JP3007161B2 - Methods and compositions for reducing cholesterol absorption - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to the field of dietetics, and more particularly to the use of bile salt-activated lipase to reduce intestinal cholesterol uptake.

The U.S. Government has granted Research Authorization Number HD-
By 23472, the present invention has certain rights.

Cardiovascular and Other Diseases Involved in High Cholesterol Hyperlipidemia, especially hypercholesterolemia and hyperlipoproteinemia, is one of the most dangerous factors causing atherosclerosis. Hyperlipoproteinosis is also involved in the development of pancreatitis. According to a long established theory, the higher the circulating level of cholesterol (usually in the form of low-density lipoprotein (LDL) containing cholesterol), the more cholesterol can penetrate into the arterial wall and cause atherosclerosis (Brown and Goldstein, "Hyperlipoproteinosis and other lipid metabolism disorders,"Harrison's Principles of Internal Medicine.)
1650-1661 (Braunwald et al., 1987)).

Cardiovascular disease is the leading cause of death in women and middle-aged American men. In 1988, more than 41,000 U.S. residents died of cardiovascular disease by the age of 50. However, atherosclerosis, a known cause of cardiovascular disease and stroke, begins to develop at a much lower age. Fat streaks are common in the arterial wall of children and a high rate of annular arterial dysfunction is found in young accidental or accidental deaths. Children and adolescents with elevated serum cholesterol levels will likely have more parents with coronary heart disease than children and adolescents with normal cholesterol levels. Higher serum cholesterol levels in childhood are associated with atherosclerosis of the vena cava in autopsy results of adolescents and adolescents, and aortic and coronary atherosclerosis in people aged 15 to 34 Both diseases are correlated with cholesterol levels at necropsy (Klag et al., New Eng. J. Med. 328 (5): 313).
-318 (February 4, 1993)).

Cholesterol is used by steroid hormones by certain endocrine glands, and a major part of the synthesis of bile acids from hepatocytes, and is an essential component of cell membranes.
It is found only in animals. Related sterols occur in plants, but plant sterols are not absorbed from the gastrointestinal tract. Most of the cholesterol in the diet is contained in egg yolk and animal fat.

Cholesterol taken up in the intestine directly from the diet,
It is derived from cholesterol-containing bile salts and bile acids, and free cholesterol synthesized in the liver and secreted into the intestine through the bile duct. Bile and dietary cholesterol esters are absorbed from the lumen of the small intestine by intestinal epithelial lining cells and are taken up by the cells and enter the chylomicrons, but in small quantities, very low density lipoproteins (VLDL) Is taken in. Both of these are secreted into the lymph and eventually merge into the bloodstream. Chylomicron and VLDL transfer some of their triacylglycerols and their cholesterol to endothelium, muscle,
And deliver to cells of adipose tissue. The cholesterol-rich chylomicron remnants and VLDL then return cholesterol to hepatocytes and other cells on the vessel wall along their pathways (Ganong, Review of Medical Physiology 24).
9-250 (Lange Medical Publications, 1985)). Enterocyte and hepatocyte-derived VLDLs release very low-density lipoproteins (LD
L). LDL contains three quarters of total plasma cholesterol.

In hypercholesterolemia, increased blood cholesterol levels are mainly associated with increased LDL levels. However, the specific causes of hypercholesterolemia are complex,
And change. At least one type of hypercholesterolemia is primarily caused by mutations in the LDL receptor gene that remove cholesterol from blood in the liver. In general, hypercholesterolemia is significantly associated with high dietary cholesterol, which results in high cholesterol uptake from the gut into the circulating blood.

Suppressing hypercholesterolemia slows the development of atherosclerosis and slows the progression of atherosclerosis, thus reducing the risk of coronary heart disease in humans and other primates. In particular,
In animals, especially primates, the progression of atherosclerosis is terminated when the relatively complex plaque induced by hyperlipidemia regresses and, furthermore, when hyperlipidemia is eliminated.
Therefore, efforts should be made to prevent atherogenesis, halt progression, and promote regression of existing lesions, possibly by reducing risk factors (Bierman,
"Vascular Disorders: Atherosclerosis and Other Forms of Atherosclerosis"Harrison's Principles
of Internal Medicine 1014-1024 (Braunwald et al., 198
7)).

Some forms of hyperlipidemia, including hypercholesterolemia, may be somewhat reversible using current techniques of prophylactic treatment. However, none of the current techniques have been completely successful and many involve undesirable side effects and complications. Taking cholesterol-lowering drugs can reduce serum cholesterol by 20%. But,
Drugs are not always reliable for hypercholesterolemia, for example, lovastatin (movastatin), cholestyramine (cholestyramin)
e) (Questran), clofibrate,
Some of the hyperlipidemic drugs, such as Probucol, and nicotinic acid, have serious side effects, including increased mortality due to liver complications, or, for example, constipation (cholestyramine), flushing skin, muscle function It may have less severe side effects such as insufficiency or may have the effect of lowering blood triacylglycerol but not blood cholesterol. Diet is usually recommended for all patients with hypercholesterolemia, but is not always effective.

Thus, there is a need for effective methods and compositions that reduce blood lipid levels (specifically, cholesterol levels), especially as they have no significant side effects themselves, and Disease states associated with high blood lipid levels, especially in patients at high risk for heart disease or who have had a heart attack already
It is effective to treat.

Accordingly, it is an object of the present invention to provide compositions and methods for use in lowering serum cholesterol in a patient in need thereof.

SUMMARY OF THE INVENTION Compositions derived from all or part of the carboxy-terminal region of human bile salt-activated lipase (BAL) are described. When they are taken orally, natural BAL
Competes for binding of cholesterol to the intestinal surface and thus suppresses the physiological role of BAL in mediating the transport of cholesterol to enterocytes, and consequently reduces the absorption of cholesterol from the intestine into the bloodstream. 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. A preferred proline-rich unit is the consensus sequence PVPPTGDSGAP
(SEQ ID NO: 6).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows C-tail O binding to lectin-like receptors.
-Binding of glycosylated carbohydrates, cholesterol and cholesterol esters to BAL, BAL to intestinal endothelial cells via hydrolysis of cholesterol esters by BAL
2 shows the proposed binding of

FIG. 2 shows the elution of the endothelial BAL lipolytic activity.
M), galactose (0.1 M), heparin (10 mg / ml), or NaCl (0.3 M) at μmol / g / h.

FIG. 3 shows nmol / g / h of radioactive cholesterol (oleate) ester in the intestine of any rat treated or not treated with ethylene bis (oxyethylene nitrilo) tetraacetic acid (EGTA) to remove BAL. 2 shows cholesterol uptake in the rat.

FIG. 4 shows a graph of cholesterol uptake rate (nmol / g / h) for 1 mM EGTA, 0.2 mg / ml C-tail, and control in rat intestine.

FIG. 5 shows [With and without C-tail, [
^{FIG. 3} is a graph showing blood [ ³ H] -cholesterol levels (nmol / mL) versus time (hour) in rats fed ³ H] -cholesterol oleate. Blood [ ³ H] in rats fed C-tail isolated from BAL in human milk
-Cholesterol levels are indicated by crosses. Blood of ^[3 H] rats not fed the C- tail - shows the cholesterol levels in a box. Blood samples were collected at hourly intervals for up to 5 hours after feeding [ ³ H] -cholesterol oleate.

FIG. 6 shows with or without (shaded) C-tail isolated from BAL in human milk (no hatched).
^[3 H] - is a graph of cholesterol levels (nmol / mL) of the mean (by 6 experiments) versus time (hour) - blood of rats fed the cholesterol oleate ^[3 H].
Blood samples were collected at hourly intervals for up to 5 hours after feeding the first tube of radiolabeled cholesterol oleate. The horizontal line at the end of the data bar is
The standard error is shown (see Table XIV).

FIG. 7 shows the intestine with 1) BAL bound before the uptake experiment.
EGTA solution containing 6 mM Taurocholate to remove urea (column 1), 2) Buffer without Taurocholate (column 2), 3) so that bound BAL is not removed
Buffer containing 6 mM Taurocholate (column 3), 30 mM
The rate of triolein uptake (μmol / mol) in rats washed with a buffer containing
g / h). Data represent the average of the results of five experiments (see Table XV). All four groups of animals received emulsified radiolabeled triolein.

FIG. 8 shows the intestine in 1) BAL bound before the uptake experiment.
Rate of taurocholate uptake (μmol / g / g) in rats washed with an EGTA solution (column 1) (shaded) just to remove lipase and a buffer solution (shaded) that does not remove bound BAL. It is a graph of h). Data represent the average of the results of five experiments (see Table XVI). Left and right column pairs are 1 mM and 6 respectively
Figure 3 shows the uptake rate with mM radiolabeled taurocholate.

DETAILED DESCRIPTION OF THE INVENTION Compositions comprising all or part of the carboxy terminal region of bile salt activated lipase (BAL), or functional equivalents thereof, are described. They compete with natural BAL in the gut for binding to the intestinal surface, and cholesterol esters (ea) in the form of free cholesterol are taken up into the bloodstream.
ter) or reduce the function of endogenous BAL to mediate the uptake of free cholesterol.

Bile salt activated lipase Warm-blooded animals synthesize many forms of lipase with different structures and activities that are secreted by mammary cells or cells in several fire extinguishers, including the pancreas, stomach and small intestine. Bile salt-activated lipase (BAL), which is substantially inactive itself on physiological substrates, is activated in the intestine by bile salts. BAL, by the pancreas,
And it is synthesized and secreted by the mammary glands of a few species, including humans, gorillas, cats and dogs. The amino acid and cDNA sequences of BAL in human milk are the same as those of pancreatic carboxylesterase and are described in the literature as lysophospholipase, cholesterol esterase, sterol ester hydrolase, non-specific lipase, lipase A, carboxyl ester lipase, and cholesterol. Closely related or identical to lipases called ester hydrolases, and vary somewhat from species to species mainly in the number of repeat units in the carboxy region (Wang and Hartsuck, Biochim. Biophys. Acta 1166: 1
-19 (1993)). Pancreatic BAL is clearly distinguished from other types of non-bile salt activated lipase, such as pancreatic lipase and phospholipase.

In the intestinal cavity, BAL attaches to the intestinal surface, presumably via specific receptors, to the surface of intestinal epithelial lining cells. BAL
Can be released from the cavity surface by EGTA, galactose and fucose, but not by heparin, isotonic buffer or sodium chloride as shown below. Where bile salts are required, BAL is essential to hydrolyze cholesterol esters to free cholesterol or to bind free cholesterol in food. Since this only known pancreatic lipolytic enzyme can mediate cholesterol uptake, both of these processes are necessary to enable cholesterol uptake. BAL can also hydrolyze the carboxylester bonds of acylglycerol, phospholipids, and vitamin esters to form fatty acids and glycerol and act on emulsified, micellar or soluble substrates. Bile salts are thought to cause BAL conformational changes, thereby providing active site access to bulky substrate molecules and providing additional lipid binding capacity in the formation of enzyme-substrate complexes. In addition, bile salts are believed to act as fatty acid acceptors during BAL catalysis.

The proposed mechanism of action of BAL is shown in FIG. BAL
Binds to lectin-like receptors on the surface of intestinal epithelial cells via C-tail O-glycosylated carbohydrates.
The catalytic unit of the enzyme remains separate from the epithelial cells with the heparin binding site and active site exposed.
The free cholesterol or cholesterol ester then binds to the active site and, in the case of cholesterol ester, is hydrolyzed to free cholesterol. The catalytic unit then binds to heparin at the cell surface and transfers cholesterol into the cell.

Cholesterol derived from lipolis by BAL in the intestinal cavity,
Fatty acids and monoacylglycerols are taken up by intestinal epithelial lining cells (mucosal cells), where these components are re-esterified to intracellular triacylglycerols. In cells, cholesterol interacts with these re-esterified triacylglycerols plus apolipoproteins and phospholipids to form kilomicrons and very low density lipoproteins. These kilomicron and very low density lipoproteins are secreted into small lymph vessels that ultimately join the vasculature for systemic circulation.

Carboxy-terminal region of the BAL molecule Complete and mature human BAL contains 722 amino acid residues (SEQ ID NO: 1). The carboxy-terminal region of BAL is
Refers to the region in the native BAL molecule that includes residues 539 to 722. BAL
This carboxy-terminal region, together with a derivative of this region that retains intestinal binding activity, is referred to herein as a "C-tail".
Called. The C-tail of the human BAL molecule has many O-linked oligosaccharide units forming a mucin-like structure. The native human C-tail amino acid sequence has 16 repetitive proline rich, each consisting of 11 amino acid residues.
Units, most of which have the consensus sequence of PVPPTGDSGAP (SEQ ID NO: 6) (Baba et al., Biochemistry 3).
0: 500-510 (1991)). By performing the β-elimination reaction, it was determined that the native C-tail was O-glycosylated, primarily with threonine and to a lesser extent when it occurred at one serine residue. Serine residues with adjacent aspartic acid are considered undesirable for O-glycosylation (Elhammer et al., J. Biol. Chem. 268: 10
029-10038 (1993)). Peptides prepared by cyanogen bromide digestion of the C-tail were found to contain most of the carbohydrates of native BAL (Baba et al. (1991)).

As shown below, "T-BA
Truncated forms of BAL lacking a C-tail junction, termed "L", are not effective at transferring cholesterol into enterocytes because the enzyme does not bind to the supersurface. Similarly, it binds to the intestinal surface only with the C-tail and can actually compete with native BAL for this binding, but cannot transfer cholesterol because the catalytic unit is neither functional nor present.

C-tail protein The carboxy-terminal region of BAL having a mucin-like structure derived from all or a part of the region containing amino acid residues 539 to 722 and containing at least three repetitive proline-rich units each having 11 amino acid residues Are referred to herein as C-tails. As used herein, a proline-rich unit, when combined with any of the 11 repetitive amino acid groups present in the naturally occurring form of BAL, or with two or more other proline-rich units, causes intestinal epithelial cells to undergo intestinal epithelial cells. Refers to derivatives of these amino acids that bind and / or become proteins that inhibit the binding of native BAL. A preferred proline-rich unit has the consensus sequence PVPPTGDSGA-P (SEQ ID NO: 6). As used herein, “C-tail protein” refers to any protein containing three or more proline-rich units as defined above, which protein binds to intestinal epithelial cells and / or BA
Suppress L binding. This can be achieved, for example, by using a BAL consisting of all or part of the mucin-like C-tail region of the native BAL. Native BAL is in the form of a C-tail protein.

The C-tail protein should contain at least three repetitive proline-rich units, each consisting of 11 amino acid residues. Rat pancreatic esterase C-tail, which has only four repeat units, also binds to the rat intestinal surface. Preferred C-tail proteins have at least 10
Most preferably, it has at least 16 proline-rich units. C-tail proteins with fewer proline-rich units are expected to bind to the intestinal surface with lower affinity. The binding affinity of any C-tail protein can be enhanced by a proline-rich unit that closely matches the consensus sequence (SEQ ID NO: 2). C-tail proteins can be constructed by linking three or more proline-rich units. Here, the proline unit has a natural amino acid sequence of a proline-rich unit from any BAL, has a common amino acid sequence of a human proline-rich unit, or has a derivative of these amino acid sequences,
As a result, the C-tail protein retains the ability to bind to intestinal epithelial cells and / or inhibit the binding of native BAL.

Amino acid sequence variants C-tail proteins are O-glycosylated to varying degrees with respect to the number of threonine and serine residues and do not significantly interfere with binding to the intestinal surface. Amino acid deletion,
It may include substitutions or additions. C- which does not change the bond
Substitutions, deletions, or additions to the tail protein are readily determined by screening assays. In a screening assay, proteins are allowed to bind to the intestinal surface and are then removed by washing with buffer at increasing salt concentrations. An example of a BAL containing a deletion that does not affect C-tail binding is a BAL lacking a heparin binding site, postulated to be between amino acid residues 56 and 62 (Baba et al. (1991)). ).

