EP0833835A1 - Intermediaires de proteines contre l'obesite, leur preparation et leur utilisation - Google Patents

Intermediaires de proteines contre l'obesite, leur preparation et leur utilisation

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Publication number
EP0833835A1
EP0833835A1 EP96921702A EP96921702A EP0833835A1 EP 0833835 A1 EP0833835 A1 EP 0833835A1 EP 96921702 A EP96921702 A EP 96921702A EP 96921702 A EP96921702 A EP 96921702A EP 0833835 A1 EP0833835 A1 EP 0833835A1
Authority
EP
European Patent Office
Prior art keywords
protein
gln
optionally replaced
glu
replaced
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP96921702A
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German (de)
English (en)
Other versions
EP0833835A4 (fr
Inventor
John Edward Hale
Warren Cameron Mackellar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP0833835A1 publication Critical patent/EP0833835A1/fr
Publication of EP0833835A4 publication Critical patent/EP0833835A4/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. Most specifically the invention relates to an intermediate used to prepare obesity proteins that when administered to a patient regulate fat tissue.
  • inclusion bodies inactive, sparingly- soluble protein aggregates, i.e., inclusion bodies.
  • the formation of such inclusion bodies is a result of the high protein concentrations in the cell arising from expression.
  • the inclusion bodies In order to obtain biologically-active proteins, the inclusion bodies must be dissolved by denaturation and reduced, and then the three-dimensional structure of the protein in its native spatial form must be formed by the adjustment of suitable conditions.
  • renaturation into the biologically active conformation is not a quantitative process -- a multitude of non-functional species and conformations of the protein may be formed.
  • conditions are selected which, both prevent the establishment of improper protein conformation and do not hinder renaturation. Because it is difficult, if not impossible, to predict how a polypeptide chain renatures into a highly, ordered conformation, the conditions that favor the formation of the biologically active protein cannot be predicted.
  • the present invention provides the penultimate intermediate in the renaturation pathway for the obesity protein.
  • the invention further provides the preparation of the intermediate and its use to prepare a biologically active obesity protein or analog thereof.
  • the intermediate also provides
  • the present invention provides a properly folded intermediate of an obesity protein or analog thereof. More particularly, the present invention is directed to a
  • A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof;
  • B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein;
  • R 1 and R 2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; provided that both R 1 and R 2 are not H.
  • the invention also provides a process of preparing a protein of Formula I, which comprises: mixing an obesity protein or analog thereof with a solution comprising a denaturant and a thiol reducing reagent at a concentration of about 1 to 100 mM at a pH from about 7 to about 12.
  • the invention further provides a process of preparing a properly folded protein of the Formula (II) :
  • A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof;
  • B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein
  • a thiol reducing reagent at a concentration of about 1 to 100 mM, at a pH from about 7 to about 12;
  • Figure 1 provides an HPLC chromatogram run on a Zorbax C-8 column with buffer A being 50 mM ammonium phosphate, 0.5% SDS, and 5 % n-propanol at pH 7.6 and buffer B being 50 mM ammonium phosphate, 0.5% SDS, and 50 % n-propanol at pH 7.6,
  • the peak at about 385 seconds represents the biologically active obesity protein and the later eluting peak at about 573 seconds represent the claimed intermediate prepared in the examples.
  • Base pair (bp) -- refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5'- monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy) thymine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and T correspond to the 5'- monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • base pair may refer to a partnership of T with U or C with G.
  • Denaturing reagent or denaturant is known to one skilled in the art. Examples of denaturing reagents are described in R. Jaenicke, Prog. Biophys. Molec. Biol. 49: 117-237 (1987). Preferred reagents include urea,
  • thiocyanate and guanidine, most preferably, 6 to 8 M urea.
  • Mixed disulfide -- refers to a group derived from a thiol reducing reagent capable of disulfide exchange between the reagent and the cysteinyl residue of the polypeptide.
  • Obesity protein refers to the protein produced from the obesity gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g., from the N-terminal valine-proline to the C-terminal cysteine of the mature protein.
  • the mouse obesity protein and human obesity protein are published in Zhang et al .
  • the rat obesity protein is published in Murakami et al., Biochemical and Biophysical Research Comm. 209(3): 944-52 (1995).
  • the numbering of amino acids in the present specification is consecutively from the amino terminus of the full length, mature protein. Such uniform numbering of amino acid residues is well accepted by those of ordinary skill in the art.
  • the cysteines associated with disulfide formation are at residues 96 and 146.
