis Treatment of hyperlipidemia, including sclerosis, cardiovascular disease, and related disease states such as pancreatitis Can be used for The C-tail protein is also expected to prevent the development of these disorders. It can also be used in normal subjects as a protective measure. Human serum cholesterol Levels are preferably maintained below 200 mg / dl; values of 240 mg are clinically high. , A value of 160 mg is considered low.   The formulations are administered in a single daily dose or in divided doses, most preferably Three times before, during or after a meal. The patient is not aware of Protein is maintained and intestinal uptake of cholesterol is reduced. Administration Conditions that should be considered when choosing quantity levels, frequency, and duration , Mainly patient severity, patient serum cholesterol levels, gastric distress Side effects, and the need for patient prophylaxis, as well as treatment efficiency. It is. For specific subjects, control individual patient needs and composition administration. Or take the time to adjust according to the professional judgment of the observer. And the range of concentrations set herein is merely exemplary. Does not limit the scope or practice of the claimed composition. Is understood. Other concentration ranges and durations of administration are determined by routine experimentation. Can be An initial estimate of the dose would be 20 μg for the 1 cm intestinal bound receptor. Based on calculations that it is necessary to block, 20 mg of C-tail protein Is enough to cover a 10 meter long intestine to block the BAL binding site It should be. Five times excessive amount (100mg) is cholesterol by intestinal cells Too much to prevent absorption. Further adjustment of the dose may be due to cholesterol in the blood. Monitoring patient's dose response to C-tail protein in reducing roll based on.   The invention will be better understood with reference to the following non-limiting examples. References In all, it is expressly incorporated herein by reference. Example 1 Proline-rich domains and glycosylation are associated with bile salt activation It is not essential for the enzyme activity of Pase.   To test the functional requirements of structural regions within the BAL molecule, a human, truncated Milk bile salt activated lipase (T-BAL) cDNA (mucin-like carboxy-terminal region (remaining Groups 539-722)) were expressed from the T7 system in Escherichia coli, purified and The properties of the truncated recombinant enzyme were studied. The method was as follows.   Abbreviation   The following abbreviations are used in this example: PANA, p-nitrophenyl acetate; PANB , P-nitrophenyl butyrate; SDS, sodium dodecyl sulfate; T-BAL, truncated bile acid Salt-activated lipase; PCR, polymerase chain reaction; TN (50 mM Tris-HCl, pH 8.0 and And 100 mM NaCl); EGTA, ethylene glycol-bis (β-aminoethyl ether B) N, N, N ', N'-tetraacetic acid; FPLC, high performance protein liquid chromatography; and B SA, bovine serum albumin.   material   A method as described in Baba et al. (1991), which is incorporated herein by reference. Thus, BAL was purified from human milk. Λgt10 cDNA library from human lactating breast tissue Bullies were purchased from Clontech (Palo Alto, CA). Glycerol tri [9,10 -^Three[H] oleate was obtained from Amersham (Arlington Heights, II). Oligo Nucleotide primer I (SEQ ID NO: 4) and primer II (SEQ ID NO: 5) , Dr. K. Jackson (Molecular Biology Resource Facility, Oklahoma Universi ty Health Science Center). All other chemicals are Sigm a Chemical Co. (St. Louis, MO).   Molecular biological methods   Standard for sequencing analysis, restriction digestion, ligation and subcloning The method is described in Sambrook et al., A commonly known and used reference technical literature. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Current P of Ausuble et al. rotocols in Molecular Bjology, Green Publishing Associates and Wiley-Int erscience, New York, NY. (1989). Tang and Wang US special No. 5,200,183, a cDNA clone from human mammary gland cDNA. G10-2 and G10-5 were isolated. United States Biochemical Corp. (Clevelan d, OH), using the DNA sequencing kit obtained from The analysis was performed. First, a double-stranded DNA template was prepared from Kraft et al., BioTechnjques 6: 544-547 (19 Denatured by the method described in 88). Then 2.5 μg of DNA, 15 ng of plasmid The immersion medium and 2 μl of reaction buffer were combined to a total volume of 10 μl, And then cooled to 25 ° C. for 30 minutes to anneal the mold. Was. The labeling mixture was diluted 5-fold with water, and Sequenase type 2.0 (Sequenase versio n 2.0) was diluted 8-fold with water. 1 μl DTT (0.1 M), 2 μl diluted labeling mixture, 5μCi [α³²P] dATP and 2 μl of diluted sequenase were annealed. Template primer. After mixing, incubate the tube at room temperature for 3 minutes did. 2.5 μl of the termination mixture was placed in a test tube. Preheat the test tube at 37 ° C for 1 minute. Was. Transfer 3.5 μl of the labeling reaction mixture to each stop tube, mix, and And then incubated at 37 ° C. for 3 minutes. Stop the reaction by adding 4 μl of stop solution I let it. The samples were then run on a 6% acrylamide gel. sample The gel was pre-migrated for 30 minutes at a constant voltage of 2000 volts before applying. Arrangement The sample was heated at 75 ° C. for 2 minutes just before loading 2 μl on the sequencing gel. 200 The gel was run at a constant voltage of 0 for 3-4 hours. After electrophoresis, convert gel to 3MM paper Transferred and then dried under vacuum at 80 ° C. for 2 hours. Subsequent reading of the DNA sequence For taking, Kodak X-Omat^TMThe PR film was exposed overnight at room temperature. This specification Innis and Gelfand PCR Protocols, A Guide to Methods and Application 3-12 (Innis et al. (Eds.), Academic Press, New York, NY) . (1990)). Invitrogen (San Diego Using a TA cloning kit obtained from Was subcloned into PCRII. For ligation with the PCRII vector, use the vector: PCR product Were set to be 1: 1 and 1: 3. 1 μl of 10 × ligation buffer , 50 ng of vector, PCR product and 4 units of T_FourAdd DNA ligase and add The volume was 10 μl. The ligation reaction was incubated at 12 ° C. overnight. Then 50 μl of INVαF ′ competent containing 2 μl of 0.5 M β-mercaptoethanol Cell 1 μl of each ligation reaction was added. The tubes were incubated on ice for 30 minutes. Next The tubes were placed in a 42 ° C. water bath for 30 seconds and then returned to ice for 2 minutes. 450 μl Room temperature SOC was added to each tube. The tubes were then incubated at 37 ° C and 225 rp m for 1 hour. The transformed cells were treated with 50 μg / ml kanamycin and 1 m Spread on LB plates containing g of X-Gal. Next, selection of transformed clones To do another, the plate was inverted and incubated overnight at 37 ° C. Promeg a (Madison, WI) provided Magic Miniprep^TMSystem for isolation of plasmids went. To pellet the cells, 3 ml of the overnight culture was harvested at 4000 rpm. For 5 minutes. The pellet was suspended in 200 μl of the cell resuspension solution . Add 200 μl cell lysate and mix by inverting the tube until the suspension is clear. I combined. 200 μl of neutralization solution was added and the tube was inverted several times to mix. Mini color Attach the syringe to the syringe barrel and insert the assembly into the vacuum manifold did. The resin / DNA mixture is transferred to a syringe and a vacuum is applied to the column. Was injected. After washing the column with 2 ml of column washing solution, the resin was dried. Centrifuge the mini-column at 14000 rpm for 20 seconds to remove any remaining wash solution. I took it apart. Add 50 μl of 70 ° C water to the column, and after 1 minute, remove the column for 20 seconds. DNA was eluted from the column by centrifugation.   Vector construction   To construct cDNA G10-6 surrounding the entire coding sequence region of BAL, The unique SmaI restriction sites of the clones G10-2 and G10-5 were used. T-BAL cDNA Use of primers I and II to express truncated BAL to prepare In both cases, PCR was used. The following E. T-BAL expression using the T7 expression system in E. coli First, the T-BAL cDNA was converted to a pET11a cloning vector (Invitrogen, San Diego, C Ligation to the NdeI / BamHI cloning site of A).   Expression of T-BAL using pET11a vector   A general approach to direct the expression of cloned genes using T7 polymerase Methods and methods are described in Studier et al., Methods Enz, incorporated herein by reference. ymol, 185: 60-89 (1990). T-BAL cDNA was converted to pET11a (Novagen, Madi son, WI) at the NdeI / BamHI cloning site. For the expression of T-BAL, This vector was used to transform E. coli BL21 (DE3). Having this vector Cells were cultured overnight in ZB medium with ampicillin (Studier et al. (1990)). The next morning, with 80 ml of the overnight culture shaken at 37 ° C until the absorbance at 600 nm reaches 0.6. Then, 4 liters of LB-ampicillin were incubated. Then isopropyl Ru-β-D-thiogalactopyranoside was added to a concentration of 0.4 mM and shaken for 3 hours. Centrifugation Harvest cells by separation and 800 ml TN buffer with 80 mg lysozyme (50 mM Tris-HCl, pH 8.0, and 100 mM NaCl). Mix 70 C., then thawed in a 37.degree. C. water bath, for 3 cycles. TN solution And centrifuged at 16,000 xg for 15 minutes. Then the pellet (Inclusion body) with Triton in TN^TMWash twice with X-100 (0.1%) Clean, collect the pellet by centrifugation and freeze at -20 ° C for further processing It was maintained in a tied state.   Regeneration and purification of T-BAL   1 mM EGTA (ethylene glycol-b (β-aminoethyl ether) N, N, N ', N'- Acetic acid), containing 10 mM β-mercaptoethanol and 5% glycerol (v / v) By stirring with 60 ml of 8M urea in 100 mM Tris-HCl (pH 12.5). The frozen inclusion bodies were further solubilized. Centrifuge this mixture at 105,000 xg for 30 minutes. After centrifugation, the supernatant was placed in a dialysis tube (exclusion MW = 10,000), and 1 M Urea, 0.1 mM β-mercaptoethanol and 5% glycerol in 10 mM Tris Dialysis was performed against 1 liter of regeneration buffer contained in -HCl (pH 8.0). 