Amino acid sequence variants of the C-tail protein fall into one or more of three classes. That is, it is a substitution variant, an insertion variant, or a deletion variant. Insertions include amino and / or carboxyl terminal fusions, as well as intrasequence insertions of single or multiple amino acid residues. The fusion can be with mature BAL, for example, in the case of human BAL,
BA, a secretory leader from another secreted human protein
Includes hybrids of L with C-tail proteins having homologous polypeptides. The fusion is a hybrid of the BAL to a polypeptide homologous to the host cell but not to the BAL, and a C-tail protein having a polypeptide that is heterologous to both the host cell and the BAL. including. Preferred fusions are amino-terminal fusions with either prokaryotic peptides or signal peptides of prokaryotic, yeast, bacterial or host cell signal sequences. It is not necessary that the signal sequence normally lack any residue mature sequence from the protein that induces its secretion, but it is preferred to avoid secretion of the C-tail protein fusion.

Insertions can also be introduced within the coding sequence of the proline-rich unit repeat region of the C-tail protein. Such insertions may include the addition of unrelated amino acids or the insertion of one or more additional proline-rich units. In the context of inserted amino acids, "unrelated" amino acids refer to amino acid sequences that are not related to the sequence of the proline-rich unit of BAL. In the case of a proline-rich unit, the inserted unit may be a consensus sequence (SEQ ID NO: 6) or a heterologous unit from a non-human BAL with additional repeats of individual human proline-rich units. However, for the insertion of an unrelated amino acid, the insertion usually consists of an insertion smaller than the amino or carboxyl terminal fusion, or an insertion of the order of 1 to 4 residues smaller than the proline-rich unit.

C-tail protein insertion amino acid sequence variant (va
riant) is one in which one or more amino acid residues have been introduced at a predetermined site in the target C-tail protein. Most commonly, insertion variants are those in which a heterologous protein or polypeptide is fused to the amino or carboxyl terminus of a C-tail protein. Preferably, these heterologous polypeptides are heterologous forms of the proline-rich unit present in human BAL. Immunogenic C-tail protein derivatives can be obtained by culturing recombinant cells in vitro with a DNA encoding the cross-linking or fusion of a polypeptide large enough to confer immunogenicity. This is done by fusing to the target sequence. Such an immunogenic polypeptide is trpL
It can be a bacterial polypeptide such as E and β-galactosidase.

Deletions are characterized by the removal of one or more amino acid residues from the C-tail protein sequence. Preferably, the deletion includes a deletion of the entire proline-rich unit. If individual amino acids in the proline-rich unit are deleted, only about 2-6 residues are deleted at any site in the C-tail protein molecule.

These variants are generally prepared by making DNA encoding the variant by site-directed mutagenesis of nucleotides in the DNA encoding the C-tail protein, and then expressing the DNA in recombinant cell culture. Prepared. However, variant C- having up to about 100-150 residues
Tail protein fragments can be conveniently prepared by in vitro synthesis. Variants typically exhibit the same biological activity as the naturally occurring analog, ie, specific intestinal binding, but as will be described in more detail below, the variants also have a C-tail It is selected to alter the properties of the protein.

The site for introducing the amino acid sequence variant is predetermined, but the mutation itself need not be predetermined. For example, random mutagenesis can be performed at the target colon or region to optimize mutation execution at a given location, and the expressed C-tail protein variant is combined with the optimal combination of desired properties. Can be screened for. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis.

Amino acid substitutions are typically of single residues. Insertions are usually of the order of about 1 to 10 amino acid residues, or the entire proline-rich unit; and deletions are of the order of 1 to 30 amino acids.
Over a single residue or a proline-rich unit;
Deletions or insertions are preferably made in adjacent pairs. That is, deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof may be combined to obtain a final construct. Obviously, mutations made in the DNA encoding the variant BAL or C-tail protein must not drive the sequence out of the reading frame and, preferably, may produce a secondary mRNA structure. Does not form complementary regions (EP75,444A).

Substitutional 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 generally involve C
-If it is desired to ultimately modify the characteristics of the tail protein or BAL, this is done according to Table I below.

Table I Examples of Original Sequence Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu By choosing less conservative substitutions than those in Table I, ie, (a) the polypeptide backbone structure in the substitution region (eg, , Sheet arrangement or spiral arrangement),
By choosing more significantly different residues in (b) the effect on the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulk of the side chains, the functional or immunological identity A substantial change is created. In BAL or C-tail protein properties,
Substitutions that are generally expected to produce the largest changes include (a) a hydrophilic residue (eg, a seryl or threonyl residue) that is a hydrophobic residue (eg, a leucyl residue,
Substitutions that replaced (or were replaced by) isoleucyl, phenylalanyl, valyl, or malanyl residues; (b) cysteine or proline replaced any other residue (or (C) a residue having a positively charged side chain (eg, a lysyl residue, an arginyl residue, or a histidyl residue) is replaced with a negatively charged residue (eg, a glutamyl residue). (D) a residue having a bulky side chain (eg, phenylalanine) is replaced with a residue having no side chain (eg, phenylalanine). , Glycine).

Using substitution or deletion mutagenesis, N-linked or O-site glycosylation sites can be created (eg,
Elimination (by deletion or substitution of asparaginyl residues in the Asn-X-Thr glycosylation site) or enhance expression of the BAL or C-tail protein or alter the half-life of the protein. Alternatively, non-glycosylated BAL or C-tail proteins can be produced in recombinant prokaryotic cell culture. Such non-glycosylated forms are expected to have lost or diminished intestinal binding activity. Deletion of cysteine or other labile residues may also be desirable, for example, in improving oxidative stability or in selecting a preferred disulfide bond configuration of BAL. Deletion or substitution of a potential proteolytic site (eg, Arg) can be accomplished, for example, by deletion of one of the basic residues or by replacement with a glutaminyl or histidyl residue.
Achieved.

To competitively inhibit cholesterol binding, full-length BAL with inactivated catalytic sites may be used. BAL cannot easily uptake cholesterol because the catalytic site is inactivated. The catalytic site may be inactivated by deleting or replacing amino acids in the active site of the recombinant BAL. The catalytic site may also be a chemical modification of the BAL at or near the active site, such as by proteolysis or other enzymatic reaction, by binding an irreversible enzyme inhibitor to the catalytic site, or by a three-dimensional arrangement of BAL catalytic units. Is inactivated by means of disrupting (since the binding of the C-tail to the intestine does not depend on its location). Examples of destruction means include:
Surfactants and heat.

Useful BAL derivatives that do not hydrolyze cholesterol esters or bind to heparin include polypeptides that may or may not be substantially homologous to BAL. These BAL derivatives do not exhibit cholesterol ester hydrolysis and / or heparin binding activity as defined above, either by recombinant or organic synthetic preparation of the BAL fragment, or by introducing amino acid sequence variants into the complete BAL. By doing so, it is generated.

BAL derivatives that do not hydrolyze cholesterol esters or bind to heparin as described above are active BAL
Is useful as an immunogen to raise antibodies against Such BAL derivatives, called "BAL protein antagonists", can be used to neutralize cholesterol uptake-mediated activity or BAL. The BAL protein antagonist may bind to the gut lining, thereby blocking the binding of native BAL. BAL protein antagonists are useful in the treatment of various cholesterol disorders, such as hypercholesterolemia, especially hypercholesterolemia exacerbated by dietary cholesterol intake.

BAL, BAL derivatives, and C-tail protein molecules can also be covalently modified. Such modifications can be made by reacting the targeted amino acid residues of the recovered or synthesized protein with an organic derivatizing agent that can react with selected side chains or terminal residues. Alternatively, the protein may be altered using post-translational modifications in the selected recombinant host cell. As is known to those skilled in the art, the resulting covalent derivatives can be used to identify residues important as immunogens or for biological activity, as well as for drug discovery of molecules such as half-life and binding affinity. Useful for changing physical properties.

Certain post-translational inductions are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often post-translationally deamidated to the corresponding glutamyl and asparatyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be used.

Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or trionyl residues, methylation of the 0-amino group of lysine, arginine, and histidine side chains (Creighto
n, Proteins: Structure and Molecular Properties, 79
−86 page (WH Freeman & Co., San Francisco, 198
3)), acetylation of the N-terminal amine, and optionally,
Amidation of the C-terminal carboxyl.

Production of C-tail protein There are several methods for the production of C-tail protein. For example, one skilled in the art can obtain a natural or recombinant BAL and use a protease (eg, trypsin,
Chymotrypsin, pepsin and others)
Alternatively, the natural C tail can be cleaved using chemical methods such as cleaving the Met-X bond with cyanogen bromide. Alternatively, one of skill in the art will appreciate that in a eukaryotic host, a gene or cDNA encoding a sequence comprising the C-tail region of BAL or any of the C-tail proteins described above, by itself or with other non-BAL DNA sequences. Can be expressed. The non-BAL DNA sequence may facilitate either expression or purification of the recombinant C-tail protein. Recombinant
BAL or C tail protein fusions can be subjected to cleavage and purification to obtain a purified C tail.

BAL can be purified from natural sources such as, for example, human and certain species milk or the intestinal or pancreatic juice of animals. However, this is impractical on a large scale. BAL
Is preferably produced by standard recombinant DNA techniques and genetic manipulation using eukaryotic host cells such as yeast or cultured mammalian or insect cells, where the C tail is appropriately glycosylated. Or produced in the milk of a non-human transgenic animal.

Methods for isolation of BAL from milk are described in Wang and Johnson, An.
al. Biochem. 133: 457 (1983), which is hereby incorporated by reference. As described herein,
"Essentially free" or "essentially pure" used to describe the state of the BAL or C tail protein produced may mean, for example, that BAL contains blood and / or
Or, when obtained by extraction and purification from tissue, means free of proteins or other substances that normally bind to BAL in its in vivo physiological environment.
Other substances include infectious organisms such as, for example, causative agents of acquired deficiency syndrome (AIDS). BAL and C tail proteins produced by the method of the present invention are greater than 95% pure.

To synthesize a recombinant BAL or C tail protein, those skilled in the art will appreciate the cDNA sequence (SEQ ID NO: 3) encoding human milk bile salt activating lipase (SEQ ID NO: 2) (Tang and Wang, US Pat. 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. This sequence is a natural
Any deletion, addition, or substitution of the sequence encoding the C tail of the BAL can be readily adapted for expression of any C tail protein as described above.

A DNA isolate is understood to mean chemically synthesized DNA, cDNA, or genomic DNA, with or without 3 'and / or 5' flanking regions. Code BAL
DNA can be obtained by: a) obtaining a cDNA library from liver, breast, pancreas, or other tissues containing BAL mRNA of a particular animal; b) detecting human clones in a cDNA library containing homologous sequences. Performing a hybridization analysis using labeled DNA encoding BAL or a fragment thereof (usually greater than 100 bp), and c) analyzing the clones by restriction enzyme analysis and nucleic acid sequencing to identify full length clones, It can be obtained from other sources than human. If the full-length clone is not present in the library, the appropriate fragments are recovered from the various clones using the nucleic acid sequences disclosed herein, and ligated at the common restriction site to the clones, The full-length clone encoding can be assembled.

“Transformation” means introducing DNA into an organism so that the DNA is replicable as an extrachromosomal element or by chromosomal integration. Unless indicated otherwise,
The method of transformation of a host cell used herein is Gr
aham and van der Eb, Virology 52: 456-457 (1973)
This is the method. However, other methods for introducing DNA into cells, such as by nucleic acid injection or by protoplast fusion, can also be used. If prokaryotic cells or cells containing rigid cell wall structures are used, suitable transfection methods are described in Cohen et al., Proc. Natl.
USA 69: 2110 (1972), calcium treatment using calcium chloride.

Standard ligation techniques are used to construct appropriate vectors containing the desired coding and control sequences. The isolated plasmid or DNA fragment is cut,
It is tailored and religated into the desired form to form the required plasmid.

Because glycosylation of the C tail is essential, prokaryotic cells are not useful as expression hosts for the C tail that bind to the receptor. However, in general, prokaryotes are used for the cloning of DNA sequences in the construction of useful vectors for expression. For example, E. coli W3110 (F, prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilus, and Salmonella typhimurium or Serratia m.
Other Enterobacteriaceae such as arcescans, various Pseudomonas species may be used.

Generally, plasmid vectors containing promoter and control sequences from a species compatible with the host cell are used with these hosts. Vectors usually have a replication site as well as one or more marker sequences. Marker sequences may provide for phenotypic selection of transformed cells. For example, E. coli is a plasmid derived from an E. coli species, pBR322.
Is typically transformed using a derivative of
Gene 2:95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance, and therefore provides a simple means of identifying transformed cells. pBR3
22 plasmids or other bacterial plasmids can also be
It contains or has been modified to contain promoters and other regulatory elemets commonly used in DNA construction.

Suitable promoters for use in prokaryotic hosts include β
Lactamase and lactose promoter system (Chang
Et al., Nature 275: 615 (1978); and Goeddel et al., Nature.
281: 544 (1979)), alkaline phosphatase, tryptophan (trp) promoter system (Goeddel et al., Nucleic
Acids Res. 8: 4057 (1980) and European Patent Application No. 36,776.
No. 2) and hybrid promoters such as the tac promoter (de Boer et al., Proc. Natl. Acad. Sci. USA 80: 21-
25 (1983)). However, other functional bacterial promoters are also suitable. These nucleotide sequences are generally known and, therefore, those skilled in the art will be able to encode these nucleotide sequences into BAL or C tail proteins using linkers or adapters to provide any necessary restriction sites. (Siebenlist et al., Cell 20: 269 (1980)). Promoters for use in bacterial systems also will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the BAL or C tail protein.

For analysis to confirm the correct sequence in the constructed plasmid, the ligation mixture was transformed with E. coli K12 strain 294 (ATCC 31446).
And successful transformants are selected for ampicillin or tetracycline resistance, as appropriate. Plasmids from transformants were prepared, analyzed by restriction, and / or
ssing et al., Nucleic Acids Res. 9: 309 (1981), or Maxam et al., Methods in Enzymology 65: 499.
(1980).

The host cell can be transformed with the expression vector and cultured in a conventional nutrient medium modified as appropriate for inducing a promoter, selecting a transformant, or amplifying a gene. Culture conditions (eg, temperature and pH) are those previously used in the host cell selected for expression, and will be apparent to those skilled in the art.

"Transfection" refers to the uptake of an expression vector by a host cell, whether or not any coding sequences are in fact expressed. Numerous transfection methods are known to those skilled in the art (eg, CaPO ₄
And electroporation). Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell.

Certain frequent methods and / or terms are described to facilitate understanding of the following examples.

"Plasmids" are designated by a lower case p preceded and / or followed by capital letters and / or numbers. The starting plasmids herein are either commercially available, publicly available without limitation, or are constructed from available plasmids according to published procedures. In addition, plasmids equivalent to the described plasmids are known in the art and will be apparent to those skilled in the art.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available, and their reaction conditions, cofactors, and other requirements used were known to those skilled in the art. For analytical purposes,
Typically, 1 μg of plasmid or DNA 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 5-50 μg of DN
A is digested in large volumes by 20-250 units of enzyme. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 ° C. are usually used, but can vary according to the supplier's instructions. After digestion, the reaction is directly applied to electrophoresis on a polyacrylamide gel and the desired fragment is isolated.