  • the phrase "A is a polypeptide
  • an obesity protein consisting essentially of amino acid residues 1 to 95 of an obesity protein
  • the phrase "B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the obesity protein” is intended to include the amino acids from the first cysteine to the C-terminal cysteine of the obesity protein. It is understood that the deletion of one or more amino acids in a natural variant or fragment as well as any amino acid additions, including additions to the Cys at position 146, that do not effect the novel and basic characteristics of the invention are included in the definitions of A and B. Renumbering the amino acid residues of the protein is unnecessary and may result in confusion. For example, particularly with the murine and human obesity protein, a desGln(28) variant has been observed. Such variant is intended to be included in the present invention.
  • Obesity protein analog refers to an obesity protein having one or more amino acid substitutions, preferably less than five and most preferably less than three substitutions, and includes proteins disclosed of the
  • Xaa at position 28 is Gln or absent
  • said protein having at least one of the following substitutions:
  • Gln at position 4 is replaced with Glu
  • Gln at position 7 is replaced with Glu
  • Thr at position 27 is replaced with Ala
  • Xaa at position 28 is replaced with Glu
  • Gln at position 34 is replaced with Glu
  • Met at position 54 is replaced with methionine sulfoxide, Leu, lle, Val, Ala, or Gly;
  • Gln at position 56 is replaced with Glu
  • Gln at position 62 is replaced with Glu
  • Met at position 68 is replaced with methionine sulfoxide, Leu, lle, Val, Ala, or Gly;
  • Asn at position 72 is replaced with Gln, Glu, or Asp; Gln at position 75 is replaced with Glu;
  • Ser at position 77 is replaced with Ala; Asn at position 78 is replaced with Gln or Asp;
  • His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp,
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val;
  • Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp
  • Gln at position 130 is replaced with Glu
  • Met at position 136 is replaced with methionine sulfoxide, Leu, lle, Val, Ala, or Gly;
  • Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lle, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu; or Gln at position 139 is replaced with Glu;
  • a properly folded obesity protein or analog thereof is the protein in the conformation or tertiary structure resulting in a biologically active protein useful in treating obesity and those conditions associated with obesity such as
  • diabetes diabetes, cardiovascular disease and cancer.
  • Ob gene refers to any nucleic acid sequence that hybridizes, and is at least 50 % homologous, preferably 70 % homologous, and most preferably 80 % homologous to the native ob gene sequences disclosed by Zhang et al . Nature 372: 425-32 (1994) and Murakami et al., Biochemical and Biophysical Research Comm. 209(3): 944-52 (1995).
  • the ob gene product is expressed specifically in adipose tissue and regulates energy balance.
  • Obesity protein inclusion bodies refers to insoluble protein aggregates or cytoplasmic aggregates containing, at least in part, the obesity protein to be recovered. Similarly, inclusion bodies when preparing an obesity protein analog refers to insoluble protein
  • aggregates or cytoplasmic aggregates containing, at least in part, the obesity protein analog to be recovered are aggregates or cytoplasmic aggregates containing, at least in part, the obesity protein analog to be recovered.
  • Plasmid an extrachromosomal self-replicating genetic element.
  • Reading frame the nucleotide sequence from which translation occurs "read" in triplets by the
  • each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame" must be maintained.
  • Recombinant DNA Cloning Vector any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • Recombinant DNA Expression Vector any recombinant DNA cloning vector in which a promoter has been incorporated.
  • Replicon A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • R 1 and R 2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed
  • a mixed disulfide is recognized and understood to be derived from a thiol reducing reagent capable of
  • Suitable thiol reagents include one or more mercapto reagents containing a free -SH that is capable of forming a mixed disulfide and operable in disulfide exchange.
  • Thiol therefore includes, but is not limited to, cysteine, 2-mercaptoethanol, glutathionine, cysteamine, S- mercaptoethanol (BME), and the like.
  • Thiol reagents such as dithiothreitol (DTT), dithioerythritol (DTE) are also included in the present invention; but are less preferred due to instability of the mixed disulfide formed with such reagents.
  • preferred moieties represented by Ri or R 2 include SO 3 , SCH 2 CH (NH 2 ) (COOH), SCH 2 CHNH 2 , SCH 2 CH 2 OH, and H 2 NCH(COOH)CH 2 CH 2 CONHCH(CH 2 S)CONHCH 2 COO _ .
  • R 1 and R 2 are not H.
  • Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • Treating describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating obesity therefore includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.
  • Vector a replicon used for the transformation of cells in gene manipulation bearing polynucleotide
  • Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using
  • Vectors include
  • Asp may rearrange to
  • amino acids are in the L configuration.
  • the present invention provides a protein of the Formula (I):
  • A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof;
  • B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein;
  • R 1 and R 2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; provided that both R 1 and R 2 are not H .