20 After dialysis for hours, as described in the following “esterase assay”, Using 1 mM PANA as quality and 2 mM Taurochol as activator, Regenerated T-BAL is assayed for esterase activity and the amount of active enzyme in the dialysis solution It was determined. The regenerated T-BAL was then replaced with 60 ml of saturated ammonium sulfate (NH_FourUsing OH Adjusted to pH 8.0). This resulted in precipitation of T-BAL. 18,000 After centrifugation at rpm for 1 hour, T-BAL was collected from the precipitate. Collected sediment Was solubilized with 40 ml of regeneration buffer and stirred at 40 ° C. for 30 minutes. Then Amic on Centriprep^TM-10 Concentrator (Beverly, MA) to reduce supernatant fraction to 3-5 ml Concentrated. After further ultracentrifugation at 105,000 × g for 30 minutes, the supernatant fraction (1 m l) for molecular sieve separation by high-performance protein liquid chromatography (FPLC) Provided. Two Superoses^TMLoad 12 columns (Pharmacia, Piscataway, NJ) with FPLC They were tandemly connected for separation. Equilibrate these columns, 1 M urea, 0.15 M NaCl, 0.1 mM β-mercaptoethanol and 50 mM Tris-HCl (pH 8.0). Elution was carried out using the buffer solution. The column was eluted at a flow rate of 0.5 ml / min. 1 ml Collecting the eluate in the fractions and measuring the absorbance at 280 nm; and Monitor by assaying esterase activity using ANA as substrate did.   N-terminal amino acid sequence analysis   Automated Edman degradation is described in Hewick et al. Biol. Chem. 256: According to 7990-7997 (1981) Equipped with Model 120A online phenylthiohydantoin amino acid analyzer Model 470A Gas Phase Protein Sequencer (Applied Biosystems, Inc., Foster City, CA). The above references are incorporated herein by reference.   Esterase assay   Kinetic studies of BAL esterase activity using short chain esters have been described by Wang, Biochem. J. 279: 297-302 (1991), as described in p-nitrophenyl acetate (P ANA) and p-nitrophenyl butyrate (PANB) were used as substrates. the above Literature is also incorporated herein by reference. Michaelis-Menten dynamics The experiment was performed at 25 ° C to derive the dynamic parameters and was performed in the final assay mixture. Substrate concentrations range from 0.4 to 2 mM for PANA and 0.1 to 0.5 mM for PANB. M and 2 mM Taurocholate was used as the activator. p-Nitrophenol Hewlett-Packard Diode Array equipped with Peltier temperature control Absorption at 418 nm using a Spectrophotometer (Hewlett Packard, Palo Alto, CA) Determined from the first part of the degree change (20 seconds). Refolding efficiency and column chromatography Monitor esterase activity and specific activity of purified enzyme in a matographic eluent For this, 1 mM PANA as substrate and 2 mM Taurocholate as activator Was used to perform the enzyme assay. 1 unit of enzyme activity is released per minute Defined as 1 micromole of product.   Thermal stability of T-BAL   0.1 mg / ml each in sodium phosphate (0.15 M, pH 7.4) to measure thermal stability Of T-BAL and natural BAL in water bath at 30 ° C, 40 ° C, 45 ° C and 50 ° C for 10 minutes. Cubbed. After the incubation, the solution was cooled with ice water. Then these Treated samples were assayed for residual activity using PANA as a substrate.   Lipase assay   BAL lipase assays were performed as described by Nilsson-Ehle and Schotz, J. Mol. Lipid Res. 17:53 Glycerol as a substrate according to a method modified from the method described in 6-541 (1976) Rutori [9,10-^Three[H] oleate. 10 ml of 50 mM NH_FourOH-HCl buffer (pH 8. 5) 28 μmol of trioleoylglycerol (specific activity 1.4 μCi / μmol) and 2.8 By emulsifying μmol of dioleoylphosphatidylcholine, a twofold concentration A contracted stock substrate solution was prepared. The mixture is placed in an ice bath for 30 seconds at 5 settings (maximum). W-380 sonicator (Heat Systems-Ultrasonics, Inc., Farming) (dale, NY). After cooling, the mixture was sonicated for an additional 30 seconds. A final volume of 100 μl of the assay mixture is 50 mM NH_FourOH-HCl buffer (pH 8.5), 1.4 mM Trioleoyl glycerol, 0.14 mM dioleoyl phosphatidyl glycerol , Taurocholate and 10 μl of enzyme solution. With stirring at 37 ° C After one and a half hour incubation, 3.2 ml of chloroform-heptane The reaction was terminated by adding ethanol (5: 4: 5.6, vol / vol / vol) and 1 ml of 0.2 M NaOH. I let it. Centrifuge the sample and add 10 ml Hydrocount^TM(J.T. Baker, Inc., Phillip burg, NJ) and emit with a Beckman scintillation counter (Fullerton, CA) Noh was measured.   Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis   Using the LKB-Pharmacia Phast system (Piscataway, NJ), Pharmacia (Piscatawa y, NJ) 8-25% polyacrylamide gel slab, SDS-polyacrylamide Midgel electrophoresis was performed. Samples at 10 ° C for 10 minutes at 10 mM β-medium for 6 minutes before electrophoresis. Treated with lucaptoethanol and 2% SDS.   Protein assay   The protein content of the enzyme preparation was determined using Lowry using serum albumin as a standard. (Lowry et al., J. Biol. Chem. 193: 265 (1951)) (Wang and Sm ith, Anal. Biochem. 63: 414-417 (1975)). This document is It is incorporated by reference in the textbook.   Fluorescence measurement of the interaction of T-BAL with taurocholate   The fluorescence measurement of T-BAL interaction with taurocholate was performed using the aminco-Bowman Series. Performed at 25 ° C. using 2 Fluorescence Spectrometer (Urbana, IL). T-BAL Tauro Use T-BAL tryptophanil fluorescence to study the interaction with cholate Was. Fluorescence was recorded at 340 nm using an excitation wavelength of 280 nm. Excitation and emission bands Both widths were set to 2 nm. The sensitivity of the device was set to 1,000 volts, the detector high pressure .   In the absence of taurocholate (activator), the fluorescence intensity of T-BAL is F °. Was. The fluorescence intensity is F at the saturation concentration of Taurocholate._∞Met. Specific tauro Based on the fluorescence (F) at the collate concentration, F is related as shown in the following equation: Determine the molar fraction (x) of T-BAL related to Taurocholate by linking did.           F = (1-x) F₀ + xF_∞   The derived molar fraction (x) is then converted to a binary complex of Taurocholate and T-BAL. Binary complex dissociation constant K_AIs used in the calculation of Conversely, K_AAnd F_∞Allowed Using Computer Least Squares Curve Fitting Procedure Treated as Variable Parameter , F, and the best agreement with the experimentally determined values Can be.   Data analysis   A kinetic analysis of the data generated in these experiments was performed using the LOTUS 1-2-3 SPREAD SHE Performed using the ET program (Worcester, MA) and an IBM-AT computer. minimum Performing a squared non-linear curve fit using Bevington, Data Reduction and Erro r Analysis for the Physical Sciences, 56-65 and 204-246, McGraw-Hill, N Performed according to the approach described in ew York (1969). This document is Incorporated in the textbook for reference.   (a) Carboxyl terminal domain, O-glycosylation and N-glycosylation It is not essential for the enzyme function of AL.   Amino acid sequence of native human milk BAL cDNA (Baba et al. (1991); Hui and Kissel, FE BS Lett. 276: 131-134 (1990); Nilsson et al., Eur. J. Biochem. 192: 543-550 (1990 )) Encodes a mature enzyme of 722 residues and is shown in SEQ ID NO: 3. cDNA structure is Pancreas B Identical to the structure of AL (Reue et al., J. Lipid Res. 32: 267-27G (1991)). This is milk It supports the notion that enzymes from the gland and the pancreas are expressed from the same gene. Carry.   As expected, T-BAL synthesized in E. coli is inert and insoluble in aqueous buffer . Active T-BAL was obtained by refolding. This procedure involves the inclusion of urea and urea. Solubilization in β-mercaptoethanol at high pH (12.5) followed by low pH (8.0) Dialysis against low concentrations of urea was involved. Based on enzyme activity measurements, The element yield was about 10 mg per 4 liters of the culture LB medium. SDS-polyacrylamide The major proteins in the urea-soluble fraction corresponding to a molecular weight of 60,000 were analyzed by gel electrophoresis. Quality occurred (> 90%). This molecular weight is very close to the estimated molecular weight of T-BAL (59,270). Similar. By analyzing the N-terminal sequence of this protein fraction, the predicted BAL N-terminal sequence Ala- Lys-Leu-Gly-Ala-Val-Tyr-Thr- (amino acids 1 to 8 of SEQ ID NO: 1) was obtained, and T-BA Successful synthesis of L and removal of the starting methionine was shown. First steps of refolding After that, the specific activity of T-BAL was about 5 to 10 units / mg. This is the specific activity of natural BAL Only 10-20%.   Recombinant T-BAL expressed in E. coli is not glycosylated. This further In addition, the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Not shown.   For purification of T-BAL, refold T-BAL by ammonium sulfate precipitation. After purification by fractionation, molecular sieving by FPLC was performed. Two column fractions A major peak was found. The first peak eluted in the excluded volume (17 ml) It represents the peak of the protein required, and mainly contains inactive T-BAL in an aggregated form. 2nd pea ク (eluted at 28 ml) contains BAL activity. Fraction 27-29 SDS-polyacrylamide gel The pattern shows that the T-BAL eluted in this peak is homogeneous. Average specific activity of 64 ± 2 units / mg against purified enzyme (fraction 28) from four different batches was gotten. This specific activity of T-BAL was previously reported (Wang and Johnson (1983 )) Is higher than the specific activity reported for native BAL (52 units / mg), Matography effectively separated the active form of the enzyme from the inactive form.   (b) Dynamics and specificity   With the availability of sufficient amounts of purified recombinant T-BAL, T-BAL and native BA The specificity and kinetics of L were compared and other properties noted.   Thermal stability of T-BAL   To compare the stability of T-BAL with the native enzyme, these two enzyme forms were Treated at a temperature in the range of 0 ° C. The thermal inactivation patterns of T-BAL and native BAL are similar In both cases, treatment at 50 ° C. for 10 minutes showed a loss of activity of about 90%. to this Furthermore, the folding of T-BAL resembles that of the catalytic domain of the native enzyme It is suggested that   Taurocholate binding dynamics of T-BAL   Dissociation constant K of T-BAL and natural BAL_AAre similar. Mutual with Tauro Corate In direct binding studies of tryptophanil fluorescence during action, the binding of bile salts In this case, the natural BAL can be obtained by using the protein tryptophanil fluorescent at a saturated concentration of taurocholate. Light was reduced by about 20% (Wang and Kloer (1983)). This is probably due to the ligand binding Arising from the conformational change of the BAL at the time. The dissociation constant of natural BAL is 0.37 mM (Wang, J. Biol. Chem. 256: 10198-10202 (1981)). Similar to tryptophanil fluorescence Was also observed with T-BAL. Where α and β are dynamic parameters, respectively. Meter K_sAnd K_catTo improve the activation effect of taurocholate. Two parameters used to represent (Segel, Enzyme Kinetics 227-23 1, John Wiley & Sons, New York (1975)). Based on 1: 1 stoichiometry, T-BAL and For the interaction with Taurocholate, the dissociation constant K_A 0.32 ± 0.03mM (n = 4) measured Was done. At a saturated concentration of taurocholate, the fluorescence intensity decreased by about 18%. Therefore, T- BAL has a higher affinity for monomeric taurocholate than the native enzyme. Slightly higher. Based on these results, T-BAL and natural BAL The microenvironment of the binding site is also involved in the deletion of the proline-rich sequence domain of T-BAL. But very similar.   Dynamic characteristics of T-BAL by PANA and PANB   Further kinetic analysis showed that PANA and PANB (p-nitrophenyl) (Butyrate) was shown to change the enzyme specificity.   Like natural BAL, T-BAL is synthesized using an esterase substrate in the absence of bile salts. The assay was found to contain basal activity. Therefore, Tauro Corate Can also be considered as a non-essential activator of T-BAL. Lineweave From r-Burke plots, power for T-BAL and PANA and PANB as substrates Parameter K_s, K_cat(For the base enzyme) and αK_s, And βk_cat(Tau Locolate activating enzyme) was obtained. Slightly less than the specific activity of the natural enzyme (52 units / mg) The fact that high T-BAL specific activity (64 units / mg) was obtained (Wang and Johnson (1983) Despite this, the derived K of T-BAL_catAnd βk_catIs the natural enzyme K_catYou And βk_catAbout 2 to 8 times lower. However, T-BAL's derived K_SAnd αK_s Is the natural enzyme K_sAnd αK_sIt was only slightly higher. Therefore, proline The presence of rich sequences plays a major role in increasing the turnover rate of enzymes, It has only a small effect on the binding affinity. In addition, preferential enzyme There is a change in response. Previously, the short chain acyl esters of p-nitrophenol Naturally, BAL is higher for PANB_catAnd βk_catWas reported to have . In contrast, when assayed in the presence of Taurocholate, T-BAL shows PAN Higher k for PANA than for B_catAnd βk_catHaving. Also in this Nevertheless, T-BAL also has a higher substrate specificity constant (k of T-BAL) in the case of PANB._cat/ K_s And βk_cat/ αK_s). This is similar to what is found in natural enzymes I have. Effect of Taurocho on BAL-catalyzed hydrolysis using PANA as substrate Due to the activating effect of the rate, the proline-rich domain of BAL No binding site is shown.   Lipase activity of T-BAL   In natural BAL-catalyzed hydrolysis of long-chain triacylglycerols, fatty acids There is an essential requirement for bile salt micelles to act as receptors (Wang et al. ( 1988)). The hydrolysis of glycerol in native BAL and T-BAL was compared. this In this regard, T-BAL is a significant factor in the hydrolysis of long chain trioleoylglycerol. It has been found that the sulphate has requirements similar to those of natural BAL. Serum al Since Bumin is not a fatty acid receptor for BAL catalysis, BAL-catalyzed reactions Is also a weak fatty acid receptor. Therefore, the lipase mixture contains fatty acid It contained only Taurocholate, not BSA.   Activation of natural BAL catalysis is not seen at taurocholate concentrations above 60 mM There is over-saturation, which is part of the natural BAL at high taurocholate concentrations. Attributed to fractional inactivation. On the other hand, micelle taurocholate (120 mM There was an apparent non-saturated activation of T-BAL (tested to locolate). one On the other hand, the apparent saturation of activation by taurocholate exceeding 60 mM This is probably due to the partial inactivation of the enzyme when the rate concentration was high. To these results Thus, the transfer of fatty acid products from the enzyme active site is likely to be a receptor-mediated process. Rather than via a process, the enzyme-fatty acid complex and taurocholate micelles Via the second order reaction kinetics. Example 2 BAL C-Tail Function Binds BAL to Cells Lining the Intestinal Hull Is Rukoto.   (a) Binding of BAL in human milk to intestinal mucosa   To determine that BAL in human milk binds to intestinal mucosa, experiment by Was done. That is, (i) purified natural human milk BAL was radiolabeled (I¹²⁵) And (ii) Incubate the radiolabeled BAL in the isolated mouse intestine and (iii) The extent of intestinal retention of BAL was determined and (iv) isotonic phosphate as a control. Using a buffer solution, mouse internalization in galactose, fucose, heparin, 0.3M NaCl The mechanism of BAL binding to the intestine by examining the release of soluble BAL .   Method   Purification of human milk BAL is described in Wang and Joh, incorporated herein by reference. nson, Anal. Biochem. 133: 457-461 (1983). Preparation of the truncated form of recombinant BAL (T-BAL) was performed as described in Example 1. .   Iodination of human milk BAL and recombinant T-BAL was performed using iodine beads (Pierce, Rockfor d, IL). Iodine¹²¹Was obtained from Amersham (Arlington Heights, IL) . 6 μl¹²⁵I was added to 94 μl of an enzyme solution (2 mg / ml in sodium phosphate buffer (pH 6.5)). ) To perform iodination. One washed bead in solution And left at room temperature for 15 minutes. Then the mixture is Bio-spin^TM6 columns (B io Rad, Hercules, CA), centrifuged at 2275 rpm for 4 minutes at 4 ° C, and The eluate was collected. Dilute the eluate with 400 μl of 0.1 M sodium phosphate buffer (pH 6.5). I shouted. The eluted fractions were combined and 1 μl of the eluate was tested for radioactivity. G Lycerol tri [9,10-^ThreeH] oleate was used as substrate and described in Example 1. Lipase assay was performed as described above. 2x concentrated stock substrate solution was added to 1 0 ml of 50 mM NH_Four28 μmol of trioleoyl glycerol in OH-HCl buffer (pH 8.5) (1.4 μCi / μmol) and 2.8 μmol of dioleoylphosphatidylcholine Was prepared by emulsification. Mix the mixture with a W-380 sonicator (Heat Systems -Ultrasonics, Inc., Farmingdale, NY), set the setting to 5 (50% of the maximum output) And emulsified in an ice bath for 30 seconds. After cooling, the mixture was sonicated for an additional 30 seconds . A final volume of 100 μl of the assay mixture is 50 mM NH_FourOH-HCl buffer (pH 8.5), 1.4  mM trioleoyl glycerol, 0.14 mM dioleoyl phosphatidyl glycerol , 30 mM taurocholate, and 10 μl of enzyme solution. 1 at 37 ° C After incubation with stirring for 3.2 hours, 3.2 ml of chloroform-heptane The reaction was terminated by adding methanol (5: 4: 5.6, vol / vol / vol) and 1 ml of 0.2 M NaOH. did. The sample is centrifuged and a 10 ml Hydrocount^TM(J.T. Baker, Inc., Phil lipsburg, NJ) and mixed with a Beckman scintillation counter (Fullerton , CA).   To the mouse small intestine¹²⁵Protocol for binding of I-labeled BAL and T-BAL   Remove the duodenum and jejunum from the small intestine of the mouse (about 20 grams each), and And cut into 12 cm segments. Three experiments were performed. Here, in parallel experiments In this case, T-BAL except C-tail was used as a control. Segment Wash once with 0.15 M NaCl and twice with 0.1 M sodium phosphate buffer (pH 6.5) did. 20 μl of the labeled sample is first mixed with 20 μl of 8M urea and then 720 μl of Diluted with distilled water and 240 μl of 20% albumin to a final volume of 1 ml. Then , 500 μl of the solution was injected into each intestinal segment ligated at both ends. Segment the room Incubated at warm for 2 hours. After the incubation, the intestines were washed with 0.1 M phosphate Wash three times with sodium buffer (pH 6.5) and add 2 to count radioactivity. Cut into small intestinal pieces of cm.   The results of these experiments are shown in Table II, Table III and Table IV, and in FIG.   In these experiments, the native BAL was found to have a T-type as shown in Tables II and III. It was retained by the intestinal mucosa in almost 12 times more amount than BAL. Natural BAL and T-BAL Differ only in the C-tail, so these results indicate that Adhesion of intestinal surface to receptors is mediated by glycosylated C-tail Indicates that   To determine whether pancreatic BAL adheres to the intestinal surface of adult animals, After the cells were completely washed with saline, BAL enzyme activity was measured in mouse intestine. . The results showed that the intestine had high BAL activity.   Wash to examine the nature of the interaction between BAL and the mouse small intestine receptor 1 cm segment of isolated intestine with fucose, galactose, heparin, NaCl and isotonic Endogenous binding from intestinal mucosa by incubation with neutral phosphate buffer An experiment to elute the BAL enzyme activity was performed. The results are shown in Table IV.   