"Recovery" or "isolation" of a given DNA fragment obtained from a restriction digest is performed by separation of the digest on a polyacrylamide or agarose gel by electrophoresis, a marker DN of known mobility and molecular weight of the fragment of interest.
Refers to the identification of the fragment of interest by comparison with the mobility of the A fragment, excision of the gel compartment containing the desired fragment, and separation of the gel from DNA. This procedure is generally known (Lawn et al., Nucleic Acids Res.
9: 6103-6114 (1981), and Goeddel et al., (198
0)).

"Dephosphorylation" refers to bacterial alkaline phosphatase (BA
Refers to removal of terminal 5 'phosphate by treatment with P). This procedure prevents "circularization" or formation of a closed circle of the two restriction-cleaved ends of the DNA fragment. "Circularization" or formation of a closed circle prevents the insertion of another DNA fragment at the restriction site. Procedures and reagents for dephosphorylation are conventional (Maniatis et al., Molecular Cloning, 13
3-134 (Cold Spring Harbor, 1982)). Reactions using BAP are performed at 68 ° C. in 50 mM Tris and suppress any exonuclease activity that may be present in the enzyme preparation. The reaction is performed for one hour. After the reaction, the DNA fragment is gel purified.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis et al.,
Ibid., 146). Unless otherwise stated, ligation can be done with 0.5 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amount of the ligated DNA fragment using known buffers and conditions.

"Packing" or "blunting" refers to a procedure by converting single-stranded and restriction-enzyme sticky-ended nucleic acids to double-stranded. This cuts out the sticky ends and forms blunt ends. This process reduces restriction cut ends, which may be sticky with the ends created by only one or a few other restriction enzymes, to ends compatible with any blunt cut restriction endonucleases or other filled sticky ends. It is a versatile means to convert to Typically, blunting is performed using 2-15 μg of target DNA in 10 mM MgCl ₂ , 1 mM
Dithiothreitol, 50 mM NaCl, 10 mM Tris (pH 7.5)
8 units of DNA polymerase I in a buffer at about 37 ° C.
Of Klenow fragment and four deoxynucleoside triphosphates in the presence of 250 μM each. Incubations are generally terminated after 30 minutes by phenol and chloroform extraction and ethanol precipitation.

Many of the above procedures for mutating, manipulating, and recombining nucleic acid molecules are also described in Sambrook et al., Molecular Clon.
ing: A Laboratory Manual (Cold Spring Harbor, 1989)
In standard laboratory manuals.

Since many eukaryotic expression system exons are correctly cleaved, both human BAL cDNA and human BAL genomic DNA can be used to direct the synthesis of recombinant human BAL protein in natural or modified form. The human BAL gene is expected to be included in its untranslated region sequence that regulates the expression of the enzyme. These regulatory sequences can be used directly even in the expression of transgenic animals.

Recombinant BAL protein can be obtained in many different ways,
It can be produced from human BAL cDNA or gene. These include, for example, T-BAL (C-tail) in E. coli, including expression of BAL in hosts such as E. coli, Bacillus, yeast, fungi, insect cells, mammalian cells, and transgenic animals. Expression of a truncated form of BAL without) is described in Downs et al., Biochemistry 33: 7980-7895 (1994). Since prokaryotic hosts cannot strip mammalian introns from mRNA, it is preferable to make appropriate modifications in prokaryotic systems to express cDNA rather than genes. However, since prokaryotes cannot glycosylate BAL, it is preferred to use eukaryotic systems for BAL expression. When a eukaryotic cell is used as a host, either the human BAL gene or cDNA can be used to direct the synthesis of the enzyme. If a "leader" or "signal" sequence is present to direct newly synthesized BAL to the interior of the rough endoplasmic reticulum, glycosylation of BAL may also be present. This is important for the C-tail to reduce cholesterol uptake as intestinal surface interactions cleave polysaccharides at the C-tail.

In all cases, the human BAL cDNA or gene contains expression control elements (eg, a transcription initiation signal,
Translation initiation signal, initiation codon, stop codon, translation termination signal, polyadenylation signal, etc.). Suitable vectors are commercially available from various companies. After transfecting host cells with recombinant vectors containing BAL cDNA or genes, they can remain as extrachromosomal DNA or integrate into the host genome. In either case, they may direct the synthesis of recombinant BAL in the host cell. Some examples of heterologous gene expression can be found in Methods in Enzym
ology, Volume 153, Chapters 23-34 (editors, Wu and Grossman,
Academic Press, 1987). Large-scale cultivation of BAL-synthesizing host cells and purification of enzymes may form a cost-effective commercial means of producing BAL or C-tail. 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 as follows: (1) Using yeast and fungi as hosts: The principle of expression of recombinant BAL in yeast is described in E. coli. Similar to expression.
Examples are described in Bitter et al., Methods in Enzymology 153: 516-
544 (1987). Like E. coli, yeast host cells can express the foreign gene either in the cytosol or, in this case, preferably, as a secreted protein. Unlike E. coli expression, secreted expression in yeast can be glycosylated. There are many vectors, promoters and leaders available for yeast expression of secreted proteins. A good example is a system based on the control of an alcohol oxidation promoter in the methylotropic yeast Pichia pastoris. The vectors pHIL-S1 and pPIC9 are commercially available (Invit
rogen), and can produce large volumes of secreted eukaryotic recombinant proteins (1-4 mg / L human serum albumin, B
arr et al., Pharmaceutical Engineering 12: 48-51 (199
2); 12 mg / L tetanus toxic fragment C, Clara et al., B
io / Technology 9: 455-460 (1991).

For expression in Saccharomyces, for example, plasmid YRp7 (Stinchcomb et al., Nature 282: 39 (1979));
ingsman et al., Gene 7: 141 (1979); Techemper et al., Gene 10: 1.
57 (1980)) is commonly used. This plasmid is
A mutant strain of yeast already lacking the ability to grow in tryptophan (eg, ATCC Accession No. 44076 or PEP4-
1 (Jones, Genetics 85:12 (1977)) containing the trpl gene which provides a selectable marker. The presence of the trpl lesion as a characterization of the yeast host cell genome then provides an effective means of selection by growth in the absence of tryptophan.

Suitable promoter sequences for using a yeast host include 3-phosphoglycerata kinas
e) (eg, enolase, glyceraldehyde-3-
Phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase) (Hitzeman J.Adv.Enzyme Re
g. 7: 149 (1968); and Holland, Biochemistry 17: 4900.
(1978)).

Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, include:
It is a promoter region of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, a degradation enzyme involved in nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and an enzyme responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are described in Hitz
Further described in eman et al., European Patent Publication No. 73,657A. Yeast enhancers are also advantageously used with yeast promoters.

There are a few fungal expression vectors that have been successfully used to express heterologous genes. Existing fungal expression vectors integrate themselves into the host genome after transfection (Cullen et al., Molecular Cloning Vect.
ors for Aspergillus and Neurospora '' in A Survey of
Molecular Cloning Vectors and their Uses (Butterw
orth Publishers, Stoneham, MA1986). If the leader is present before the expressed protein codon, the secreted recombinant protein can be glycosylated. Some examples of successful expression are bovine chymosin (Cullen et al.,
Bio / Technology 5: 369-378 (1987)) and acid proteases from different fungi (Gray et al., Gene 48: 41-53 (198
7)) Including.

(2) Insect cells as hosts: Baculovirus expression vectors for the synthesis of foreign genes in insect cells have been successfully used to express many mammalian and viral proteins, and have described rat pancreatic BAL. (DiPersio et al., Protein E
xpr. Purif. 3: 114-120 (1992)). This system can be glycosylated and can also express recombinant proteins at high levels. The use of this system has been reviewed in some detail (Luckow and Summers, "Trends in the Devel
opment of Baculovirus Expression Vectors "Bio / Tech
nology, September 11, 1987). Many vectors derived from baculovirus expression systems are commercially available (eg, pBlueBacHi
s A, B, C and pEBVHis A, B, C) and insect host cells
Available from Invitrogen.

(3) Mammalian cells as hosts: Expression of many heterologous genes in mammalian cells has been successfully accomplished for commercial purposes. A commercial product of recombinant human tissue plasminogen activator is one example. Most of these expression vectors contain either a mammalian or viral promoter, a polyadenylation signal, and appropriate regulatory elements for cloning into E. coli, including the antibiotic resistance genes. After inserting DNA encoding BAL or C-tail downstream of the promoter, the vector can be first cloned into E. coli, isolated, and transfected into mammalian cells. Either neomycin or a similar resistance selectable marker can be co-transfected into mammalian cells. Either neomycin or a similar resistance selectable marker can be co-transfected in another vector or the same vector. For high level expression,
A gene amplification system is advantageous. Transformed clones secreting BAL or C-tail protein can be identified by enzyme assays or Western blots. Successful approaches to this approach using other mammalian proteins include glycosylated recombinant proteins (Poorman et al., Prote
ins 1: 139-145 (1986)) and human immune interferon (Scahill et al., Proc. Natl. Acad. Sci. USA 80: 4654-46).
58 (1983)).

Preferred promoters for controlling transcription from a vector in a mammalian host cell are available from a variety of sources: eg, polyoma, simian virus 40
(SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably from a viral genome such as cytomegalovirus, or from a heterologous mammalian promoter (eg, β-actin promoter). The early and late promoters of the SV40 virus are easily obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature 2).
73: 113 (1978)). The immediate early promoter of the human cytomegalovirus is easily obtained as a HindIII E restriction fragment (Greenaway et al., Gene 18: 355-360 (198
2)). Of course, promoters from the host cell or related species are also useful herein.

Transcription of a DNA encoding a BAL or C-tail protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp
And acts on the promoter to increase its translation initiation ability. Enhancers are relatively direction and position independent and are 5 'of the transcription unit (Laimins et al., Proc. Natl. A
cad. Sci. USA 78: 993 (1981)) and 3 '(Lusky et al., Mo.
l.Cell.Bio.3: 1108 (1983)) in the intron (Banerji
Cell 33: 729 (1983)), as well as within the coding sequence itself (Osborne et al., Mol. Cell Bio. 4: 1293 (1984)). Many enhancer sequences from mammalian genes (globulin, elastase, albumin, α-fetoprotein, and insulin) are known. Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer behind the origin of replication (nucleotides 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and adenovirus enhancers.

Eukaryotic host cells (yeast, fungi, insects, plants, animals,
Expression vectors used in humans, or nucleated cells) can also contain sequences necessary for termination of transcription, which can affect mRNA expression. These regions are BAL or C-
Transcribed as a polyadenylation segment in the untranslated portion of the mRNA encoding the tail. The 3 'untranslated region also contains a transcription termination site.

An expression vector can include a selection gene (also referred to as a selectable marker). Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, or neomycin. When such a selectable marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive under selective pressure. There are two widely used different categories of selection regimes. The first category is based on the metabolism of cells and the use of mutant cell lines that lack the ability to grow independent of supplemented media. Two examples are CHO DHFR cells and mouse LTK cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to media supplementation is to introduce an intact DHFR or TK gene into cells lacking the respective gene, thus altering their growth requirements. Individual cells that have not been transformed with the DHFR or TK genes cannot survive in unsupplemented media.

The second category is dominant choice. This refers to the selection scheme used in any cell type, and does not require the use of mutant cell lines. Typically, these schemes use a drug to stop the growth of the host cell. Cells with the new gene express proteins that confer drug resistance and survive selection. Examples of such dominant selection include neomycin (Southern and Berg. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan and Berg, Science 209: 1422 (198)
0)), or hygromycin (Sugden et al., Mol. Cell.
Biol. 5: 410-413 (1985)).
The three examples listed above use bacterial genes under eukaryotic control to confer resistance to appropriate drugs such as G418 or neomycin (Geneticin), Xgpt (mycophenolic acid), or hygromycin, respectively.

"Amplification" refers to the amplification or replication of an isolated region within the chromosomal DNA of a cell. Amplification is performed using a selection agent (eg, DH
Achieved using methotrexate (MTX), which inactivates FR. Amplification of the DHFR gene or generation of a continuous copy results in greater DTX being produced against greater MTX.
This produces HFR. Amplification pressure is applied despite the presence of endogenous DHFR by adding even more MTX to the medium. Amplification of the desired gene can be accomplished by co-transfecting a mammalian host cell with a plasmid having DNA encoding the desired protein and a DHFR gene or an amplified gene that allows for co-integration. By selecting only cells that can grow in the presence of even higher MTX concentrations, cells will have more DH
Ensure that FR is required (this need is met by duplication of the selected gene). As long as the gene encoding the desired heterologous protein has been co-integrated with the selection gene, replication of this gene will result in replication of the gene encoding the desired protein. As a result, the increased copy of the gene (ie, the amplified gene) encoding the desired heterologous protein will express more of the desired heterologous protein.

Suitable suitable host cells for expressing the disclosed vectors encoding BAL or C-tail proteins in higher eukaryotes include the following: monkey kidney CVI strain (COS-7, ATCC transformed with SV40) CRL1651); human embryonic kidney strain (293, Graham et al., J. Gen. Virol. 36:59 (197
7)), baby hamster kidney cells (BHK, ATCC CCL10);
Chinese hamster ovary cells-DHFR (CHO, Urlaub
And Chasin, Proc. Natl. Acad. Sci. USA 77: 4216 (198
0)) Mouse Sertoli cells (TM4, Mather, Biol. Repro
d.23: 243-251 (1980)), monkey kidney cells (CVI, ATCC CCL)
70); African green monkey kidney cells (VERO-76, ATCC CRL
-1587); human cervical cancer cells (HELA, ATCC CCL2),
Canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human hepatocytes (hep G2,
HB8065); mouse breast tumor (MMT060562, ATCC CCL51);
And TRI cells (Mather et al., Annals NYAcad. Sci. 38
3: 44-68 (1982)).

(4) Expression of C-tail protein or BAL in transgenic animals: Techniques for transferring the human BAL gene into the genome of other animals for tissue-specific expression already exist (Jaenisch, Science 240 : 1468-1474
(1988); Westphal. FASEB J. 3: 117-120 (1989)). Several studies are already underway to alter milk composition by using transgenic techniques (for a review, see Bremel et al., J. Dairy Sci. 72: 2826
-2833 (1989)). The general approach summarized in the three reviews listed above is that the promoter of a secretory mammary gland (milk) protein (eg, casein or milk lysozyme), the DNA encoding the BAL or C-tail protein, and the appropriate complement To construct a vector containing the elements. The cloned vector is then microinjected into newly fertilized bovine or ovine ova, and the ova are transferred to a "nanny" for fetal development and birth. Transgenic progeny are analyzed for gene transfer by Southern blot and for BAL or C-tail protein production in milk. It is important to note that cattle and sheep do not produce BAL in their milk. Transgenic animals can be bred to produce high yielding strains. C-tail proteins secreted into milk are completely glycosylated. Because cows and sheep do not make milk BAL, recombinant products can be easily distinguished from host milk proteins.

Sugar or sugar analog containing compounds that mimic the C-tail structure can also be synthesized by chemical reactions. They are,
It can be bound to the intestinal surface and used to compete with endogenous BAL in the same manner as the C-tail itself. The following examples demonstrate that galactose and fucose can elute endogenous bound BAL from rat intestinal surfaces, which indicates that these sugar-containing synthetic mimetics of C-tail or their Figure 4 shows that structural analogs can be used to affect binding to the intestinal surface. Since the C-tail contains repetitive sequences and multiple glycosylation sites, synthetic mimetics can include multiple sugar-containing sites. The sugar chemical bonds may be modeled based on the oligosaccharide structures of the C-tail, or structural analogs of these oligosaccharide structures may include essential properties for efficient binding to intestinal BAL receptors . Sugars can be chemically attached to polymers to create repeating units. Examples of suitable polymers are
Includes polypeptides, polyethylene glycols, dextra-like sugar polymers, and other synthetic polymers with appropriate functional groups for chemical attachment to sugar.