  • A is a polypeptide of the formula: (SEQ ID NO : 2 )
  • R 3 is absent, Met, Met-R 4 , or a leader sequence
  • R 4 is any amino acid except Pro
  • Xaa at position 28 is Gln or absent
  • Gln at position 4 is optionally replaced with Glu
  • Gln at position 7 is optionally replaced with Glu
  • Asn at position 22 is optionally replaced with Gln or Asp;
  • Thr at position 27 is optionally replaced with Ala;
  • Xaa at position 28 is optionally replaced with Glu;
  • Gln at position 34 is optionally replaced with Glu;
  • Met at position 54 is optionally replaced with
  • methionine sulfoxide Leu, lle, Val, Ala, or Gly;
  • Asn at position 72 is optionally replaced with Gln, Glu, or Asp;
  • Gln at position 75 is optionally replaced with Glu
  • Ser at position 77 is optionally replaced with Ala;
  • Asn at position 78 is optionally replaced with Gln or Asp; or
  • Asn at position 82 is optionally replaced with Gln or Asp.
  • A is a polypeptide of the formula: (SEQ ID NO: 2)
  • Xaa at position 28 is Gln or absent.
  • Yet additional preferred embodiments include proteins wherein B is a polypeptide of the Formula:
  • His at position 97 is optionally replaced with Gln,
  • Trp at position 100 is optionally replaced with Ala, Glu, Asp, Asn, Met, lle, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
  • Ala at position 101 is optionally replaced with Ser,
  • Ser at position 102 is optionally replaced with Arg; Gly at position 103 is optionally replaced with Ala; Glu at position 105 is optionally replaced with Gln; Thr at position 106 is optionally replaced with Lys or
  • Trp at position 138 is optionally replaced with Ala
  • Gln at position 139 is optionally replaced with Glu.
  • N-terminal extension is optionally cleaved from the protein prior to administration.
  • Preferred N-terminal extensions are Met or Met-R4- wherein R4 is any amino acid except Pro.
  • the present invention also includes proteins wherein A optionally includes one or more amino acid leader sequence that may be used for purification or other
  • leader sequences include Gly-Ser- His-Met (SEQ ID NO: 4), Met-Gly-Ser-Ser-His-His-His-His- His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met (SEQ ID NO: 5), Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO: 6), Glu-Ala- Glu-Ala (SEQ ID NO: 7), Leu-Glu-Lys-Arg- (SEQ ID NO: 8),
  • the compounds of Formula (I) are folding intermediates of the biologically active obesity protein.
  • Preferred obesity proteins are the native sequences and analogs such as those described in Basinski et al., in U.S. applications serial number 08/383,638, filed February 6,
  • Xaa at position 28 is Gln or absent.
  • Gln at position 4 is replaced with Glu
  • Gln at position 7 is replaced with Glu
  • Thr at position 27 is replaced with Ala
  • Gln at position 28 is replaced with Glu
  • Gln at position 34 is replaced with Glu
  • Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala;
  • Gln at position 56 is replaced with Glu
  • Gln at position 62 is replaced with Glu
  • Met at position 68 is replaced with methionine sulfoxide, or Leu; Asn at position 72 is replaced with Gln or Asp;
  • Gln at position 75 is replaced with Glu
  • Gln at position 130 is replaced with Glu
  • Met at position 136 is replaced with methionine sulfoxide, Leu, lle; or
  • Gln at position 139 is replaced with Glu.
  • Thr at position 27 is replaced with Ala
  • Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala;
  • Met at position 68 is replaced with methionine sulfoxide, or Leu;
  • Thr at position 27 is replaced with Ala
  • Met at position 54 is replaced with Leu, or Ala;
  • Met at position 136 is replaced with Leu, or lle.
  • other preferred embodiments are specific substitutions to amino acid residues 97 to 111 and/or 138 of the proteins of SEQ ID NO: 1.
  • Xaa at position 28 is Gln or absent
  • said protein having at least one substitution selected from the group consisting of:
  • His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp,
  • Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp;
  • Trp at position 138 is replaced with Ala, Glu, Asp,
  • Preferred proteins are proteins of the Formula
  • said protein having at least one substitution selected from the group consisting of: His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lle, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
  • Ala at position 101 is replaced with Ser, Asn, Gly,
  • Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp;
  • Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lle, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu; or a pharmaceutically acceptable salt thereof.