Only low activity BAL elutes with isotonic phosphate buffer and 0.3 M NaCl. This indicated that BAL was not bound to the intestine through ionic interactions. Sa In addition, heparin was activated by isotonic phosphate buffer and BAL activity eluted by NaCl. Did not elute more BAL activity. This is mediated by heparin The intestinal binding activity of BAL (known to bind to heparin) Was shown. However, elution was achieved using either galactose or fucose. If so, a large amount of BAL activity eluted. These results indicate that the C-tail oligo Fig. 2 shows that BAL binds to the intestinal lumen surface via a sugar group. In addition, elution is very high Being specific, the result is specific binding to oligosaccharides in the B-C-tail Indicates that a receptor (CT receptor) is present.   Mouse pancreas BAL contains four repetitive motifs, similar to the C-terminal region of the human enzyme. C-terminal region. Because human BAL binds to mouse intestine, , Similarities in the structures of the oligosaccharides were confirmed.   Example 3: BAL elution from intestine   (a) Flat endogenous intestinal BAL, galactose, fucose, methyl-α-mannoside With EGTA, EGTA, heparin, and isotonic phosphate buffer as a control   The intestines from mice are too small in size, so that Used to demonstrate the elution of BAL. Initial experiments were performed on the intestine of mice. Similar to the observations made, rat endogenous BAL also uses galactose and fucose. To demonstrate that it could be eluted as well. Riga C-type lectin Calcium ions are required for binding to the gland, so that Testing for calcium requirement included EGTA elution.   Table V shows that the bound rat intestinal BAL showed galactose (0.1 M), fucose (0.1 m). ) And three experiments showing elution by EGTA (1 mM). You. Elution with heparin (10 mg / ml) was performed using an isotonic phosphate buffer (pH 7.4). As well as the level of output, it was not effective because of the low activity in the eluate. this In these experiments, a 1 cm segment of washed rat intestine was prepared using isotonic phosphate buffer, pH Incubated for 30 minutes at room temperature with the eluted compounds dissolved in 7.4. this BAL activity in the eluate was assayed as described in Example 1.   The inability of heparin to elute intestinal bound BAL indicates that BAL is not It was not bound to the intestine. These results indicate that BAL from mouse intestine Is consistent with the results obtained for the elution of (Table IV). Galactose or The successful elution of bound BAL by any of the fucoses It shows that attachment is through an oligosaccharide group in the C tail. EGTA That BAL tail was bound to the intestine by calcium It has a cofactor requirement.   (b) BAL release from intestinal epithelial cells decreased from cholesterol oleate Associated with cholesterol uptake.   Experiment   As shown below, BAL intestinal lining cells An experiment was performed to demonstrate that adhesion to was required.   A cholesterol (oriate) ester emulsion solution was prepared as follows. A 2-fold concentration of cholesterol oleate stock solution was added to 6 μmol [^ThreeH] Cholesterol Oleate (1.6 μCi / μmol) (Amersham, Arlington Heights, IL) 15 ml isotonic phosphate buffer with μmol dioleoylphosphatidylcholine, Prepared by emulsification in pH 7.4. This mixture was set to the setting value 5 (maximum output Of a W380 sonicator (Heat System-Ultrasonics, Inc.) in an ice bath for 30 seconds in an ice bath. , Farmingdale, NY). After cooling, the mixture is left for an additional 30 seconds. Further sonication was performed.   Excision of two 12 cm segments of rat small intestine in each of four experiments And washed once with an isotonic phosphate buffer, pH 7.4. Each animal-derived cell The control (not washed by EGTA) and the experiment (EGTA (Washed) group. Copy pairs of segments from the same animal The control and experimental groups are used to determine the amount of BAL in the intestine. All differences between objects have been reduced. For infusion or removal of the solution from the lumen, the intestinal Segments are ligated at each end and secured to the catheter at one end. Specified.   The control segment was first injected with 1 ml of isotonic phosphate buffer, pH 7.5 . To release BAL bound to the intestinal surface, the experimental segment contains 1 mM EGTA. Received the same isotonic buffer containing. Intestinal segment with 5 ml of the same isotonic buffer Contains a polystyrene weighing dish (14 cm^Two) And gently shake for 30 minutes Was. The experimental segment was then washed once with isotonic phosphate EGTA solution and in isotonic buffer. And washed twice. Control intestines were washed three times with isotonic buffer.   To allow uptake of cholesterol by intestinal segments, , 0.2 mM [^ThreeH] -cholesterol oleate (Amersham, Arlington Heights, I L) and 6 mM Taurocholate (Sigma Chemical Co., St. Louis. MO) (BAL A 1 ml aliquot containing a cofactor for each) was placed in each intestine. After incubation for 60 minutes with gentle shaking in a polystyrene weighing dish The contents in the intestinal segment were removed. Transfer these segments with isotonic buffer Washed 3 times with.   Radioactive cholesterol from control and experimental intestinal segments Take the central 10 cm of each intestinal segment to measure It was removed and further cut into four segments of about 2.5 cm. That segment Each was mixed with 1 ml of 2% sodium dodecyl sulfate + 8 M urine for tissue solubilization. Was placed in a scintillation vial containing nitrogen. 10 m after solubilization process l Hydrocount_TM(JT Baker, Inc., Phillipsburg, NJ) And in the intestinal segment^ThreeThe amount of radioactivity from H-cholesterol And counting using a Beckman Scintillation Counter (Fullerton, CA).   The results obtained from these four experiments are shown in Table VI and FIG.   As shown in Table VI, EGTA treatment caused cholesterol uptake by intestinal epithelial cells. , The control uptake was reduced to about 16%. Raw data (p <0.005) and The statistical significance of data calculated as% of control (p <0.0001) Showed a very high confidence level of the difference. These results indicate that BAL (adhered to the intestinal surface) ) Uptake of cholesterol from the intestinal lumen by intestinal lining epithelial cells. Mediating.   (c) Role of BAL in cholesterol uptake from cholesterol ester In addition, BAL mediates the direct uptake of or free cholesterol.   About 85% of dietary cholesterol is in the form of free cholesterol, It is important to establish a role for BAL in uptake of isolated cholesterol.   Bound BAL in control gut compared to gut with bound BAL removed by EGTA In vitro experiments were performed to compare the uptake of radioactive cholesterol . Table VII shows the results obtained from three experiments. Control experiment The average was 16.38 nmol / g intestine / hour uptake. Mean values for EGTA-treated gut Was 4.40 nmol / g intestine / hour. Removal of BAL results in uptake of free cholesterol Resulted in a reduction of about 70%. Despite the relatively large variation between the three rats However, the difference between these two groups is very significant (p <0.005).   A 2-fold concentration of cholesterol stock solution was added to 6 μmol (specific activity = 1.6 μCi / −μm ol) cholesterol and 0.6 μmol of dioleoylphosphatidylcholine Prepared by emulsification in 5 ml isotonic phosphate buffer, pH 7.4. This mixture The object was placed in an ice bath at a setting of 5 (50% of maximum output) for 30 seconds in a W-380 sonicator (Heat). -System-Ultrasonics, Inc.). After cooling, the mixture is Ultrasonic crushing was continued for another 30 seconds.   Two segments (11 cm) of the rat intestine were each treated with isotonic phosphate buffer (pH 7. After washing once in 4), these segments were ligated at both ends. Then 1 mM EGTA 1 ml of isotonic phosphate buffer containing 1 ml of isotonic phosphate buffer without 1 mM EGTA The buffer was injected into each intestinal segment. Transfer this segment to 5 ml isotonic The sample was placed on a polystyrene weighing dish (14 cm square) containing a phosphate buffer. Inn After incubation, 1 ml of the solution in the intestine was^ThreeH] cholesterol (0.2 mM) and Replace with 6 mM Taurocholate and replace these segments in an ice bath as described above. Placed on the incubation tray above. Then these intestinal segments Were washed three times with isotonic phosphate buffer and 8 cm sections of each intestinal segment (Approximately 0.7 g) were cut into 2 cm fragments, and each of those fragments was Placed in 1 ml 2% SDS-8 M urea for solubilization. Add 10 ml of Hydro to this mixture ocount^TMWas added for radioactivity counting as described in Example 3a.   (d) Heparin inhibits the uptake of free cholesterol by rat intestine.   By rat intestine^Three[H] cholesterol oleate uptake It was studied in vitro in the presence of phosphorus (10 mg / ml). This procedure is essentially The procedure is the same as described in Example 3b. Table VIII shows the controls (column 1, tau Average presence up to 20.65 nmol / g / Hour. In the presence of heparin (column 2), 2.62 nmol / g / It is time. This difference is significant (p <.05). The value from heparin inhibition is -Near background uptake in the absence of rate.   The experimental procedure is the same as the procedure described in Example 3b. Intestines not containing EGTA Bound BAL was not removed from the intestinal surface as it was washed with tonic phosphate buffer. The concentration of heparin (ICN Chemicals, Costa Mesa, CA) in the uptake solution was 10 mg / m l.   (e) Addition of BAL to EGTA-treated intestine restored cholesterol uptake Materials and reagents   As described in Wang and Johnson (1983), which is incorporated herein by reference. Thus, natural human milk bile salt active lipase (N-BAL) was purified. BAL recombination short The truncated form (T-BAL, residues 1-538 lacking the carboxy terminus) is described in Example 1. As reported by Downs et al., Biochemistry 33: 7979-7985 (1994). It was prepared as follows.   [Iα-2α (n)-^ThreeH] cholesterol oleate (^ThreeH-cholesterol) with Amersham (A rlington Heights, IL). Other chemical reagents were purchased from Sigma Chemical Co. St. Louis, purchased from MO.   2x concentrated cholesterol oleate stock solution was added to 6 μmol^ThreeH-Cholesterol (1.6 μCi / μmol) and 0.6 μmol of dioleoyl-phosphatidylcholine 4 Prepared by emulsification in 15 ml isotonic sodium phosphate buffer, pH 7.4 . The mixture was used with a W-380 sonicator (Heat-system Ultrasonics, Inc., Farmingdale). , (NY) using an ice bath for 30 seconds at a set value of 5 (50% of maximum output). After cooling, mix The mixture was further sonicated for an additional 30 seconds.   Method   In each of the five experiments, three segments (11 cm each) of the rat small intestine were Wash once with isotonic buffer (pH 7.4) and place the catheter in each intestinal tract for infusion of the solution. The segment was ligated at both ends by insertion at one end of the fragment. Then execute As shown in Example 1 (a), 1 mM EGTA was included to release BAL attached to the intestinal surface. 1 ml of the isotonic phosphate buffer was injected into each intestinal segment. Maintain moisture Polystyrene weighing dish containing 14 ml of isotonic phosphate buffer (14 cm^Two) To that seg The sample was placed and incubated on an ice bath for 30 minutes with gentle shaking. After incubation, intestinal segments were washed once with EGTA-containing isotonic buffer and 0.2 times. Washed twice with isotonic buffer containing mM calcium chloride. Then isotonic phosphoric acid Buffer and 0.2 mM Ca⁺²At an average enzyme concentration of 0.8 mg / ml in the presence of Inject 1 ml of purified N-BAL into one and 1 ml of T-BAL into the second segment Was. The third intestinal segment without BAL was the control. Then Again, place these intestinal segments again in the incubation tray and let sit for 30 minutes And incubated with shaking. After incubation, the solution in the segment The solution is removed and [^ThreeH] cholesterol oleate (0.2mM) And 1 ml of 6 mM Taurocholate. Incubate with shaking as described above. After washing, the segments were then washed three times with isotonic phosphate buffer and An 8 cm segment of the intestinal segment (approximately 0.7 g) was cut into 2 cm fragments, each of which was Placed in 1 ml of 2% SDS-8 M urea for solubilization. Add 10 ml of Hydrocou to this mixture nt^TMWas added and the radioactivity weighed as described in Example 1 (b).   The results are shown in Table IX.   As shown in Table IX, cholesterol uptake was determined by EGTA-washed intestine without BAL. Segment (control), EGTA-washed segment treated with truncated BAL (+ T-BAL) And EGTA-washed segments (+ N-BAL) treated with purified native human BAL did. Measured in nmol cholesterol uptake per g intestine per hour Cholesterol uptake is shown in the left column of each group. Average uptake value of 5 experiments Were control 6.76, + T-BAL 7.32, and + BAL 14.41. Control Uptake in the urea segment, for example, Fusion of intestine and residual intestinal BAL remaining after EGTA washing And non-specific trapping of radioactive cholesterol in the   Due to large deviations within each group due to biological differences between the five rats, Data were calculated as a percentage of the + N-BAL group and are shown in the right column for each group. percentage Data means mean uptake in controls is 49.67% of + N-BAL group (100%) In the case of + T-BAL, it was 55.92%. + Incorporation of N-BAL The difference between + T-BAL uptake is statistically significant (p <0.005).   When the control data is subtracted from the + T-BAL data and + N-BAL data, The average net uptake is as follows:   + T-BAL: 7.32-6.76 = 0.56 nmol / g / h   + N-BAL: 14.41-6.76 = 7.65 nmol / g / h The net cholesterol uptake for + N-BAL is the net cholesterol uptake for + T-BAL. It is about 14 times cholesterol uptake. The percentage difference between these two groups is also Statistically highly significant (p <0.005).   These results indicate that (a) cholesterol uptake by EGTA- or EDTA- The reduction in uptake is mediated by BAL loss and Not due to impairment of the cholesterol mechanism; and (b) restoration of cholesterol uptake Caused the binding of the BAL carboxy-terminal tail to the intestinal surface, which was missing in T-BAL. It indicates that   Example 4: Addition of C-tail to intestinal contents releases bound endogenous BAL   C-tail binds to intestinal surface CT-receptor resulting in translocation of bound endogenous BAL Experiments were performed to show that they could compete for binding.   (a) Preparation of C tail   A procedure was devised for preparing a pure BAL C tail from BAL. C tail spirit Is very effective, so that the starting BAL does not need to be completely homogeneous It should be noted. The BAL used in these C tail isolation experiments was described previously. Heparin-Sepharose as described (Wang and Jonson (1983))^TMColumn (bed capacity 14 X 1.5 cm) concentrated by chromatography. 300 ml of human skim milk, First, it was centrifuged at 20,000 rpm for 2 hours. The supernatant is filtered, and 50 mM Tris-HCl buffer ( Heparin-Sepharose pre-equilibrated with pH 8.0)^TMApplied to the column. After loading, The column is washed with 200 ml of equilibration buffer and BAL is washed with 0.3 M NaC in equilibration buffer. eluted in 1 l and collected in 5 ml fractions.   Fractions were purified using BAL Esterer using p-nitrophenyl acetate as substrate. Assayed for zealase activity. Fractions containing BAL activity were pooled and pooled against distilled water. Dialyzed and lyophilized. The yield is about 100mg-150mg dry from each batch Substance. For C-tail purification, three batches of partially purified BAL (about 350 mg) These pools were treated with 8 M urea (10 mg / ml) for 2 hours at room temperature and overnight at 50 mM Tris. Dialyzed against -HCl. Denatured BAL at 37 ° C for 4 hours, trypsin and chymotryp Digested with syn (substrate: protease ratio, 50: 1, w / w). Equal amounts of trypsin and Motrypsin is added a second time to the mixture and incubated at 37 ° C overnight. Continued. The digest was then dialyzed against distilled water and lyophilized . Dissolve the dry powder in 5 ml 70% formic acid and 50 mg cyanogen bromide and pack in a glass tube. Closed and incubated overnight at room temperature. Dilute the solution 10 times with distilled water and freeze It was dried. The material is further dissolved in distilled water, dialyzed, and the insoluble material is centrifuged. Removed by separation. The soluble fraction was then lyophilized. Then two tongues Sepharose linked to dem^TMFPLC (fast protein liquid chromato For gel permeation chromatography using (g raphy), dry material was Was solubilized using 3 ml of 50 mM Tris-HCl buffer containing. 1 ml sample The column was applied for each round of matography. Carbohydrate analysis of the eluate fraction is (D ubois et al., Anal. Chem. 28: 350-356 (1956)), in fractions with a tail of 21-25 (1 ml / fraction). It was eluted.   b) Structure and composition of C tail   Analysis of the N-terminal sequence and amino acid composition of substances from the carbohydrate-containing FPLC fraction Corresponded to the human BAL region containing residues 528-712. table As shown in X, the amino acid composition of the purified C tail and the composition based on the known sequence Very similar. Β-elimination using alkali for release of O-linked oligosaccharides From experiments and in further amino acid composition analysis of samples, 8-10 residues The intervening threonine is broken within each molecule of the C tail while the serine up to one residue Was destroyed by the same procedure. Therefore, not all, but most It was concluded that most oligosaccharides were attached to C-tail threonine residues.   Gas-liquid chromatography of alditol acetate derivatives (Griggs et al., Anal. B. iochem. 43: 369-381 (1971)) and the carbohydrate composition of the C-tail of human milk BAL. The data from the subsequent analyses, as shown in Table XI, show the fucose, galactose in C tail. It indicated the presence of toose, galactosamine and glucosamine. Galactosamine is Because it is the sugar that anchors the carbohydrate to attach it to the polypeptide chain, From the ratio of gin to galactosamine, there are about 8 to 10 oligosaccharides per C tail molecule. It is estimated that there is.   c) C tail causes release of endogenous BAL from rat intestine.   BAL with isolated C-tail domain of human milk BAL bound from rat intestinal surface An experiment was performed to determine if could be replaced. In these experiments, Different amounts of C tail were placed in the solution contained inside the ligated rat intestine . BAL activity released after 30 minutes was measured.   Incubation of BAL intestine with EGTA and BAL C tail and B in eluate The measurement of AL lipolytic activity was as described in Examples 3a and 3b.   The data is shown in Table XII. This result indicates that the isolated C tail was not found in rat intestine. It was shown to be effective in replacing the combined endogenous BAL. Example 5: Competitive inhibition of cholesterol uptake by BAL C-tail   The preceding example demonstrates that C-tail binds endogenously bound BAL from rat intestinal surfaces. This indicates that it can be eluted. The following study shows that isolated C-tails Cholesterol uptake can be competitively inhibited.   a) In vitro inhibition of cholesterol uptake by C-tail of BAL.   