Pharmaceutical Applications and Compositions As described in the examples below, BAL is found to bind to lectin-like receptors on the intestinal surface via its C-tail oligosaccharide. This binding is an essential step in mediating intestinal cholesterol uptake. In addition, it has been found that the isolated C-tail of BAL can compete with BAL for binding to receptors on the intestinal surface. In addition, the C tail has BA bound to the intestine.
It has been shown that reducing L can 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 may also hydrolyze other fatty acid esters, thus facilitating the uptake of other fats. However, the C-tail reduction of intestinal uptake of non-cholesterol fat is not specific. This is because other lipases in the intestine can also digest non-cholesterol fats. Thus, as defined above, the C-tail protein is administered as a therapeutic to a solid in need of a specific reduction in cholesterol uptake and / or a more general reduction in the uptake of other fats, Treat diseases associated with hyperlipoproteinosis, hypercholesterolemia, and atherosclerosis.

In a preferred embodiment, the C-tail protein is
It is administered orally in an amount effective to reduce cholesterol uptake from food, as measured by a reduction in cholesterol levels in the blood. Dosage will vary depending on individual variability, such as formulation, excretion rate, number of intestinal surface receptors, levels of cholesterol to be reduced, and frequency of administration, and other factors usually optimized by a physician.

Intestinal cholesterol uptake from food is approximately 200 mg per person per day. The body synthesizes about 500 mg of cholesterol per person per day. The pancreas secretes about 500 mg of cholesterol per person per day, which is reabsorbed through the intestine. Therefore, C at the intestinal surface
The tail composition must be effective in reducing the amount of cholesterol from uptake of about 700 mg cholesterol per person per day. When the reduction is below 500 mg a day, the body decreases the body's cholesterol.

A C-tail protein designed to enhance the pharmacological activity of the C-tail protein when administered to a patient in an amount effective to reduce intestinal cholesterol uptake, thereby reducing cholesterol levels in the blood. The pharmaceutical composition comprises a suitable pharmaceutical stabilizing compound, delivery vehicle, carrier, inert diluent, and / or other additives suitable for enteral (oral) administration by methods well known in the art. It can be prepared in combination. The formulation is usually released into the stomach or intestines. C-tail proteins include lipids, pastes, suspensions, gels, powders, tablets,
Formulated in capsules, food additives, or other standard forms. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Examples include binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogen ^™ , or corn starch; Lubricating agents; aglidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; and / or flavoring agents such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Other dosage unit forms may additionally contain sugar, shellac coatings, or other enteric agents. The C-tail protein may be administered as a fluid, such as an elixir, suspension, beverage, liquid food supplement or substitute, or syrup; or as a component of the individual, such as a cachet or candy. The C-tail protein may be mixed with other active materials that do not impair the desired effect, such as other blood lipid lowering pharmaceutical compositions, or with materials that supplement the desired effect.

In one preferred embodiment, the C-tail protein is encapsulated in a carrier that is released into the small intestine, such as microparticles, microcapsules or microspheres prepared from synthetic or natural polymers such as proteins, polyhydroxy acids or polysaccharides. You. Suitable systems are known to those skilled in the art. Some microsphere formulations have been proposed as a means for oral drug delivery. These formulations generally protect the encapsulated compound and serve to deliver the compound to the bloodstream. Enteric formulations protect orally administered drugs,
It has been used extensively for many years to delay release. Other formulations designed to deliver the compound to the bloodstream and protect the encapsulated drug include PCT / US90 / 064
Hydrophobic proteins such as zein described in US Pat. No. 30,979 and PCT / US90 / 06433; “proteinoids” described in Steiner US Pat. No. 4,976,968;
Or UAB Research Foundation and Southern Researc
formed from synthetic polymers described in European Patent Application 0 333 523 by the h Institute. EPA 0 333
No. 523 describes microparticles less than 10 microns in diameter containing antigens used for oral administration of vaccines. Larger sizes are preferred for the applications described herein to prevent incorporation of the encapsulated C-tail protein into the blood and lymphatic systems.

Microparticles are rapid bioerodible polymers such as poly [lactide-co-glycolide], polyanhydrides, and polyorthoesters, whose carboxyl groups are exposed on the outer surface when their smooth surface erodes; zein, modified zein, Natural polymers such as casein, gelatin, gluten, proteins such as serum albumin or collagen, and polysaccharides such as cellulose, dextran, polyhyaluronic acid, acrylic acid and methacrylic acid ester and alginic acid polymers; polyphosphofazine (polyphage)
osphazines), poly (vinyl alcohol), polyamide, polycarbonate, polyalkylene, polyacrylamide, polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate, polyvinyl ether, polyvinyl ester, polyvinyl halide,
It can be formed of synthetic polymers such as polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane and copolymers thereof; and synthetically modified natural polymers such as alkylcellulose, hydroxyalkylcellulose, cellulose ethers, cellulose esters, and nitrocellulose. Representative polymers include methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate,
Cellulose acetate phthalate, carboxymethylcellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl) (Methacrylate), poly (uralyl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate, poly (isobutyl acrylate), poly (octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glycol), poly (Ethylene oxide), poly (ethylene terephthalate), poly (vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyro Don, and polyvinyl phenyl and the like. Particularly Representative bioerodible polymers, polylactides, polyglycolides and copolymers thereof, poly (ethylene terephthalate), poly (butyric acid),
Poly (valeric acid), poly (lactide-co-caprolactone), poly [lactide-coglycolide], polyanhydrides, polyorthoesters, blends, and copolymers thereof.

These polymers are available from Sigma Chemical Co., St. Louis,
Mo., Polysciences, Warrenton, PA, Aldrich, Milwaukee, W
It can be obtained from sources such as I, Fluka, Ronkonkoma, NY, and BioRad, Richmond, CA, or synthesized from monomers obtained from these suppliers using standard techniques.

Pharmaceutical compositions containing the C-tail protein must be stable under the conditions of manufacture and storage and include vitamin E and ethoxyquin and bacteriostats listed on the list of compounds approved for use by the Food and Drug Administration Can be used to protect against contamination by microorganisms such as bacteria and fungi.

As noted above, the rate of C-tail protein inactivation and elimination, as well as other factors known to those of skill in the art, affect the amount of drug administered to a patient. The decisive criterion is whether the amount is effective in reducing cholesterol in the blood. Cholesterol levels in the blood are assayed by standard techniques used in clinical laboratories.

C-tail protein reduces intestinal cholesterol uptake, thereby hyperlipidemia, including hypercholesterolemia, hypertriglyceridemia and related disease states such as atherosclerosis, cardiovascular disease, and pancreatitis. Can be used to treat The C-tail protein can also be used in normal subjects as a preventive measure to prevent the occurrence of these disorders. Human serum cholesterol levels are preferably maintained below 200 mg / dl, a value of 240 mg is considered clinically high and 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. Patients keep C-tail protein unnoticed and reduce intestinal cholesterol uptake. Conditions that should be considered when choosing a dose level, frequency, and duration are primarily
The severity of the patient's disease, the patient's serum cholesterol level, side effects such as gastric distress, and the need for the patient's prophylactic therapy, and the efficacy of treatment. For a particular subject, it must be adjusted over time according to the needs of the individual patient and the professional judgment of the person who administers or monitors the administration of the composition, and the range of concentrations set herein. It is understood that the examples are merely illustrative and do not limit the scope or practice of the claimed compositions. Other concentration ranges and durations of administration can be determined by routine experimentation. An initial estimate of dosage is based on the calculation that 20 μg is required to block 1 cm of intestinal bound receptor, and 20 mg of C-tail protein is equivalent to BAL
It should be enough to cover a 10 meter long intestine to block the binding site. 5 times excessive amount (10
0 mg) is too much to prevent the absorption of corsterols by enterocytes. Further dose adjustments are based on monitoring the dose response to the patient's C-tail protein in reducing cholesterol in the blood.

The invention will be better understood with reference to the following non-limiting examples. All references are expressly incorporated herein by reference.

Example 1: Proline-rich domains and glycosylation are not essential for the enzymatic activity of bile salt-activated lipase.

To test the functional requirements of structural regions within the BAL molecule, human truncated lactate activated lipase (T-BAL) cDNA (mucin-like carboxy-terminal region (residue 5
39-722) None) was expressed from the T7 system in Escherichia coli, purified, and the properties of the truncated recombinant enzyme were studied. The method was as follows.

Abbreviations The following abbreviations are used in this example: PANA, p-nitrophenyl acetate; PANB, p-nitrophenyl butyrate; SD
S, sodium dodecyl sulfate; T-BAL, truncated bile salt activated lipase; PCR, polymerase chain reaction; TN (50 mM T
ris-HCl, pH 8.0 and 100 mM NaCl); EGTA, ethylene glycol-bis (β-aminoethyl ether) N, N,
N ', N'-tetraacetic acid; FPLC, high performance protein liquid chromatography; and BSA, bovine serum albumin.

Materials Purification of BAL from human milk was performed as described in Baba et al. (1991), which is incorporated herein by reference. A λgt10 cDNA library from human lactating breast tissue was purchased from Clontech (Palo Alto, CA). Glycerol tri [9,10- ³ H] oleate, Amersham
(Arlington Heights, Il). Oligonucleotide primer I (SEQ ID NO: 4) and primer
II (SEQ ID NO: 5) was obtained from Dr. K. Jackson (Molecular Biolog
y Resource Facility, Oklahoma University Health Sc
ience Center). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Molecular Biological Methods Standard methods for sequencing analysis, restriction digestion, ligation, and subcloning are described in Mole, Sambrook et al., A commonly known and used reference technical literature.
cular Cloning, A Laboratory Manual, Cold Spring Harb
or Laboratory Press, Cold Spring Harbor, NY (198
9) and Current Protocols in Ausuble et al.
Molecular Biology, Green Publishing Associates and
Wiley-Interscience, New York, NY (1989). CDNA clones G10-2 and G10-5 were isolated from human mammary gland cDNA by a method as described in US Pat. No. 5,200,183 to Tang and Wang. United States
DNA obtained from Biochemical Corp. (Cleveland, OH)
DNA sequencing analysis was performed using the sequencing kit as follows. First, a double-stranded DNA template was prepared using Kraft et al.'S BioT
echniques 6: 544-547 (1988). Then 2.5 μg DNA, 15 ng primer and 2 μl reaction buffer were combined to make a total volume of 10
μl, which was heated to 65 ° C. for 2 minutes and then cooled to 25 ° C. in 30 minutes to anneal the mold. The labeling mixture was diluted 5-fold with water and sequenase form 2.0
(Sequenase version 2.0) was diluted 8-fold with water. 1μ
Of DTT (0.1 M), 2 μl of the diluted labeling mixture, 5 μCi of [α ³² P] dATP and 2 μl of diluted sequenase
Added to the annealed template primer. After mixing, the tubes were incubated at room temperature for 3 minutes. 2.5
μl of the termination mixture was placed in a test tube. Test tube is 1 at 37 ℃
Preheated for a minute. Transfer 2.5 μl of the labeling reaction mixture to each termination tube, mix, and then
For 3 minutes. The reaction was stopped by adding 4 μl of stop solution. The samples were then run on a 6% acrylamide gel. Before applying the sample, 20
The gel was pre-run for 30 minutes at a constant voltage of 00 volts. The sample was heated at 75 ° C. for 2 minutes just before loading 2 μl on the sequencing gel. 3 hours at 2000 steady voltage
The gel was run for 4 hours. After electrophoresis, the gel was transferred to 3MM paper and then dried under vacuum at 80 ° C for 2 hours. For subsequent reading of the DNA sequence, Kodak X-Omat
^{The TM} PR film was exposed overnight at room temperature. Innis and Gelfand's PCR Prot, herein incorporated by reference.
ocols, A Guide to Methods and Application 3-12 (Inn
is et al. (eds.), Academic Press, New York, NY (1990))
PCR was performed by the method described in. Invitroge
PCR cloned DNA was subcloned into PCR II as follows using a TA cloning kit from San Diego, CA. Ligation with the PCR II vector is performed using the vector: PCR
The product molar ratio was set to be 1: 1 and 13.
1 μl of 10 × ligation buffer, 50 ng of vector, PCR product and 4 units of T ₄ DNA ligase were combined to make a total volume of 1
0 μl was used. The ligation reaction was incubated at 12 ° C. overnight. Next, 1 μl of each ligation reaction was added to 50 μl of INVαF ′ competent cells containing 2 μl of 0.5 M β-mercaptoethanol. The tubes were incubated on ice for 30 minutes. The test tube was then placed in a 42 ° C water bath.
Placed for 30 seconds, then returned on ice for 2 minutes. 450 μl of room temperature SOC was added to each tube. The tubes were then incubated at 37 ° C. and shaken at 225 rpm for 1 hour. The transformed cells were transformed with 50 μg / ml kanamycin and 1 mg X
-Spread on LB plate containing Gal. The plate was then turned over and incubated at 37 ° C. overnight for selection of transformed clones. Promega (Madis
on, WI) The plasmid was isolated using the Magic Miniprep ^™ system provided. To pellet cells,
3 ml of the overnight culture was centrifuged at 4000 rpm for 5 minutes. The pellet was suspended in 200 μl of the cell resuspension solution. 200 μl of cell lysate was added and the tubes were inverted and mixed until the suspension was clear. 200 μl of neutralization solution was added and the tube was inverted several times to mix. The mini-column was attached to a syringe barrel and the assembly was inserted into a vacuum manifold. The resin / DNA mixture was transferred to a syringe and injected onto the column by applying a vacuum. After washing the column with 2 ml of column washing solution, the resin was dried. The mini-column was centrifuged at 14000 rpm for 20 seconds to remove any remaining washings. Add 50 μl of 70 ° C. water to the column, and after 1 minute,
DNA was eluted from the column by centrifuging the column for 20 seconds.

Vector Construction For construction of cDNA G10-6 surrounding the entire coding sequence region of BAL, overlapping clones G10-2 and G10
-5 unique Sma I restriction sites were used. T-BAL cDN
To prepare A, PCR was used with the use of primers I and II to express truncated BAL. For subsequent T-BAL expression using the T7 expression system in E. coli, the T-BAL cDNA was transferred to a pET11a cloning vector (In
In vitrogen, San Diego, CA).

Expression of T-BAL Using pET11a Vector The general approach and method of directing the expression of a cloned gene using T7 polymerase is described in Studier et al., Methods Enzymol, 1, incorporated herein by reference.
85: 60-89 (1990). T-BAL cDNA
It was ligated into the NdeI / BamHI cloning site of pET11a (Novagen, Madison, WI). This vector was used to transform E. coli BL21 (DE3) for T-BAL expression.
Cells containing this vector were transformed into ZB with ampicillin.
Cultured overnight in medium (Studier et al. (1990)). The next morning, 6
Four liters of LB-ampicillin were incubated with 80 ml of the overnight culture shaken at 37 ° C. until the absorbance at 00 nm was 0.6. Then, isopropyne-β-D-
Thiogalactopyranoside was added to a concentration of 0.4 mM and shaken for 3 hours. Harvest cells by centrifugation and 80 mg
800 ml of TN buffer (50 mM Tris
-HCl, pH 8.0, and 100 mM NaCl). The mixture is frozen at 70 ° C. and then thawed in a 37 ° C. water bath,
This was performed for three cycles. The solution was diluted to 2,400 ml with TN and centrifuged at 16,000 xg for 15 minutes. The pellet (inclusion body) was then transferred to Triton in TN.
Wash twice with ^TM X-100 (0.1%), collect the pellet by centrifugation and at -20 ° C for further processing.
And kept frozen.

Regeneration and purification of T-BAL 1 mM EGTA (ethylene glycol-b (β-aminoethyl ether) N, N, N ′, N′-tetraacetic acid), 10 mM β-mercaptoethanol and 5% glycerol (v / v)
8M urea in 100 mM Tris-HCl (pH 12.5) containing
The frozen inclusion bodies were further solubilized by stirring with 60 ml. The mixture is centrifuged at 105,000 xg for 30 minutes, after which the supernatant is dialyzed (exclusion MW =
10,000), 1 M urea, 0.1 mM β-mercaptoethanol and 5% glycerol were added to 10 mM Tris-HCl
Dialysis was performed against 1 liter of regeneration buffer contained in (pH 8.0). After dialysis for 20 hours, as described in the “esterase assay” below,
Regenerated T-BAL was assayed for esterase activity using 1 mM PANA and 2 mM Taurochol as activator to determine the amount of active enzyme in the dialysis solution. The regenerated T-BAL was then replaced with 60 ml of saturated ammonium sulfate (NH ₄
PH was adjusted to 8.0 with OH). This resulted in precipitation of T-BAL. After centrifugation at 18,000 rpm for 1 hour, T-BAL was collected from the precipitate. The collected precipitate was solubilized using 40 ml of regeneration buffer, and
For 30 minutes. Then, Amicon Centriprep ^TM -10
In a concentrator (Beverly, MA), remove the supernatant fraction from 3 ml to 5 ml.
Concentrated. After a further ultracentrifugation at 105,000 xg for 30 minutes, the supernatant fraction (1 ml) was subjected to molecular sieving by high performance protein liquid chromatography (FPLC). Two Superose ^™ 12 columns (Pharmacia, Piscatawa)
y, NJ) were tandemly connected to perform FPLC separation. The columns were equilibrated and 1M urea, 0.15M NaCl, 0.1M
mM β-mercaptoethanol and 50 mM Tris-HCl
(PH 8.0). The column was eluted at a flow rate of 0.5 ml / min. The eluate in the 1 ml fraction was collected and monitored by measuring the absorbance at 280 nm and assaying for esterase activity using PANA as a substrate.

N-terminal amino acid sequence analysis Automated Edman degradation was performed according to Hewick et al., J. Biol. Chem. 256: 79.
Model 470A gas phase protein sequencer equipped with a model 120A online phenylthiohydantoin amino acid analyzer (Applied Biosyste) according to 90-7997 (1981).
ms, Inc., Foster City, CA). The above references are incorporated herein by reference.

Esterase Assay Kinetic studies of BAL esterase activity using short-chain esters were performed as described in Wang, Biochem. J. 279: 297-302 (1991) using p-nitrophenyl acetate (PA
NA) and p-nitrophenyl butyrate (PANB) as substrates. The above references are also incorporated herein by reference. To derive the Michaelis-Menten kinetic parameters, the experiments were performed at 25 ° C. and the range of substrate concentrations in the final assay mixture was 0.4-2 mM for PANA and 0.1-0.5 mM for PANB, 2 mM Taurocholate was used as the agent.
The production rate of p-nitrophenol was determined using a Hewlett-Packard Diode Array Spectroph equipped with Peltier temperature control.
Using otometer (Hewlett Packard, Palo Alto, CA)
It was determined from the first part of the absorbance change at 418 nm (20 seconds). To monitor the refolding efficiency, as well as the esterase activity in the column chromatography eluent and the specific activity of the purified enzyme, an enzyme assay was performed using 1 mM PANA as substrate and 2 mM taurocholate as activator. One unit of enzyme activity was defined as one micromole of product released per minute.

Thermal stability of T-BAL To measure thermal stability, sodium phosphate (0.15
M, pH 7.4) at 0.1 mg / ml each of T-BAL and native BAL.
Incubated at 30 ° C, 40 ° C, 45 ° C and 50 ° C for 10 minutes in a water bath. After incubation, the solution was cooled with ice water. These treated samples were then assayed for residual activity using PANA as a substrate.

Lipase Assay BAL's lipase assay was performed using Nilsson-Ehle and Scho.
tz, J.Lipid Res.17: according to the method was modified method described in 536-541 (1976), was performed using glycerol tri [9,10- ³ H] oleate as substrate. 10 ml of 50 mM
28 µmol of trioleoylglycerol (specific activity 1.4 µCi / µmol) and 2.8 µm in NH ₄ OH-HCl buffer (pH 8.5)
A 2-fold concentrated stock substrate solution was prepared by emulsifying mol dioleoylphosphatidylcholine. The mixture was placed in an ice bath for 30 seconds at 5 settings (maximum output).
50%), W-380 sonicator (Heat Systems-Ultraso)
nics, Inc., Farmingdale, NY). After cooling, the mixture was sonicated for an additional 30 seconds. Final capacity 10
0 μl of the assay mixture is 50 mM NH ₄ OH-HCl buffer (p
H8.5), 1.4 mM trioleoylglycerol, 0.14 mM
Dioleoylphosphatidylglycerol, taurocholate and 10 μl of enzyme solution. After incubating for 1 hour and a half with shaking at 37 ° C,
3.2 ml of chloroform-heptanemethanol (5: 4: 5.6,
vol / vol / vol) and 1 ml of 0.2 M NaOH were added to terminate the reaction. Samples were centrifuged, Hydrocount of 10 ml ^TM
(JTBaker, Inc., Phillipsburg, NJ) and mix with Beckma
Radioactivity was measured with an n scintillation counter (Fullerton, CA).

Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis using an LKB-Pharmacia Phast system (Piscataway, NJ) with an 8-25% polyacrylamide gel slab manufactured by Pharmacia (Piscataway, NJ) Was done. 6 minutes before electrophoresis
At 0 ° C., the sample was treated with 10 mM β-mercaptoethanol and 2 mM.
% SDS.

Protein Assay The protein content of the enzyme preparation was determined by the procedure of Lowry using serum albumin as a standard (Lowry et al., J. Biol. Chem.
193: 265 (1951)) (Wang and Smith, A
nal. Biochem. 63: 414: 417 (1975)).
This document is incorporated herein by reference.

Fluorescence Measurement of Interaction of T-BAL with Taurocholate Fluorescence measurement of the interaction of T-BAL with Taurocholate was performed using aminco-Bowman Series 2 Fluorescence Spectrome.
Performed at 25 ° C. with ter (Urbana, IL). To study the interaction of T-BAL with taurocholate, T-BAL was
Tryptophanyl fluorescence was used. Fluorescence was recorded at 340 nm using an excitation wavelength of 280 nm. Both the excitation and emission bandwidths were set at 2 nm. The sensitivity of the device
Set to 1,000 volts.

When no taurocholate (activator) is present, T
The fluorescence intensity of -BAL was F ゜. At the saturated concentration of taurocholate, the fluorescence intensity was _F∞ . Based on the fluorescence (F) at a particular taurocholate concentration, the molar fraction (x) of T-BAL associated with taurocholate was determined by associating F as shown in the following equation.

F = (1-x) F ₀ + xF _{∞ The} derived molar fraction (x) is then used in the calculation of the binary complex dissociation constant K _A of taurocholate and T-BAL . Conversely, a computer least squares curve fitting procedure that treats K _A and F _{可 変} as variable parameters can be used to experiment with the best match between the calculated and experimentally measured values of F.

Data analysis Dynamic analysis of the data generated in these experiments
LOTUS 1-2-3 SPREAD SHEET program (Worcester, M
A) and using an IBM-AT computer. Perform least-squares nonlinear curve fitting, Bevington, Dat
a Reduction and Error Analysis for the Physical Sc
iences, 56-65 and 204-246, McGraw-Hill, New Yor
k (1969). This document is incorporated herein by reference.

(A) Carboxyl-terminal domain, O-glycosylation and N-glycosylation are not essential for the enzymatic function of BAL.

Amino acid sequence of natural human milk BAL cDNA (Baba et al. (19
91); Hui and Kissel, FEBS Lett. 276: 131-134 (199).
0); Nilsson et al., Eur. J. Biochem. 192: 543-550 (199
0)) encodes a mature enzyme of 722 residues and is shown in SEQ ID NO: 3. The cDNA structure is identical to that of pancreatic BAL (Reu
e et al., J. Lipid Res. 32: 267-27G (1991)). This supports the concept that enzymes from the mammary gland and from the pancreas are expressed from the same gene.

As expected, T-BAL synthesized in E. coli is inert and insoluble in aqueous buffers. Active T-BAL was obtained by refolding. This procedure involved solubilizing the inclusion bodies with urea and β-mercaptoethanol at high pH (12.5), followed by dialysis at low pH (8.0) against low concentrations of urea. Based on enzyme activity measurements, the yield of active enzyme was about 10 mg per 4 liters of culture LB medium. SDS-polyacrylamide gel electrophoresis yielded the major protein (> 90%) in the urea soluble fraction corresponding to a molecular weight of 60,000. This molecular weight was very close to the estimated molecular weight of T-BAL (59,270). By analyzing the N-terminal sequence of this protein fraction, the predicted BAL N-terminal sequence Ala-Ly
s-Leu-Gly-Ala-Val-Tyr-Thr (amino acids 1-8 of SEQ ID NO: 1) was obtained, indicating successful synthesis of T-BAL and removal of the starting methionine. The specific activity of T-BAL after the first step of refolding was about 5-10 units / mg. This is only 10-20% of the specific activity of natural BAL.

Recombinant T-BAL expressed in E. coli is not glycosylated. This further indicates that the highly glycosylated C-terminal region of BAL is not essential for catalytic function.

The purification of T-BAL was performed by partially purifying T-BAL refolded by ammonium sulfate precipitation, and then molecular sieving by FPLC. Two major peaks were found in the column fraction. The first peak, eluting at the exclusion volume (17 ml), represents the major protein peak and contains mainly the aggregated form of inactive T-BAL. The second peak (eluted at 28 ml) contains BAL activity. Fraction 27-29 SDS
-The polyacrylamide gel pattern shows that the T-BAL eluted in this peak is homogeneous. 4
64 batches of purified enzyme (fraction 28) from two different batches
An average specific activity of ± 2 units / mg was obtained. This specific activity of T-BAL has been previously reported (Wang and Johnson (198
3)) Specific activity reported for natural BAL (52 units / mg)
Because of the higher, FPLC column chromatography effectively separated the active form of the enzyme from the inactive form.

(B) Kinetics and specificity The availability and availability of sufficient amounts of purified recombinant T-BAL compared the specificity and kinetics of T-BAL and native BAL, and noted other properties.

Thermostability of T-BAL To compare the stability of T-BAL with the native enzyme, these two enzyme forms were treated at temperatures ranging from 30 ° C to 50 ° C. The thermal inactivation patterns of T-BAL and native BAL were similar, and both showed about a 90% loss of activity upon treatment at 50 ° C for 10 minutes. This further suggests that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme.

Dissociation constant K _A of T-BAL data scales over rate binding kinetics T-BAL and natural BAL are similar.
In a direct binding study of tryptophanil fluorescence upon interaction with taurocholate, native BAL upon binding to bile salts reduced protein tryptophanyl fluorescence by about 20% at saturated concentrations of taurocholate (Wang and Kl
oer (1983)). This is probably BAL during ligand binding
Arising from a conformational change. The dissociation constant of native BAL is 0.37 mM (Wang, J. Biol. Chem. 256: 10198-102.
02 (1981)). A similar decrease in tryptophanyl fluorescence is due to T
-Also observed with BAL. Here, α and β, respectively, by modifying the kinetic parameters K _s and K _cat, a two parameters used to represent the activation effect of taurocholate over rate (Segel, Enzyme Kine
tics 227-231, John Wiley & Sons, New York (197
Five)). Based on a 1: 1 stoichiometry, for the interaction of T-BAL with taurocholate, the dissociation constant K _A 0.32 ± 0.03
mM (n = 4) was measured. At a saturated concentration of taurocholate, the fluorescence intensity decreased by about 18%. Therefore, T-BAL is
Affinity to Taurocholate in monomer form
Slightly higher than natural enzyme. With these results,
The microenvironment of the taurocholate binding site of T-BAL and native BAL is very similar despite the deletion of the proline-rich sequence domain of T-BAL.

Kinetic properties of T-BAL by PANA and PANB Further kinetic analysis showed that PANA and
It was shown that the use of PANB (p-nitrophenyl butyrate) changed the enzyme specificity.

Like native BAL, T-BAL was found to contain basal activity when assayed with an esterase substrate in the absence of bile salts. Thus, taurocholate can also be considered as a non-essential activator of T-BAL. From Lineweaver-Burke plot, T-BAL
And the kinetic parameters K _s , K _cat (for the base enzyme) and αK _s for PANA and PANB as substrates
And βK _cat (taurocholate activating enzyme) were obtained. Despite the fact that a specific activity of T-BAL (64 units / mg) was obtained (Wang and Johnson (1983)) slightly higher than that of the native enzyme (52 units / mg), TB
The derived K _cat and βk _cat of AL are the natural enzymes K _cat
And about 2-8 times lower than βk _cat . However, T-BA
K _s and .alpha.k _s derived of L was only slightly higher than the K _s and .alpha.k _s native enzyme. Thus, the presence of the proline-rich sequence plays a role primarily in increasing the turnover rate of the enzyme, but has only a small effect on the substrate binding affinity. In addition, there is a change in the preferential reactivity of the enzyme. Previously, among the short-chain acyl esters of p-nitrophenol, natural BAL had higher k in the case of PANB.
It was reported to have _cat and βk _cat . In contrast, T-BAL, when assayed in the presence of taurocholate, has a higher k in the case of PANA than in the case of PANB.
_cat and βk _cat . Despite this, TB
AL also has a higher substrate specificity constant (T
Having a k _cat / K _s and βk _cat / αK _s) of -BAL. This is similar to that found in the native enzyme. The activating effect of taurocholate on the BAL-catalyzed hydrolysis using PANA as substrate indicates that the proline-rich domain of BAL does not represent the bile salt binding site of the enzyme.

Lipase activity of T-BAL There is an essential requirement for bile salt micelles to act as fatty acid acceptors in the native BAL-catalyzed hydrolysis of long-chain triacylglycerols (Wang et al. (19)
88)). The hydrolysis of glycerol in native BAL and T-BAL was compared. In this regard, T-BAL was found to have requirements similar to those of natural BAL for bile salts in the hydrolysis of long chain trioleoylglycerol. Since serum albumin is not a fatty acid receptor for BAL catalysis, it is also a weak fatty acid receptor in BAL-catalyzed reactions. Therefore, the lipase mixture contained only taurocholate, not BSA, as the fatty acid receptor.

Natural BAL when taurocholate concentration exceeds 60 mM
There is apparent saturation in the activation of catalysis,
Attributable to the partial inactivation of natural BAL at high taurocholate concentrations. In contrast, there was apparent desaturated activation of T-BAL by micellar taurocholate (tested to 120 mM taurocholate). On the other hand, 60m
The apparent saturation of activation by taurocholate above M is believed to be due to partial inactivation of the enzyme at high taurocholate concentrations. These results indicate that the transfer of the fatty acid product from the enzyme active site is probably not through a receptor-mediated process, but through a second-order kinetics involving the enzyme-fatty acid complex and taurocholate micelles. Is shown.

Example 2: The function of the C-tail of BAL is to bind BAL to cells lining the intestinal tract.

(A) Binding of BAL in human milk to intestinal mucosa To determine that ABL in human milk binds to intestinal mucosa, an experiment was performed as follows. That is, (i) radioactively labeling (I ¹²⁵ ) the purified natural human milk BAL, (ii) incubating the radiolabeled BAL in the isolated mouse intestine, and (iii) retaining the labeled BAL in the intestine And (iv) galactose, fucose, heparin, 0.3M using isotonic phosphate buffer as a control.
The mechanism of BAL binding to the intestine was examined by examining the elution of mouse endogenous BAL by NaCl.