In this study, three intestinal segments were washed with isotonic phosphate buffer (pH 7.4). Was cleaned. One of the intestinal segments was used as a substrate^ThreeH] Cholesterol Incubated only with oleate (0.2 mM). The control intestinal segment Higher because endogenous BAL on the gut surface is available for cholesterol transport Should have a high cholesterol uptake rate. The second intestinal segment [^ThreeH] Cholesterol oleate and EGTA (1 mM) were incubated. EGT A is known to elute endogenous BAL from the intestinal surface. Third intestinal segment And the same [^ThreeH] cholesterol oleate and isolated C-tail (0.2 mg / ml) Incubated.   The data shown in Table XIII below is from four separate experiments. Average day The data is shown in FIG. These results show that when C-tail is present, Entrapment indicates only about 10% of control intestinal uptake. this is , Uptake of cholesterol in the presence of EGTA (about 18% of control ). These results indicate that C-tail is not intrinsic for intestinal surface binding. 4 shows that it can compete effectively with sexually bound BAL. This inhibition is due to the C-tail Cholesterol from human diet using C-tail due to replacement of endogenous BAL on intestinal surface 4 shows that terol uptake can be reduced.   b) Background of in vivo inhibition of cholesterol uptake by C-tail of BAL.   FIG. 1 shows the physiological function of BAL in cholesterol uptake by the intestine. The scheme was constructed based on data obtained from in vitro experiments. This In chimes, BAL initially binds to the gut surface by putative receptors. Next Is cholesterol or cholesterol ester a hydrophobic food vehicle? Transported to the active site of the enzyme. Cholesterol esters in the BAL active site Hydrolyzed. Free cholesterol in the active site (hydrolysate or (Whether transported directly from the vehicle) is transported to enterocytes. This Later stages are assisted by cell surface heparin, which may act as an orientation factor. Helped. The above results indicate that BAL not bound to the intestinal surface has cholesterol uptake It is not effective to intervene only.   This scheme assumes that the supply of isolated C-tail to surviving animals is Compete for different receptors and therefore competitively inhibit cholesterol uptake Foresee what to do. The following results confirm this prediction. Experimental conditions   Eighteen hours before the experiment, food (but not water) was given to a pair of adult white rats (mean body weight). Weight 259 ± 27 g). For experimental rats, 4 mg in isotonic phosphate buffer (pH 7.4) Of 1% (wt./vol) glucose containing 1% (wt./vol) glucose of the isolated C-tail (from BAL in human milk) The solution was supplied in a tube. Control animals contained the same volume without C-tail. An amount of glucose-isotonic phosphate buffer was given. One hour later, both animals received 10 μCi [^Three H] -cholesterol oleate + non-radioactive cholesterol oleate (final concentration 0.2 mM), 4 mg of isolated C-tail, 0.2 mM triolein and 0.02 mM diol 1 ml of the emulsified mixture containing oil phosphatidylcholine was given in a tube. Emulsification The procedure was performed as described in section e) of Example 3 above. Again, control Animals received the same mixture (but no C-tail). In short-term experiments, Blood samples were taken at hourly intervals for a period of 5 hours. Aliquot (100 μl) this Take from blood and 2 ml of hepataneisopropanol ( 3: 7, vol / vol) and 1.25 ml of 0.033 N H_TwoSO_FourAcidified with. this The mixture was vortexed for 30 seconds and centrifuged at 4 ° C. for 10 minutes. Next The upper half (250 μl) was then used to count the radioactivity.   The procedure for C-tail isolation from BAL in human milk is described in Section 4 of Example 4 above. The procedure was as described in section a). Choles in cholesterol oleate emulsified mixture Based on the measured specific activity of terol oleate, the radioactivity in blood Converted to the amount of cholesterol carried to the blood compartment. result.   Rats were fasted for 18 hours to reduce small intestinal contents. 4 mg for each experimental rat 1 hour before further administration of radioactive cholesterol oleate with C-tail In addition, 4 mg of the isolated C-tail was provided in a tube. Control rats are C-tay The animals were treated in the same manner as the experimental rats, except that no rats were given. Cholester with blood sample Samples were taken at hourly intervals after roll administration and radioactivity was measured.   Figure 5 shows the results from a representative experiment of one experimental and control rat. The result is shown. The results were obtained from a total of 6 control rats and 6 experimental rats. Obtained. Table XIV shows data from all six experiments, and FIG. Fig. 7 shows the average cholesterol uptake of the trol group and the experimental group fed with C-tail. . Inhibition of cholesterol uptake by administration of C-tail The measurements obtained 1, 2, 3, 4 and 5 hours after administration of the , 35%, 48.8%, 55.7%, 62.2% and 55.5%. As shown in Table XIV, 2 Data between control and experimental groups at 3, 4, and 5 hours were statistically significant. Are highly significant (p <0.01). Significant difference   The following conclusions can be drawn from these results:   (1) Inhibition of cholesterol uptake by C-tail in surviving rats Supports the conclusion that BAL conjugated to mediated cholesterol uptake.   (2) that cholesterol uptake is inhibited by the isolated C-tail In fact, (a) C-tail (either on BAL or isolated as a fragment itself) Binding to the same receptor on the surface, (b) binding of C-tail and receptor Must be very specific to achieve such significant inhibition. And (c) indicate that the number of receptors on the intestinal surface is limited. This is heavy It is important. Because it is a reasonable level of C-tail This is because it indicates that it can have a significant effect.   (3) These data from surviving animals are based on C-tails (BAL from different animal species). Equivalent), recombinantly produced C-tails and chemically synthesized C-tails. That yl and its analogs can effectively reduce cholesterol uptake Confirms the in vitro results confirmed. This is because the use of C-tails in humans By reducing serum cholesterol levels due to dietary cholesterol Supports the conclusion that cardiovascular disease can be reduced.   (4) Many factors are optimal to reduce cholesterol uptake more effectively Can be For example, the dose of C-tail can be optimized and the pattern of C-tail Can be modified to maximize its effect and in relation to cholesterol intake The time of administration of the C-tail can be varied. Chemical modification and physical modification of the C-tail Modifications (additions and formulations) may also be made to maximize inhibition.   Example 6: BAL uptake of triglycerides by isolated rat intestinal tissue But not the uptake of taurocholate.   The above example demonstrates that taurocholate uptake was due to BAL bound to the intestinal surface. It is proof that it is mediated. BAL also hydrolyzes triglycerides And activated by Taurocholate, the following examples , BAL bound to the intestine also uptakes triglycerides and taurocholate Deals with the question of whether to mediate. Experiment   ^ThreeH radiolabelled trioleylglycerol and¹⁴C-Tauro Cholate Except for the use of sterols, the above cholesterol uptake Triolein and taurocholate uptake experiments were performed in the same manner as Was. For triolein uptake experiments, 6 mM or 30 mM non-radiolabeled Tauroco All of the rates contained radiolabeled triolein. Experimental system is isolated Because of the intestine, Taurocholate was supplied to activate BAL. [^ThreeH] -G Liolyl glycerol (Amersham, Arlington Heights, IL) was identified as described (D owns et al. (1994)). 15 in 7.5 ml of isotonic phosphate buffer (pH 7.4) Double the concentration by emulsifying μmol of dioleyl phosphatidylcholine A prepared substrate solution was prepared. W-380 Sonicator (Heat Systems-Ultrasonics, Inc. .) In an ice bath at a setting of 5 (50% maximum power) for 30 seconds. Thus, the mixture was emulsified. After cooling, the mixture was sonicated for an additional 30 seconds. Destination A volume of 1 ml of this emulsified solution was isolated for uptake measurements as described in They were placed in the intestine of rats. For studies of taurocholate uptake, 2 μCi Tauro (¹⁴5 ml of CO) colate and 30 μmol of taurocholate (Sigma) A stock solution of Taurocholate by adding to an isotonic phosphate buffer (6 mM). result   As described for the cholesterol uptake experiment in section b) of Example 2. Using isolated rat intestinal system, triglyceride and Taurochole The uptake of fatty acids from the sample was measured. EGTA elution from each of the five experiments Therefore, when BAL is removed from the intestinal surface, the absorption of radioactive triolein is statistically high. About 75% (Table XV and FIG. 7). Table XV shows 6 mM Tauro The addition of cholate increases the maximum triolein uptake rate in buffer-washed intestines. Indicates that it is enough to maintain. Because the 30mM taurocholate is This results in qualitatively the same uptake values (the last two columns in Table XV and FIG. 7). Compare columns 3 and 4). If Taurocholate was not added , Only the baseline is accepted and for the uptake, the process is Mediated and demonstrated that bile salt activation was required for uptake. BAL Remove The removed EGTA-washed intestine showed very low uptake (first data table in Table XV). Ram and column 1 of FIG. 7). This uptake is close to the baseline (Table XV Second data column at 0 mM Taurocholate and column 2 in FIG. 7). these The results indicate that BAL mediates triolein uptake.   On the other hand, taurocholate uptake was significant even after removing bound BAL from the intestinal surface. (Table XVI and FIG. 8). Wash the intestine with EGTA solution Even if the bound BAL was removed before the experiment, the uptake of radiolabeled taurocholate was minimal. Only diminished. Slight effects from EGTA elution are not statistically significant Was. Significance   These results indicate that BAL C-tails, recombinant C-tails, and natural or chemical Oral Administration of Synthetic C-Tail Analogs Has Medical or Nutritional Benefits Should be useful in reducing triglyceride absorption in humans You.   