Methods Purification of human milk BAL was performed according to Wang and Johnson, Anal. Biochem. 133: 457, which is incorporated herein by reference.
-461 (1983). Preparation of a truncated form of recombinant BAL (T-BAL) is described in Example 1.
Performed as described in.

Iodination of human milk BAL and recombinant T-BAL was performed using iodobeads (Pierce, Rockford, IL). Iodine ¹²¹ was obtained from Amersham (Arlington Heights, IL).
By adding 6 μl of ¹²⁵ I to 94 μl of enzyme solution (2 mg / ml in sodium phosphate buffer, pH 6.5)
Iodination was performed. One washed bead was placed in the solution and left at room temperature for 15 minutes. The mixture is then Bi
Passed through an o-spin ^™ 6 column (Bio Rad, Hercules, Calif.), centrifuged at 2275 rpm for 4 minutes at 4 ° C., and collected eluate. The eluate was diluted with 400 μl of 0.1 M sodium phosphate buffer (pH 6.5). Combine the eluted fractions,
One microliter of the eluate was tested for radioactivity. Glycerol tri [9,10- ³ H] oleate is used as a substrate, the lipase assay was performed BAL as described in Example 1. Two-fold concentrated stock substrate solution is added to 10 ml of 50 mM N
In a H ₄ OH-HCl buffer (pH 8.5), 28 μmol of trioleoylglycerol (specific activity 1.4 μCi / μmol) and 2.8 μmol
It was prepared by emulsifying mol dioleoylphosphatidylcholine. The mixture was washed with a W-380 sonicator (Heat Systems-Ultrasonics, Inc., Farmingdale, NY).
The emulsion was emulsified in an ice bath for 30 seconds with a setting of 5 (50% of maximum power). After cooling, the mixture was sonicated for an additional 30 seconds. An assay mixture with a final volume of 100 μl contains 5
0mM NH ₄ OH-HCl buffer (pH 8.5), 1.4 mM trioleoylglycerol contained 0.14mM dioleoylphosphatidylglycerol, 30 mM taurocholate over rate, and 10μl of the enzyme solution. After incubation with shaking at 37 ° C. for 1 hour, 3.2 ml of chloroform-heptanemethanol (5: 4: 5.6, vol / vol / vol) and 1 ml of 0.2 M Na
The reaction was terminated by adding OH. Centrifuge the sample, and
10 ml of Hydrocount ^TM (JTBaker, Inc., Phillipsburg, N
J) and radioactivity was measured in a Beckman scintillation counter (Fullerton, CA).

Protocol for binding of ¹²⁵ I-labeled BAL and T-BAL to mouse small intestine The duodenum and jejunum were removed from the small intestine of mice (about 20 grams each) and cut into 12 cm segments. Three experiments were performed. Here, in a parallel experiment, T-BAL except C-tail was used as a control. Segments are once with 0.15M NaCl and 0.1M
The plate was washed twice with a sodium phosphate buffer (pH 6.5). 20
μl of the labeled sample is first mixed with 20 μl of 8M urea,
It was then diluted with 720 μl of 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. The segments were incubated for 2 hours at room temperature. After incubation, the intestine was washed with 0.1 M sodium phosphate buffer (pH
Washed 3 times with 6.5) and cut into 2 cm intestinal pieces for radioactivity counting.

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 in Table II and Table I.
As shown in II, almost 12-fold more than T-BAL was retained by the intestinal mucosa. Natural BAL and T-BAL
Differ only in the C-tail, these results indicate that adhesion of the intestinal surface, possibly to the intestinal surface, to the receptor is mediated by the glycosylated C-tail.

To determine whether pancreatic BAL adheres to the intestinal surface of adult animals, BAL enzyme activity was measured in mouse intestine after the segments were thoroughly washed with saline. The results showed that the intestine had high BAL activity.

To examine the nature of the interaction between BAL and the mouse small intestinal receptor, the 1 cm segment of washed intestine was incubated with fucose, galactose, heparin, NaCl and isotonic phosphate buffer to inoculate the intestinal mucosa. An experiment was performed to elute endogenously bound BAL enzyme activity. The results are shown in Table IV.

Only low-activity BAL contains isotonic phosphate buffer and 0.3
Eluted with M NaCl, indicating that BAL was not bound to the intestine through ionic interactions. In addition, heparin did not elute more BAL activity than isotonic phosphate buffer and BAL activity eluted with NaCl. This indicated that the intestinal binding activity of BAL (known to bind heparin) mediated by heparin was not important. However, when elution was performed with either galactose or fucose, large amounts of BA
L activity eluted. These results indicate that BAL binds to the intestinal lumen surface via the oligosaccharide group of the C-tail. Furthermore, the elution is very specific, so the result is
This shows that there is a receptor (CT receptor) that specifically binds the oligosaccharide in the C-tail of BAL.

The mouse pancreatic BAL has a C-terminal region containing four repeat motifs, similar to the C-terminal region of the human enzyme. Since human BAL binds to mouse intestine, the above results confirmed similarities in oligosaccharide structure.

Example 3: Elution of BAL from intestine (a) Elution of rat endogenous intestinal BAL from mouse using galactose, fucose, methyl-α-mannoside, EGTA, heparin, and isotonic phosphate buffer as a control Since the intestine was too small in size, rat intestine was used to demonstrate elution of BAL by various compounds. Initial experiments were to demonstrate that, similar to the observations made on the intestine of mice, rat endogenous BAL could also be eluted using glassose and fucose. Since calcium binding is required for ligand binding of C-type lectin, EGTA elution was included in the test for calcium requirement for intestinal surface BAL binding.

Table V shows that the bound rat intestinal BAL showed galactose (0.1
(M), fucose (0.1 mM) and the results of three experiments showing elution by EGTA (1 mM). Elution with heparin (10 mg / ml) was performed using an isotonic phosphate buffer (pH 7.
Similar to the level of elution according to 4), the activity in the eluate was low and therefore not effective. In these experiments, a 1 cm segment of washed rat intestine was washed at room temperature with eluted compounds dissolved in isotonic phosphate buffer, pH 7.4, at room temperature.
Incubated for minutes. The BAL activity in this eluate was
Assays were performed as described in Example 1.

Heparin's inability to elute intestinal bound BAL
AL was not bound to rat intestine via heparin. These results are entirely consistent with those obtained for elution of BAL from mouse intestine (Table IV).
Bound by either galactose or fucose
Successful elution of BAL indicates that binding of BAL to the intestinal lumen surface is via an oligosaccharide group on the C tail. The discovery of BAL eluted by EGTA
Tail binding to the intestine was shown to have a calcium cofactor requirement.

(B) BAL release from intestinal epithelial cells was associated with reduced cholesterol uptake from cholesterol oleate.

Experiments As shown below, experiments were performed to demonstrate that BAL uptake of BAL to intestinal lining cells was required for cholesterol uptake.

Cholesterol (oriate) ester emulsion solution,
It was prepared as follows. Double concentration cholesterol oleate stock solution was added to 6 μmol [ ³ H] cholesterol oleate (1.6 μCi / μmol) (Amersham, Arlington H
eights, IL) was prepared by emulsification in 15 ml isotonic phosphate buffer, pH 7.4, with 0.6 μmol dioleoylphosphatidylcholine. This mixture was set to a setting of 5
(50% of maximum output) for 30 seconds in an ice bath with a W380 sonicator (Heat System-Ultrasonics, Inc., Farmingdale, N.
Emulsified using Y). After cooling, the mixture was further sonicated for an additional 30 seconds.

In each of the four experiments, two of the rat small intestine
Excision of 12 cm segment, isotonic phosphate buffer, pH 7.4
Washed once with. A segment from each animal was divided into a control (not washed with EGTA) group,
They were randomly assigned to the experiment (washed by EGTA) group. The use of paired segments from the same animal in the control and experimental groups showed that intestinal bound BA
All animal differences in the amount of L were reduced. For infusion or removal of the solution from the lumen, segments of the intestine were ligated at each end and secured to the catheter at one end.

The control segment was first injected with 1 ml of isotonic phosphate buffer, pH 7.5. The experimental segments received the same isotonic buffer containing 1 mM EGTA to release BAL bound to the intestinal surface. The intestinal segment was placed in a polystyrene weighing dish (14 cm) containing 5 ml of the same isotonic buffer.
² ) and gently shake for 30 minutes. Then
Experimental segments were washed once with isotonic phosphate EGTA solution and twice with isotonic buffer. Control intestines were washed three times with isotonic buffer.

To enable the segments by cholesterol uptake intestinal then the 0.2mM ^[3 H] - Cholesterol oleate (Amersham, Arlington Heights, IL)
And 6 mM Taurocholate (Sigma Chemical Co., S
A 1 ml aliquot containing t. Louis, MO) (a cofactor for BAL) was placed in each intestine. After incubation for 60 minutes in a polystyrene weighing dish with gentle shaking, the contents in the intestinal segment were removed. These segments were washed three times with isotonic buffer.

To measure the uptake of radiocholesterol by the control and experimental intestinal segments, remove the central 10 cm of each intestinal segment,
It was further cut into four segments of about 2.5 cm. Each of those segments is 1m for tissue solubilization.
1 in 2% sodium dodecyl sulfate + 8 M urea in scintillation vials. After the solubilization process, 10 ml of Hydrocount _™ (JTBaker, Inc., Phillipsbu
rg, added NJ) to each vial and the amount of radioactivity from ³ H- cholesterol in segments of the intestine,
Counting was performed 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 reduced cholesterol uptake by intestinal epithelial cells by about
Reduced to 16%. The statistical significance of the raw data (p <0.005) and the data calculated as% of control (p <0.0001) indicated a very high confidence level of this difference. These results indicate that BAL (attached to the intestinal surface)
Mediates the uptake of cholesterol from the intestinal lumen by intestinal lining epithelial cells.

(C) In addition to the role of BAL in cholesterol uptake from cholesterol esters, BAL also mediates direct uptake of free cholesterol.

Since about 85% of dietary cholesterol is in the form of free cholesterol, it is important to establish a role for BAL in free cholesterol uptake.

In vitro experiments were performed to compare the uptake of radioactive cholesterol in control intestine as compared to intestine where bound BAL was removed by EGTA. table
VII shows the results obtained from three experiments. The mean in the control experiment was 16.38 nmol / g intestine / hour uptake. The mean for EGTA treated intestine is 4.40 nm
ol / g intestine / hour. Removal of BAL resulted in about a 70% reduction in free cholesterol uptake. Despite the relatively large variation between the three rats, the difference between these two groups is very significant (p <0.005).

2 μm cholesterol stock solution was added to 6 μmol
(Specific activity = 1.6 μCi / −μmol) cholesterol and
Prepared by emulsifying 0.6 μmol of dioleoylphosphatidylcholine in 15 ml of isotonic phosphate buffer, pH 7.4. Set this mixture at a setting of 5 (50% of maximum output)
W-380 ultrasonic crusher (Heat-System) in ice bath for 30 seconds
-Ultrasonics, Inc.). After cooling, the mixture was further sonicated for an additional 30 seconds.

Two segments (11 cm) of the rat intestine were each washed once with isotonic phosphate buffer (pH 7.4), and these segments were ligated at both ends. Then 1 ml containing 1 mM EGTA
Isotonic phosphate buffer and 1 ml of isotonic phosphate buffer without 1 mM EGTA were injected into each intestinal segment. This segment was placed on a polystyrene weighing dish (14 cm square) containing 5 ml of isotonic phosphate buffer. After incubation, the solution in the gut, and replaced with ^[3 H] cholesterol (0.2 mM) and 6mM of taurocholate over rate of 1 ml, these segments, as described above, on the incubation tray on ice bath Placed. Then
The intestinal segments were washed three times with isotonic phosphate buffer and 8 cm sections of each intestinal segment (approximately 0.7
g) was cut into 2 cm fragments and each of those fragments was placed in 1 ml of 2% SDS-8M urea for tissue solubilization. To this mixture, 10 ml of Hydrocount ^™ was added for radioactivity counting as described in Example 3a.

(D) Heparin inhibits uptake of free cholesterol by rat intestine.

[ ³ H] Cholesterol oleate uptake by rat intestine was studied in vitro in the presence of added heparin (10 mg / ml). This procedure is essentially an example
The procedure is the same as described in 3b. Table VIII shows that the controls (column 1, presence of taurocholate and absence of heparin) have an average uptake of 20.65 nmol / g / h. In the presence of heparin (column 2), 2.62
nmol / g / hr. This difference is significant (p <.05).
Values from heparin inhibition are close to those of background uptake in the absence of taurocholate.

The experimental procedure is the same as the procedure described in Example 3b.
Since the intestine was washed with an isotonic phosphate buffer solution without EGTA,
Bound BAL was not removed from the intestinal surface. Heparin in uptake solution (ICN Chemicals, Costa Mesa, CA)
The concentration was 10 mg / ml.

(E) Addition of BAL to EGTA-treated intestine restored cholesterol uptake Materials and reagents Wang and Johnso, incorporated herein by reference.
n (1983), natural human milk bile salt active lipase (N-BAL) was purified. Recombinant truncated form of BAL (T-BAL, residue 1 lacking the carboxy terminus)
538) as described in Example 1 and Downs
Prepared as reported by Biochemistry 33: 7979-7985 (1994).

[1α-2α (n) - 3 H] cholesterol oleate and ⁽³ H- cholesterol) Amersham (Arlington Height
s, IL). Sigma Chemical
Co. St. Louis, purchased from MO.

2x concentrated cholesterol oleate stock solution
6 μmol of ³ H-cholesterol (1.6 μCi / μmol) and
0.6 μmol of dioleoyl-phosphatidylcholine 4 in 15
Prepared by emulsification in ml of isotonic sodium phosphate buffer, pH 7.4. The mixture was subjected to W-380 sonication (Heat-system Ultrasonics, Inc., Farmingdale, N.
Y) in an ice bath for 30 seconds at a set value of 5 (5
0%) emulsified. After cooling, the mixture was further sonicated for an additional 30 seconds.

Methods In each of the five experiments, three segments (11 cm each) of the rat small intestine were washed once with isotonic buffer (pH 7.4), and a catheter was placed at one end of each intestinal segment for infusion of the solution. And the segments were joined at both ends. Then, as shown in Example 1 (a), 1 ml of an isotonic phosphate buffer containing 1 mM EGTA was injected into each intestinal segment to release BAL attached to the intestinal surface. The segment was placed in a polystyrene weighing dish (14 cm ² ) containing 5 ml of isotonic phosphate buffer to maintain moisture and incubated on an ice bath for 30 minutes with gentle shaking. After incubation, the intestinal segments were washed once with EGTA-containing isotonic buffer and twice with isotonic buffer containing 0.2 mM calcium chloride. Then, in the presence of an isotonic phosphate buffer and 0.2 mM Ca ⁺² , 0.1.
At an average enzyme concentration of 8 mg / ml, inject 1 ml of purified N-BAL in one of the segments and add 1 ml of T-BAL to the second segment.
-BAL was injected. The third intestinal segment without BAL was the control. These intestinal segments were then placed back on the incubation tray and incubated for 30 minutes with gentle shaking. After incubation, the solution in the segment was removed and ^[3 H] cholesterol oleate in isotonic dried solution (0.2 mM)
And 1 ml of 6 mM Taurocholate. After incubation with shaking as described above, the segments are then washed three times with isotonic phosphate buffer and the 8 cm segment (about 0.7 g) of each intestinal segment is cut into 2 cm fragments, For 1% of 2% SDS- for tissue solubilization
Placed in 8M urea. To this mixture, add 10 ml of Hydrocount ^™
Was 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
EGTA-washed intestine segment without control (control), EGTA-washed segment treated with truncated BAL (+
T-BAL) and EGTA-washed segment (+ N-BAL) treated with purified native human BAL. Cholesterol uptake, measured in nmol cholesterol uptake per g intestine per hour, is shown in the left column of each group. The average uptake value of the five experiments was control 6.7.
6, + T-BAL 7.32, and + BAL 14.41. Uptake in the control segment represented, for example, fusion of cholesterol micelles with intestinal cell membranes and non-specific trapping of radioactive cholesterol in intestinal segments caused by residual intestinal BAL remaining after EGTA washing.