All of the carboxy terminal (C tail) region of bile salt activated lipase (BAL) Describes a composition comprising a part or their functional equivalent (C-tail peptide). In the gut, it competes with natural BAL for binding to the gut surface And binds to the biologically active composition. The BAL C-tail molecule is delivered Is bound to the substance to be sought, and therefore is delivered specifically to the intestine by oral administration of the conjugate. Can be reached. In the intestine, these compositions bind to the intestinal surface and treat the therapeutic compound in the intestinal lining. Delivery and / or long-term presence. Join   Any of a variety of bioactive agents can bind to the C-tail, Enables the delivery of For example, the bioactive agent may be a C-tail carboxyl group or The C tail can be modified by covalent attachment to an amino group. Share to C tail Modify the C tail by using any of a number of different binding agents that bind I can do it.   One useful protocol is a non-proton such as DMSO, acetone, or THF Of hydroxyl groups on C-tail carbonyl diimidazole (CDI) in aprotic solvents "Activation". CDI is free of biologically active ligands such as proteins It can be substituted by attachment of an amino group. Imidazolyl carbamate with hydroxyl group Form a complex. The reaction is an N-nucleophilic substitution, and the C tail is ligated. Resulting in a stable N-alkyl carbamate linkage of the compound. Generated ligand-C The complex is stable and resistant to hydrolysis for extended periods of time.   Another conjugation method is the conjugation with N-hydroxylsulfosuccinimide (sulfo NHS). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) Alternatively, use "water-soluble CDI" to bioactive the exposed carboxyl groups of the C tail. Binding to the free amino group of the ligand. EDAC and sulfo NHS Forms an activated ester with the carboxylic acid group of the C tail. This acid group Reacts with the amino terminus of the ligand to form a C-tail bond. Generated peptide bond Is resistant to hydrolysis. 10-fold increase in EDAC binding efficiency when using sulfo NHS in the reaction Very gentle conditions to ensure the viability of the ligand-C tail complex. Offer. These protocols are based on hydroxyl or carboxyl groups on the C tail. Enables activation of any of the groups and binding of the desired bioactive ligand.   Binds free hydroxyl and carboxyl ligands to C-tail A useful coupling procedure for involves the use of a crosslinker, divinyl sulfone. This one The method is useful for attaching sugars or other hydroxyl compounds to hydroxyl groups on the C tail . Activation is based on vinylsulfonylethyl of divinylsulfone and hydroxyl group of C tail. Includes reaction to ether. Vinyl groups include alcohols, phenols, and alcohols. Binds to Min. Activation and binding occurs at pH 11. Binding is pH 1 to 8 And is suitable for transport through the gut. Any known to those skilled in the art These appropriate binding methods are also used to bind a bioactive ligand to the C tail. obtain.   Therapeutic compounds can be linked directly or indirectly to C- It can be covalently linked to a tail protein. Between C-tail protein and therapeutic compounds If more flexibility or space is needed in between, the linker molecule Typically used. Can bind to both C-tail proteins and therapeutic compounds Any suitable molecule can be used as the linker molecule. Examples of linkers include , Peptides or molecules with straight carbon chains. C-tail composition in the intestine A binding or binding that binds the C-tail protein and the therapeutic compound for use. The linker must be stable in the intestinal environment. Bioactive agent   Any type of bioactive agent can be attached to the C-tail using standard techniques . The resulting conjugate of C-tail and bioactive agent is referred to herein as a C-tail composition or Is referred to as the C-tail-drug conjugate. C-tail fragments are biologically active It can be conjugated to any drug. The term "biologically active material" refers to proteins, Organic compounds such as carbohydrates, nucleic acids, lipids, drugs, or combinations thereof When administered to animals, including humans, in vivo, it produces a biological effect.   Examples include antigens, enzymes, hormones, receptors, peptides, proteins, Sugars, nucleic acids, nucleosides, nucleotides, liposomes, vitamins, minerals, Examples include, but are not limited to, inorganic compounds and viruses. C-tail also , Prokaryotic and eukaryotic cells (eg, bacterial, yeast, and mammalian cells). (Including human cells), and their components (eg, cell walls, and cellular components). Conjugate).   Examples of useful proteins include insulin, growth hormone (somatomethine) (including somatometin), transforming growth factors, and other growth factors) Hormones like oral vaccine antigens, enzymes like lactase or lipase And digestive agents such as pancreatin.   Examples of useful drugs include Carafate from Marion Pharmaceuticals^TMlike Ulcer treatment, L-DOPA, antihypertensives or salt excretors (eg, Searle Pharmaceuti metolazone from cals), carbonic anhydrase inhibitors (eg, Acetazolamide from Lederle pharmaceuticals), insulin-like drugs (eg , Glyburide), a sulfonylurea class blood glucose lowering drug, synthetic formo (Eg, Android F and ICN Pharmaceuticals from Brown Pharmaceuticals) s-derived Testred (methyltestosterone)), an anthelmintic (eg mebendazole) (Vermox^TM, Jannsen Pharmaceutical).   C-tail drug conjugates are useful for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease It is particularly useful for the treatment of In ulcerative colitis, inflammation is restricted to the large intestine, In Horn disease, inflammatory foci can be found throughout the gastrointestinal tract, from the oral cavity to the rectum. Sur Fasalazine (sulfasalazin) is one of the drugs used to treat the above diseases. You. Sulfasalazine is degraded by bacteria in the large intestine and sulfapyridine ( Antibiotics) and 5-aminosalicylic acid (anti-inflammatory). 5-aminosalicylic acid Is an active drug and is needed locally. Decomposition product (5-aminosalicylic acid) May be more beneficial. Protein-drug delivery systems are Retention of the drug for an extended period of time may improve treatment. A place for Crohn's disease In that case, retention of 5-aminosalicylic acid in the upper part of the intestine is quite important. Because Bacteria degrade sulfasalazine in the large intestine. Treat upper intestinal inflammation The only way is by topical administration of 5-aminosalicylic acid.   The antigen may be conjugated to the peptide to provide a vaccine. Producing vaccines, It may have different retention times in the digestive tract. This retention time allows peptides to be used in vaccines. It depends on the strength of the covalent bond to be bonded. When holding this different among other factors Can stimulate the production of more than one type of antibody (IgG, IgM, IgA, IgE, etc.) You.   The term “antigen” is any that stimulates the formation of antibodies or induces a cell-mediated response. Proteins, polysaccharides, nucleoproteins, lipoproteins, Includes, but is not limited to, a synthetic polypeptide or small molecule that binds to a protein. Not. Specific antigens that can bind to the peptide include attenuated viruses or killed bacteria Viruses, toxoids, polysaccharides, cell walls, and virus and bacterial surfaces Or coat protein. These also include conjugates, adjuvants, Or it can be used in combination with other antigens. For example, many refined capsules Hemophilus influenzae (Hib) in saccharide form can be used alone or as a diphtheria ibis Can be used as a conjugate with a pseudo. Organism from which these antigens are derived Examples of poliovirus, rotavirus, type A, type B, hepatitis C, influenza Enza, rabies, HIV, measles, mumps, rubella, Bordetella pertussus, Str eptococcus pneumoniae, Diphtheria, Tetanus, Cholera, Salmonella, Neisser ia, Shigella, and Enterotoxigenic E. coli.   C-tail also provides water-soluble or water-insoluble drugs (eg, non-steroidal anti- Inflammatory compounds, anesthetics, chemotherapeutics, immunosuppressants, steroids, antibiotics, anti- Drugs, antifungals, steroidal anti-inflammatory drugs, and anticoagulants). Can be used.   Imaging agents can also be attached to the C-tail. These are metals, radioisotopes , Radiopaque agents, fluorescent dyes, and radiolucent agents. Radioisotopes and Radiopaque agents include gallium, technetium, indium, strontium, Contains iodine, barium, and phosphorus.   Therapeutic compounds that bind to the C-tail (ie, biologically active agents) It can be any compound that has a useful effect when delivered to the inner layer. Treatment The substance acts to reduce uptake of the compound taken up by the individual or It can either enhance or degrade harmful compounds. example For example, the therapeutic compound can be an enzyme, or a non-enzymatic binding molecule, or a ligand . Enzymes that catalyze unwanted compounds that are ingested by the individual, or such Antibodies or receptors specific for the compound are useful as therapeutic compounds. C -Because the tail composition is used in the intestine, the therapeutic compound is stable and active in the intestinal environment Must.   Preferred therapeutic compounds are catabolic enzymes, which catalyze the breakdown of certain molecules. Especially active Enzymatic enzymes are not normally present in the intestine. Suitable therapeutic compounds of this type are Kutase. C-tail / lactase compositions can be used in individuals lacking lactase, or Or for delivering lactase to the intestinal lining of individuals with reduced lactase activity Can be done. C-tail / lactase binds to the intestinal lining and Maintains protease activity and degrades ingested lactose. Therefore, lactose intolerance Relieve the symptoms of the disease. Oral administration   In a preferred embodiment, the C-tail-drug conjugate is effective for a particular therapeutic application. Oral administration in an appropriate amount. Dosage depends on formulation, rate of excretion, and number of receptors on intestinal surface. Changes in the individual, type of treatment, and number of doses, and usually by a physician Varies depending on other factors being optimized. In one embodiment, BAL C- The tail composition reduces or enhances the uptake of certain compounds from the food, Alternatively, the compound is orally administered in an amount effective to reduce the intestinal concentration of the undesired compound.

[Procedure amendment] [Date of submission] February 18, 1998 [Content of amendment] Claims 1 . A composition for reducing intestinal absorption of cholesterol, comprising a C-tail protein comprising part of the carboxyl region of human bile salt lipase, or a derivative thereof that binds to a specific receptor on intestinal cells. or when administered to an animal, in an amount effective to inhibit cholesterol uptake, it is administered in combination with a suitable pharmaceutical carrier for oral administration, the composition. 2 . 2. The composition of claim 1 , wherein said C-tail protein contains all or a portion of amino acid residues 539 to 722 of SEQ ID NO: 1. 3 . 2. The composition of claim 1 , wherein the C-tail protein further comprises a region of bile salt activated lipase that includes a catalytic site that has been inactivated. 4 . 2. The composition of claim 1 , wherein the C-tail protein further comprises at least three repeat regions of a primate, human cat, dog, or rodent bile salt-activated lipase. 5 . 2. The composition according to claim 1 , wherein the C-tail protein contains a consensus motif PVPPTGDSGAV. 6 . 6. The composition of claim 5 , wherein the carrier is a polymer or an intestinal encapsulation composition, and wherein the C-tail protein is incorporated on or in the carrier. 7 . 2. The composition of claim 1 , wherein the C-tail protein is formed by substitution, deletion, or addition of one or more amino acid sequences of SEQ ID NO: 1. <8 . The composition of claim 1 , wherein the composition further comprises another compound that is effective in lowering blood cholesterol. 9 . The compound comprising: (i) a chemically synthesized mimetic of the carboxy-terminal region of bile salt-activated lipase, which competes for binding of bile salt-activated lipase on the human intestinal surface; and (ii) a human BAL Bile from other animals selected from the group of compositions consisting of all or part of the carboxyl-terminal region having sequence homology to the carboxy-terminal region, but which competes for binding of bile salt-activating lipase on the human intestinal surface 9. The composition of claim 8 , wherein the composition is derived from a salt activated lipase. 10 ． 2. The composition of claim 1 , wherein the C-tail protein is a dietary formulation for oral administration. 11 . 2. The composition of claim 1 , wherein the C-tail protein is in milk of a transgenic cow or sheep. 12 . A composition for inducing a therapeutic compound comprising : when combined with a suitable pharmaceutical carrier for oral administration, a therapeutically effective amount of a C -tail protein comprises a therapeutic composition. Is a therapeutic compound without lipase activity that binds to a substance, wherein the C-tail protein has the basic structure of Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro Or a composition comprising three or more repeats having a basic structure with a small number of substitutions, wherein the composition binds to human enterocytes. 13 . 13. The composition of claim 12 , wherein said therapeutic compound is selected from the group consisting of proteins, carbohydrates, nucleic acids, nucleosides, nucleotides, liposomes, inorganic compounds, vitamins, drugs, and minerals. 14 . The therapeutic compound is a group consisting of a nonsteroidal anti-inflammatory compound, an anesthetic, a chemotherapeutic agent, an immunosuppressant, a steroid, an antibiotic, an antiviral, an antifungal, a steroid anti-inflammatory, and an anticoagulant. 13. The composition of claim 12 , wherein the composition is selected from: 15 . 13. The composition of claim 12 , wherein the carrier is a polymer or an intestinal encapsulation composition, and wherein the C-tail protein is incorporated on or in the carrier.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI A61K 47/48 C12N 15/00 ZNAA C12N 15/09 ZNA A61K 37/54 ADN (31) Priority claim number 482,262 (32) Priority Date June 7, 1995 (33) Priority Country United States (US) (81) Designated State EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AU, CA, JP

Claims (1)

[Claims] 1. A method for reducing intestinal absorption of cholesterol that requires treatment , A C-tay containing a part of the carboxyl region of human bile salt lipase Protein, or a derivative thereof that binds to a specific receptor on enterocytes, Suitable for oral administration in an amount effective to inhibit uptake of resterol. And administering the combination in an appropriate pharmaceutical carrier. 2. The C-tail protein comprises all of amino acid residues 539 to 722 of SEQ ID NO: 1. Or the method of claim 1, wherein the method comprises a portion. 3. The C-tail protein further contains a catalytic site that has been inactivated. 2. The method of claim 1, comprising a region of bile salt activated lipase. 4. The C-tail protein further comprises a primate, human, cat, dog, or ginger. Containing at least three repeat regions of rodent bile salt-activating lipase, The method of claim 1. 5. The C-tail protein contains a consensus motif PVPPTGDSGAV The method of claim 1. 6. The C-tail protein is administered before administration of the C-tail protein. Administered an effective amount to lower blood cholesterol levels compared to the bell The method of claim 5, wherein 7. The C-tail protein comprises one or more amino acid sequences of SEQ ID NO: 1. 2. The method of claim 1, wherein the method is formed by substitution, deletion, or addition of a sequence. 8. Part of the carboxyl region of human bile salt lipase, or specific on enterocytes Reduces intestinal absorption of cholesterol, including its derivatives that bind to receptors For administration to humans or animals. Pharmaceutical capsules suitable for oral administration in an amount effective to inhibit uptake. A composition for administration in combination using a rear. 9. The C-tail protein comprises all of amino acid residues 539 to 722 of SEQ ID NO: 1. 9. The composition according to claim 8, wherein the composition comprises at least a portion. 10. The C-tail protein further comprises a catalytic site that has been inactivated. 9. The composition of claim 8, wherein said composition comprises a region of bile salt activated lipase. 11. The C-tail protein further comprises a primate, human, cat, dog, or Contains at least three repeat regions of rodent bile salt-activated lipase The composition of claim 8. 12. The C-tail protein contains a consensus motif PVPPTGDSGAV 9. The composition of claim 8, wherein 13. The carrier is a polymer or an intestinal encapsulation composition; and The C-tail protein is incorporated on or into the carrier; The composition according to claim 12. 14． The C-tail protein comprises one or more amino acids of SEQ ID NO: 1 9. The composition of claim 8, wherein the composition is formed by substitution, deletion, or addition of a sequence. 15. The composition may further comprise another effective cholesterol-lowering composition. 9. The composition of claim 8, wherein the composition comprises: 16. The composition competes for (i) binding of bile salt activated lipase on the human intestinal surface Chemically synthesized mimic of the carboxy-terminal region of bile salt-activated lipase And (ii) a carboxyl having sequence homology to the carboxy-terminal region of human BAL Selected from the group of compositions consisting of all or part of the terminal region, Salt activation from other animals competes for binding of bile salt-activating lipase in mice 16. The composition of claim 15, wherein the composition is derived from a lipase. 17． The C-tail protein is a dietary regimen for oral administration The composition of claim 8. 18. The C-tail protein is a transgenic cow or sheep milk 9. The composition of claim 8, wherein the composition is in a juice. 19. A composition for deriving a therapeutic composition comprising: When combined with a suitable pharmaceutical carrier for oral administration, the C- Lipase activity at which a therapeutically effective amount of tail protein binds to a therapeutic composition Therapeutic composition without   The C-tail protein is Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro Containing a basic structure, or a repetition having a basic structure in which a small number is substituted three or more times, A composition that binds to human enterocytes. 20. The therapeutic composition comprises a protein, a carbohydrate, a nucleic acid, a nucleoside, a nucleoside. Consists of leotide, liposomes, inorganic compounds, vitamins, drugs, and minerals 20. The composition according to claim 19, wherein the composition is selected from the group. 21. The therapeutic composition comprises a non-steroidal anti-inflammatory compound, an anesthetic, a chemotherapeutic agent. , Immunosuppressants, steroids, antibiotics, antivirals, antifungals, antisteroids 20. The composition of claim 19, wherein the composition is selected from the group consisting of an inflammatory agent and an anticoagulant. . 22. The carrier is a polymer or an intestinal encapsulation composition; and The C-tail protein is incorporated on or into the carrier; The composition according to claim 19.