Due to the large deviations within each group due to biological differences between the 5 rats, the data was calculated as a percentage of the + N-BAL group and is shown in the right column for each group. The average of the percentage data is the uptake in the control + N-
This is 49.67% in the BAL group (100%), and 55.92% in + T-BAL. The difference between + N-BAL uptake and + T-BAL uptake is statistically significant (p <0.005).

Control data is + T-BAL data and +
Subtracting from the N-BAL data, the average net incorporation 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 + N The net cholesterol uptake for -BAL is about 14 times that of + T-BAL. The percentage difference between these two groups is also statistically highly significant (p <0.005).

These results indicate that (a) the reduction of cholesterol uptake by EGTA- or EDTA-treated intestines is mediated by BAL loss and not by impairment of the cholesterol uptake mechanism; and (b) cholesterol uptake Recovery is due to the binding of the BAL carboxy-terminal tail to the intestinal surface that was lacking in T-BAL.

Example 4: Addition of C-tail to intestinal contents is bound endogenous
Experiments were performed to show that the C tail, which releases BAL, can compete for binding to intestinal surface CT-receptors resulting in translocation of bound endogenous BAL.

(A) Preparation of C tail A procedure was devised for preparing pure BAL C tail from BAL. Purification of the C tail is very effective,
It should be noted that the starting BAL does not need to be completely homogeneous. Used in these C tail isolation experiments
BAL is described as previously described (Wang and Jonson (198
3)) Heparin-Sepharose ^™ column (14 x 1.5 bed capacity)
cm) Concentrated by chromatography. 300 ml of human skim milk was first centrifuged at 20,000 rpm for 2 hours. The supernatant is filtered and a 50 mM Tris-HCl buffer (pH 8.0)
On a Heparin-Sepharose ^™ column that had been equilibrated in advance. After loading, the column was washed with 200 ml of equilibration buffer and BAL was eluted with 0.3 M NaCl in equilibration buffer and collected in 5 ml fractions.

Fractions were assayed for BAL esterase activity using p-nitrophenyl acetate as substrate. Fractions containing BAL activity were pooled, dialyzed against distilled water and lyophilized. The yield was about 100-150 mg of dry material from each batch. For C tail purification, pools from three batches of partially purified BAL (about 350 mg) were first treated with 8 M urea (10 mg / ml) for 2 hours at room temperature and dialyzed overnight against 50 mM Tris-HCl. . Modified BAL
Was digested with trypsin and chymotrypsin (substrate: protease ratio, 50: 1, w / w) at 37 ° C. for 4 hours. Equal amounts of trypsin and chymotrypsin are added to this mixture for 2 hours.
A second addition and incubation continued at 37 ° C. overnight. The digest was then dialyzed against distilled water and lyophilized. Dry powder with 5ml 70% formic acid and 50m
g cyanide bromide, sealed in a glass tube and incubated overnight at room temperature. The solution was diluted 10 times with distilled water and lyophilized. The material was further dissolved in distilled water, dialyzed, and insoluble material was removed by centrifugation. The soluble fraction was then lyophilized. Then 2
FPL using Separose ^™ column connected to two tandems
For gel permeation chromatography using C (fast protein liquid chromatography), dry matter is 0.15M
Solubilization was performed using 3 ml of 50 mM Tris-HCl buffer containing NaCl. A 1 ml sample was applied to the column for each chromatographic run. Carbohydrate analysis of eluate fractions (Du
Bois et al., Anal. Chem. 28: 350-356 (1956)), tail 2
It was shown that eluted in fractions 1 to 25 (1 ml / fraction).

b) C-tail structure and composition N-terminal sequence and amino acid composition analysis of the material from the carbohydrate-containing FPLC fraction showed that this material corresponded to the human BAL region containing residues 528-712. As shown in Table X, the amino acid composition of the purified C tail and the composition based on the known sequence are very similar. From a β-elimination experiment using alkali for release of 0-linked oligosaccharides,
And in further amino acid composition analysis of the sample,
It was determined that threonine between 8-10 residues was destroyed in each molecule of the C-tail, while up to one serine was destroyed by the same procedure. Therefore, it was concluded that most, if not all, oligosaccharides were attached to threonine residues in the C tail.

Gas-liquid chromatography of alditol acetate derivatives (Griggs et al., Anal. Biochem. 43: 369-381 (1971)).
Further data from the analysis of the carbohydrate composition of the C-tail of human milk BAL based on the TAL showed the presence of fucose, galactose, galactosamine and glucosamine in the C-tail, as shown in Table XI. Since galactosamine is a sugar that anchors a carbohydrate to bind to a polypeptide chain, it was estimated from the ratio of lysine to galactosamine that about 8 to 10 oligosaccharides exist per C tail molecule.

Experiments were performed to determine whether the isolated C-tail domain of human milk BAL could displace BAL bound from rat intestinal surface. In these experiments, different amounts of C tail were placed in a 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 measurement of BAL lipolytic activity in the eluate were as described in Examples 3a and 3b.

The data is shown in Table XII. This result indicated that the isolated C tail was effective in placing endogenous BAL bound from rat intestine.

Example 5: Competitive inhibition of cholesterol uptake by C-tail of BAL The previous example shows that C-tail can elute endogenously bound BAL from rat intestinal surface. The following studies show that isolated C-tails can competitively inhibit cholesterol uptake in rat intestine.

a) In vitro inhibition of cholesterol uptake by BAL C-tail.

In this study, three intestinal segments were washed with isotonic phosphate buffer (pH 7.4). One of the intestinal segments was incubated with the substrate [ ³ H] cholesterol oleate (0.2 mM) alone as a control. The control gut segment should have a higher cholesterol uptake rate because endogenous BAL on the gut surface is available for cholesterol transport. A second intestinal segment was incubated with the same [ ³ H] cholesterol oleate and EGTA (1 mM). EGTA is known to elute endogenous BAL from the intestinal surface. The third intestinal segments were incubated with the same ^[3 H] cholesterol oleate and isolated C- tail (0.2mg / ml).

The data shown in Table XIII below is from four separate experiments. The averaged data is shown in FIG. These results indicate that when C-tail is present, cholesterol uptake is only about 10% of control intestinal uptake. This compares favorably with cholesterol uptake in the presence of EGTA (about 18% of controls). These results indicate that C-tail can effectively compete with endogenous bound BAL for intestinal surface binding. This inhibition is due to the replacement of endogenous BAL on the intestinal surface by C-tail, indicating that C-tail can be used to reduce cholesterol uptake from human diet.

The scheme of the physiological function of BAL in intestinal cholesterol uptake, shown in FIG. 1, was constructed based on data obtained from in vitro experiments. In this scheme, BAL initially binds to the intestinal surface via putative receptors. Cholesterol or cholesterol esters are then transported from the hydrophobic food vehicle to the active site of the enzyme. Cholesterol esters are hydrolyzed in the BAL active part. Free cholesterol in the active site (whether delivered by the hydrolyzate or directly from the vehicle) is delivered to enterocytes. This last step is aided by cell surface heparin, which may act as an orientation factor. The above results indicate that BAL not bound to the intestinal surface is not effective in mediating cholesterol uptake.

This scheme predicts that the supply of the isolated C-tail to surviving animals would compete for putative receptors on the intestinal surface and thus competitively inhibit cholesterol uptake. The following results confirm this prediction.

Experimental conditions Eighteen hours before the experiment, food (but not water) was taken from a pair of adult white rats (mean weight 259 ± 27 g). 4 mg in an isotonic phosphate buffer (pH 7.4)
1% of isolated C-tail (derived from BAL in human milk) (wt.
/ vol) 1 ml of a solution containing glucose was supplied in a tube. Control animals received the same volume of glucose-isotonic phosphate buffer without C-tail. One hour later, both animals received 10 μCi [ ³ H] -cholesterol oleate + non-radioactive cholesterol oleate (final concentration
0.2 ml), 1 ml of an emulsified mixture containing 4 mg of isolated C-tail, 0.2 mM triolein and 0.02 mM dioleoylphosphatidylcholine was given in a tube. The emulsification procedure was performed as described in section e) of Example 3 above. Again, the same mixture (but C
-Tail not included). For short-term experiments, blood samples were taken at hourly intervals for 5 hours. An aliquot (100 μl) was taken from this blood and 2 ml of hepataneisopropanol (3: 7,
After mixing vol / vol) and was acidified with 0.033NH ₂ SO ₄ in 1.25 ml. The mixture was vortexed for 30 seconds and centrifuged at 4 ° C. for 10 minutes. The upper half (250 μl) was then used for radioactivity counting.

The procedure for C-tail isolation from BAL in human milk was performed as described in section a) of Example 4 above. Based on the measured specific activity of cholesterol oleate in the cholesterol oleate emulsified mixture, the radioactivity in the blood was converted to the amount of cholesterol transported from the intestine to the blood compartment.

result.

Rats were fasted for 18 hours to reduce small intestinal contents. One hour prior to further administration of radioactive cholesterol oleate with 4 mg of C-tail to each of the experimental rats,
4 mg of the isolated C-tail was given in a tube. Control rats were treated in the same manner as the experimental rats except that they did not receive the C-tail. Blood samples were taken at one hour intervals after cholesterol administration and radioactivity was measured.

FIG. 5 shows the results from a representative experiment of one experimental and control rat. The results were obtained from a total of 6 control rats and 6 experimental rats.
Table XIV shows data from all six experiments, and FIG. 6 shows the mean cholesterol uptake of the control group and the experimental group receiving the C-tail. Inhibition of cholesterol uptake by administration of C-tail was 35%, 48.8, respectively, for measurements obtained 1, 2, 3, 4, and 5 hours after administration of radiolabeled cholesterol.
%, 55.7%, 62.2% and 55.5%. As shown in Table XIV, the data between the control and experimental groups at 2, 3, 4 and 5 hours are statistically highly significant (p <0.01).

Table XIV: Appearance of orally given radiolabeled cholesterol in rat blood with and without C-tail fragment administration.

Significant Differences These results provide the following conclusions: (1) Inhibition of cholesterol uptake by C-tail in surviving rats supports the conclusion that intestinal bound BAL mediates cholesterol uptake.

(2) The fact that cholesterol uptake is inhibited by the isolated C-tail indicates that (a) C-tail (BA
L, or isolated as a fragment itself) binds to the same receptor on the intestinal surface; (b) C-
Tail and receptor binding indicates that to achieve such significant inhibition, it must be very specific, and (c) the number of receptors on the intestinal surface is limited. This is important. Because it indicates that reasonable levels of C-tail can have a significant effect on cholesterol uptake.

(3) These data from surviving animals include C-tail (its equivalent from BAL of different animal species), recombinantly produced C-tail and chemically synthesized C-tail.
-Confirm in vitro results confirming that the tail and its analogs can effectively reduce cholesterol uptake. This supports the conclusion that the use of C-tail in humans can reduce cardiovascular disease by reducing serum cholesterol levels due to dietary cholesterol.

(4) Many members can be optimized to reduce cholesterol uptake more effectively. For example, the dose of C-tail can be optimized, the pattern of C-tail administration can be modified to maximize its effect, and the time of administration of C-tail in relation to cholesterol intake can be varied. Also, chemical and physical modifications (additions and formulations) of the C-tail can be made to maximize inhibition.

Example 6: BAL mediates uptake of triglyceride but not of taurocholate by isolated rat intestinal tissue.

The above examples support that taurocholate uptake is mediated by BAL bound to the intestinal surface. BAL also hydrolyzes triglycerides,
And since it is activated by Taurocholate,
The following examples address the question of whether intestinal BAL also mediates the uptake of triglycerides and taurocholate.

Experiment ³ H radiolabeled trioleylglycerol and ¹⁴ C-
Triolein and taurocholate uptake experiments were performed in a similar manner to the cholesterol uptake experiments described above, except that taurocholate was used instead of cholesterol. In the triolein uptake experiment,
Both 6 mM or 30 mM non-radiolabeled taurocholate contained radiolabeled triolein. Since the experimental system was an isolated intestine, Taurocholate was supplied to activate BAL. ^[3 H] - trioleyl glycerol (Amersham, Arlington Heights, IL) were prepared in the same manner as previously described (Downs et al. (1994)). A two-fold concentration of the substrate solution was prepared by emulsifying 15 μmol of dioleyl phosphatidylcholine in 7.5 ml of isotonic phosphate buffer (pH 7.4). W-380 Sonicator (Heat Sy
stems-Ultrasonics, Inc.) with 5 settings (50
The mixture was emulsified by using it in an ice bath for 30 seconds at a maximum power (% power). After cooling, the mixture was sonicated for an additional 30 seconds. As described above, a 1 ml volume of this emulsified solution was placed in the intestine of isolated rats for uptake measurements. For studies of taurocholate uptake, 2 μCi of tauro ( ¹⁴ CO) cholate and 30 μCi
A stock solution of taurocholate (6 mM) was made by adding mol of taurocholate (Sigma) to 5 ml of isotonic phosphate buffer.

Results The fatty acid uptake from triglycerides and taurocholate was measured using the isolated rat intestinal system as described for the cholesterol uptake experiment in section b) of Example 2. From each of the five experiments, removing BAL from the intestinal surface by EGTA elution,
Radioactive triolein absorption is about 75% with high statistical significance
(Table XV and FIG. 7). Table XV is
4 shows that the addition of 6 mM Taurocholate is sufficient to maintain the maximum uptake rate of triolein in buffer-washed intestine. Because 30 mM Taurocholate yields essentially the same uptake value (Table XV
Compare the last two columns with columns 3 and 4 in FIG. 7). When no taurocholate was added, only a baseline was observed, indicating that the uptake was mediated by the BAL and that bile salt activation was required for uptake. BAL-depleted EGTA-washed intestines showed very low uptake (first data column in Table XV and column 1 in FIG. 7).
This uptake is close to baseline (second data column at 0 mM Taurocholate in Table XV and column 2 in FIG. 7). These results indicate that BAL mediates triolein uptake.

On the other hand, removal of bound BAL from the intestinal surface did not significantly alter taurocholate uptake (Table XVI and FIG. 8). Washing the intestine with EGTA solution to remove bound BAL prior to the uptake experiments did not significantly reduce the uptake of radiolabeled taurocholate. The slight effect of EGTA elution was not statistically significant.

Significance These results indicate that oral administration of BAL C-tails, recombinant C-tails, and natural or chemically synthesized C-tail analogs may reduce triglyceride in humans that would have medical or nutritional benefits. Should be useful in reducing absorption.

The carboxy terminus of the bile salt-activated lipase (BAL) (C
Compositions containing all or part of the (tail) region or their functional equivalents (C-tail peptides), but in the gut compete with native BAL for binding to the gut surface, and Binds to biologically active compositions. The C-tail molecule of BAL is conjugated to the substance to be delivered and therefore can deliver the substance specifically to the intestine upon oral administration of the conjugate. In the gut, these compositions bind to the gut surface, resulting in the delivery and / or long-term presence of the therapeutic compound in the lining of the gut.

Binding Any of a variety of bioactive agents can bind to the C-tail, allowing delivery of the bioactive molecule to the intestine. For example,
The C-tail can be modified by covalently attaching a bioactive agent to the carboxyl or amino group of the C-tail. The C tail can be modified by using any of a number of different binding agents that covalently attach to the C tail.

One useful protocol is DMSO, acetone, or
Includes "activation" of hydroxyl groups on C-tail carbonyl diimidazole (CDI) in an aprotic solvent such as THF. CDI can be replaced by the attachment of a free amino group of a biologically active ligand such as a protein. An imidazolyl carbamate complex having a hydroxyl group is formed. The reaction is an N-nucleophilic substitution and results in a stable N-alkyl carbamate linkage of the ligand to the C tail. The resulting ligand-C tail complex is stable and resistant to hydrolysis for extended periods of time.

Another conjugation method involves coupling 1-ethyl-3 in N-hydroxylsulfosuccinimide (sulfo NHS).
-(3-dimethylaminopropyl) carbodiimide (ED
AC) or using "water soluble CDI" to attach the exposed carboxyl group of the C-tail to the free amino group of the bioactive 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. The resulting peptide bond is resistant to hydrolysis. Using sulfo-NHS in the reaction increases the efficiency of EDAC binding by a factor of 10 and provides very mild conditions to ensure the viability of the ligand-C tail complex. These protocols allow for activation of either hydroxyl or carboxyl groups on the C-tail and binding of the desired bioactive ligand.

Useful coupling procedures for coupling ligands having free hydroxyl and carboxyl groups to the C tail include the use of a crosslinker, divinyl sulfone. This method is useful for attaching sugars or other hydroxyl compounds to hydroxyl groups on the C-tail. Activation involves the reaction of divinyl sulfone with the hydroxyl group of the C tail to vinyl sulfonyl ethyl ether. Vinyl groups attach to alcohols, phenols, and amines. Activation and binding is pH
Occurs at 11. The linkage is stable in the pH range from 1 to 8 and is suitable for transport through the intestine. Any suitable conjugation method known to those of skill in the art can
Can be used to attach to the tail.

Therapeutic compounds can be covalently attached to the C-tail protein, either directly or indirectly, using a linker molecule. Where additional flexibility or space is required between the C-tail protein and the therapeutic compound, a linker molecule is typically used. Any suitable molecule that can bind to both the C-tail protein and the therapeutic compound can be used as a linker molecule. Examples of linkers are peptides or molecules with straight carbon chains. Since the C-tail composition is used in the intestine,
The bond or linker connecting the C-tail protein and the therapeutic compound must be stable in the intestinal environment.

Bioactive Agents 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 C-tail composition.
-Called tail-drug conjugate. The C-tail fragment can be conjugated to any biologically active agent. The term "biologically active material" refers to organic compounds such as proteins, carbohydrates, nucleic acids, lipids, drugs, or combinations thereof, and when administered to animals, including humans, in vivo, has the biological effect. Bring.

Examples include, but are not limited to, antigens, enzymes, hormones, receptors, peptides, proteins, polysaccharides, nucleic acids, nucleosides, nucleotides, liposomes, vitamins, minerals, inorganic compounds and viruses.
C-tails also deliver prokaryotic and eukaryotic cells, such as bacteria, yeast, and mammalian cells, including human cells, and components thereof, such as cell walls, and conjugates of cellular components. Can be used to

Examples of useful proteins include hormones such as insulin, growth hormone (including somatometin, transforming growth factor, and other growth factors), antigens for oral vaccines, enzymes such as lactase or lipase, and Digestives such as pancreatin.

Examples of useful drugs include ulcer treatments such as Carafate ^™ from Marion Pharmaceuticals, L-DOPA, antihypertensives or salt excretors (eg, Seale Pharmaceuticals
Methanolazone), a carbonic anhydrase inhibitor (eg, Ace from Lederle pharmaceuticals)
tazolamide), insulin-like drugs (eg, glyburide), sulfonylurea class blood glucose lowering drugs, synthetic hormones (eg, Android F from Brown Pharmaceuticals and Testre from ICN Pharmaceuticals)
d (methyltestosterone)) and anthelmintics (for example, mebendazole (Vermox ^TX , Jannsen Pharmaceutical)).

C-tail drug conjugates are particularly useful for treating inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. In ulcerative colitis, inflammation is restricted to the large intestine, whereas in Crohn's disease, inflammatory foci can be found throughout the gastrointestinal tract from the oral cavity to the rectum. Sulfasalazin is one of the drugs used to treat the above diseases. Sulfasalazine is broken down by bacteria in the large intestine into sulfapyridine (an antibiotic) and 5-aminosalicylic acid (an anti-inflammatory agent). 5-Aminosalicylic acid is an active drug and is needed locally. Direct administration of the degradation product (5-aminosalicylic acid) may be more beneficial. Protein-drug delivery systems may improve treatment by retaining drugs for a prolonged time in the intestinal tract. In the case of Crohn's disease, retention of 5-aminosalicylic acid in the upper intestine is of significant importance. This is because bacteria break down sulfasalazine in the large intestine. The only way to treat intestinal inflammation is by topical administration of 5-aminosalicylic acid.

The antigen may be conjugated to the peptide to provide a vaccine. Vaccines can be produced and have different retention times in the digestive tract. This retention time depends on the strength of the covalent bond that binds the peptide to the vaccine. This different retention time, among other factors, can result in more than one type (IgG, I
gM, IgA, IgE, etc.).

The term “antigen” encompasses any chemical structure that stimulates antibody formation or induces a cell-mediated response and binds to proteins, polysaccharides, nucleoproteins, lipoproteins, synthetic polypeptides, or small molecules that bind to proteins Including, but not limited to. Specific antigens that can bind to the peptide include attenuated or killed virus,
There are toxoids, polysaccharides, cell walls, and viral and bacterial surface or coat proteins. They can also be used in combination with conjugates, adjuvants, or other antigens. For example, Hemophilus influenzae (Hib) in the form of a purified capsule polysaccharide,
It can be used alone or as a conjugate with diphtheria toxoid. Examples of organisms from which these antigens are derived include poliovirus, rotavirus, type A, B
Type, hepatitis C, influenza, rabies, HIV, measles,
Mumps, rubella, Bordetella pertussus, Streptoc
occus pneumoniae, Diphtheria, Tetanus, Cholera, Sa
lmonella, Neisseria, Shigella, and Enterotoxigen
ic E.coli.

C-tail also provides water-soluble or water-insoluble drugs (eg, non-steroidal anti-inflammatory compounds, anesthetics, chemotherapeutics, immunosuppressants, steroids, antibiotics, antivirals, antifungals, steroidal anti-inflammatory drugs). Inflammatory agents, and anticoagulants).

An imaging agent can also be attached to the C-tail. These are metals, radioisotopes, radiopaque agents, fluorescent dyes,
And a radiolucent agent. Radioisotopes and radiopaque agents include gallium, technetium, indium,
Includes strontium, iodine, barium, and phosphorus.

The therapeutic compound that binds to the C-tail (ie, a biologically active agent) can be any compound that has a useful effect when delivered to the intestinal lining. Whether the therapeutic compound can act to either reduce or enhance the uptake of the compound taken by the individual;
Alternatively, it can degrade harmful compounds. For example, the therapeutic compound can be an enzyme, or a non-enzymatic binding molecule, or a ligand. Enzymes that catabolize unwanted compounds, or antibodies or receptors specific for such compounds, that are ingested by an individual are useful as therapeutic compounds. Because the C-tail composition is used in the intestine, the therapeutic compound must be stable and active in the intestinal environment.

Preferred therapeutic compounds are catabolic enzymes, which catalyze the breakdown of certain molecules. Particularly active enzymes are not normally present in the intestine. A preferred therapeutic compound of this type is lactase. C-tail / lactase compositions can be used to deliver lactase to the intestinal lining of individuals that lack lactase or that have reduced lactase activity. C
-Tail / lactase binds to the intestinal lining and maintains lactase activity for a long time, breaking down ingested lactose. Thus, it alleviates the symptoms of lactose intolerance.

Oral Administration In a preferred embodiment, the C-tail-drug conjugate is administered orally in an amount effective for the particular therapeutic application. Dosage will vary depending on prescribing, rate of excretion, individual changes such as the number of receptors on the intestinal surface, the type of treatment, and the number of doses, and other factors usually adjusted by a physician. In one embodiment, the BAL C-tail composition is administered orally in an amount effective to reduce or enhance the uptake of a particular compound from the food, or to reduce the intestinal concentration of an undesired compound.

Sequence Listing (1) General Information: (i) Applicant: Oklahoma Medical Research Foundation (ii) Title of the Invention: Method for Reducing Intestinal Absorption of Cholesterol (iii) Number of Sequences: 6 (iv) Contact Address: (A) Name: Patrea El Pabst (B) Address: 2800 One Atlantic Center 1
201 West Peachtree Street (C) City: Atlanta (D) State: Georgia (E) Country: United States (F) Zip Code: 30309-3450 (v) Computer Read Format: (A) Media Type: Floppy Disk (B) Computer: IBM PC compatible (C) OS: PC-DOS / MS-DOS (D) Software: Patent In Release 1.0
Version 1.25 (vi) Prior application data: (A) Application number: (B) Application date: (C) Classification: (vii) Current application data: (A) Application number: US 08 / 479,160 (B) Application Date: June 7, 1995 (C) Classification: (vii) Current application data: (A) Application number: US 08 / 347,718 (B) Filing date: December 1, 1994 (C) Classification: (viii) ) Agent / office information: (A) Name: Patrea El Pabst (B) Registration number: 31,284 (C) Inquiry / Record number: OMRF150CIPPCT (ix) Telephone line information: (A) Telephone: (404) 873-8794 (B) Telefax: (404) 873-8975 (2) Information of SEQ ID NO: 1 (i) Sequence features: (A) Length: 722 residues (B) Type: amino acid (D) Topology: linear (Ii) Sequence type: protein (iii) Hypotical sequence: NO (v) Fragment type: middle fragment (xi) Sequence: sequence Number 1: (2) Information of SEQ ID NO: 2 (i) Sequence features: (A) Length: 742 residues (B) Type: amino acid (D) Topology: linear (ii) Sequence type: protein (iii) Hypotical sequence: NO (v) Fragment type: Intermediate fragment (ix) Characteristic of sequence: (A) Symbol indicating characteristic: Modified-Site (B) Location: 186..187 (D) Other information: / Note = "Position 187 represents the N-glucoside binding site." (Ix) Sequence features: (A) Feature designation: Modified-Site (B) Location: 193..194 (D) Other information: / Note = "The serine at position 194 represents the active serine site." (Ix) Sequence features: (A) Symbol representing feature: misc.feature (B) Location: 1..742 (D) Other information : / Function = “amino acid structure of bile salt-activating lipase in human milk” (xi) Sequence: SEQ ID NO: 2 (2) Information of SEQ ID NO: 3 (i) Sequence features: (A) Length: 3018 base pairs (B) Type: nucleic acid (C) Number of strands: single strand (D) Topology: linear ( ii) Sequence type: cDNA (iii) Hypotical sequence: NO (iv) Antisense: NO (ix) Feature of sequence: (A) Symbol indicating feature: misc.feature (B) Location: 1..742 (D) Other information: / function = "nucleotide on sequence 2904
679 encodes the amino acid of bile salt activating lipase in human milk "(xi) Sequence: SEQ ID NO: 3 (2) Information of SEQ ID NO: 4 (i) Sequence features: (A) Length: 21 base pairs (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear ( ii) Sequence type: DNA (iii) Hypotical sequence: NO (iv) Antisense: NO (ix) Sequence characteristics: (A) Symbol representing characteristics: misc.feature (B) Location: 1..21 (D) Other information: / function = “primer” (xi) Sequence: SEQ ID NO: 4: (2) Information of SEQ ID NO: 5: (i) Characteristics of sequence: (A) Length: 25 base pairs (B) Type: nucleic acid (C) Number of chains: single-stranded (D) Topology: linear ( ii) Sequence type: DNA (iii) Hypotical sequence: NO (iv) Antisense: NO (ix) Sequence features: (A) Symbol indicating feature: misc.feature (B) Location: 1..25 (D) Other information: / function = “primer” (xi) Sequence: SEQ ID NO: 5 (2) Information of SEQ ID NO: 6: (i) Sequence features: (A) Length: 11 residues (B) Type: amino acid (D) Topology: linear (ii) Sequence type: protein (iii) Hypotical sequence: NO (v) Fragment type: Intermediate fragment (ix) Sequence characteristics: (A) Symbol representing characteristics: misc.feature (B) Location: 1..11 (D) Other information: / Function = "consensus sequence" (xi) Sequence: SEQ ID NO: 6:

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI // C12N 15/09 ZNA C12P 21/02 C12P 21/02 C12N 15/00 ZNAA (31) Priority claim number 482,262 (32 ) Priority Date June 7, 1995 (June 7, 1995) (33) Priority Country United States (US) (72) Inventor One, Chi San United States of America 73162, Oklahoma City, Woodbridge Road 11808 ( 58) Fields surveyed (Int.Cl. ⁷ , DB name) A61K 38/46 A61K 31/70 C12P 21/02 A61K 45/00 A61K 47/48 C12N 15/00 CAPLUS (STN) REGISTRY (STN)

Claims (15)

(57) [Claims] 1. A composition for reducing intestinal absorption of cholesterol, comprising a portion of the carboxyl region of human bile salt lipase, or a C-tail comprising a derivative thereof that binds to a specific receptor on intestinal cells. Including protein,
A composition which, when administered to a human or animal, is administered in an amount effective to inhibit cholesterol uptake in combination with a suitable pharmaceutical carrier for oral administration. 2. The method according to claim 2, wherein the C-tail protein is SEQ ID NO: 1.
The composition of claim 1, comprising all or part of amino acid residues 539 to 722 of the formula (I). 3. The composition of claim 1, wherein said C-tail protein further comprises a region of bile salt activated lipase that includes a catalytic site that has been inactivated. 4. The composition of claim 1, wherein said C-tail protein further comprises at least three repeat regions of a primate, human, cat, dog, or rodent bile salt-activating lipase. object. 5. The composition according to claim 1, wherein said C-tail protein contains a consensus motif PVPPTGDSGAV. 6. The method according to claim 1, wherein the carrier is a polymer or an intestinal encapsulation composition, and the C-tail protein is incorporated on or in the carrier.
A composition according to claim 5. 7. The composition of claim 1, wherein said 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 said composition further comprises another compound effective in lowering blood cholesterol.
A composition according to claim 1. 9. The compound comprising: (i) a chemically synthesized mimetic of the carboxyl terminal region of bile salt activated lipase, which competes for binding of bile salt activated lipase on the human intestinal surface; ii) selected from the group of compositions consisting of all or part of the carboxyl-terminal region having sequence homology to the carboxy-terminal region of human BAL, 9. The composition of claim 8, wherein the composition is derived from a bile salt-activated lipase from an animal of the invention. 10. The composition of claim 1, wherein said C-tail protein is a dietary formulation for oral administration. 11. The method of claim 1, wherein said C-tail protein is in the milk of a transgenic cow or sheep.
A composition according to claim 1. 12. A composition for inducing a therapeutic compound comprising: a therapeutically effective amount of C-tail protein when combined with a suitable pharmaceutical carrier for oral administration. Is a therapeutic compound without lipase activity that binds to a therapeutic composition, wherein the C-tail protein is Pro-Val-Pro-Pro-Thr
A composition comprising at least three times the basic structure of Gly-Asp-Ser-Gly-Ala-Pro or a repeating structure having a basic structure with a small number of substitutions, and binds to human enterocytes. 13. The composition according to 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 method of claim 10, wherein the therapeutic compound is a non-steroidal 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. Selected from the group consisting of blood agents,
13. The composition according to claim 12. 15. The composition of claim 12, wherein said carrier is a polymer or an intestinal encapsulation composition, and wherein said C-tail protein is incorporated on or in said carrier.