  • More preferred proteins of the Formula (V) are those wherein:
  • His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lle, Phe, Tyr, Ser, Thr, Gly, Gln or Leu;
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr or Val;
  • Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp;
  • Trp at position 138 is replaced with Ala, Glu, Asp,
  • Trp at position 100 is replaced with Ala, Gly, Gln,
  • Val, lle, or Leu Val, lle, or Leu; Ala at position 101 is replaced with Thr; or
  • Trp at position 138 is replaced with Ala, lle, Gly, Gln, Val or Leu.
  • Trp at position 100 is replaced with Ala, Gln or Leu; Ala at position 101 is replaced with Thr; or
  • Trp at position 138 is replaced with Gln.
  • Most preferred species of Formula (V) include species of SEQ ID NO: 16 and 17:
  • the present invention provides an intermediate, which leads to a very efficient conversion to the biologically active protein conformation of the obesity protein. Therefore, by preparing the intermediate of the present invention, the teriary structure of the biologically active protein is formed first. Thus, allowing almost quantitative conversion of the disulfide bonds. The formation of S-S linked dimer or other multimers is
  • the protein of the present invention is prepared by recombinant DNA technology or well known chemical
  • a protein of Formula (III), or SEQ ID NOs:11, 12, 13, or 14 is prepared by recombinant synthesis.
  • Recombinant methods are preferred if a high yield is desired.
  • the basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the protein,
  • Synthetic genes the in vitro or in vivo transcription and translation of which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be
  • a convenient protease sensitive cleavage site e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the fusion protein construct.
  • Techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis.
  • the mutations that might be made in the DNA encoding the present anti-obesity proteins must not place the sequence out of reading frame and preferably will not create complementary regions that could produce
  • the gene encoding the protein may also be created by using polymerase chain reaction (PCR).
  • the template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue.
  • the obesity proteins may be made either by direct expression or as fusion protein comprising the protein followed by enzymatic or chemical cleavage.
  • a variety of peptidases e.g. trypsin
  • which cleave a polypeptide at specific sites or digest the peptides from the amino (e.g. diaminopeptidase) or carboxy termini of the peptide chain are known.
  • particular chemicals e.g. cyanogen bromide
  • the skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi- synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., Carter P., Site Specific Proteolysis of Fusion
  • Plasmids containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are digested by restriction enzymes, tailored, and religated to form the plasmids required.
  • a synthetic coding sequence is designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification plasmids.
  • the isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art.
  • the particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed.
  • the choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein.
  • plasmid vectors which contain promoters and control sequences which are derived from species compatible with the host cell.
  • E. coli is typically transformed using pBR322, a plasmid derived from an ⁇ . coli species (Bolivar, et al., Gene 2: 95 (1977)). Plasmid pBR322 contains genes for ampicillin and
  • the pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology.
  • the desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed and translated from a promoter and ribosome binding site, both of which should be functional in the host cell.
  • An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No. 5,304,493, the teachings of which are herein incorporated by reference.
  • the gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes Ndel and BamHI.
  • the genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette.
  • procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention.
  • E. coli K12 strain 294 ATCC No.
  • E. coli B and E. coli X1776 (ATCC No. 31537). These examples are illustrative rather than
  • prokaryotes are used for expression of recombinant proteins.
  • the aforementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various pseudomonas species may be used.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase (vector
  • PGX2907 contains the replicon and ⁇ -lactamase gene) and lactose promoter systems (Chang et al., Nature, 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATH1 [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC- 37282).
  • a DNA sequence encoding the protein of SEQ ID NO: 14 with a Met-Arg N-terminal extension was obtained using standard PCR methodology.
  • a forward primer (5'-GG GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC, SEQ ID NO: 14
  • PCR product is cloned into PCR-Script (available from STRATAGENE) and sequenced.
  • a plasmid containing the DNA sequence encoding the desired protein is constructed to include Ndel and BamHI restriction sites.
  • the plasmid carrying the cloned PCR product is digested with Ndel and BamHI restriction enzymes.
  • the small ⁇ 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding sequence for A-C-B proinsulin is deleted.
  • the ligation products are
  • GIBCO-BRL colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 ⁇ g/mL of tetracycline are analyzed. Plasmid DNA is isolated, digested with Ndel and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ⁇ 450bp Ndel to BamHI fragment are kept. E. coli B BL21 (DE3) (commercially available from NOVOGEN) are transformed with this second plasmid expression suitable for culture for protein production.
  • DIFCO tryptone-yeast
  • thermoinducible promoter-operator regions such as the c1857 thermoinducible lambda-phage promoter-operator region, require a temperature shift from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis.
  • E. coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, E. coli K12 L201, L687, L693, L507, L640, L641, L695, L814 (E. coli B).
  • the transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid.
  • the cultures are then incubated for a time and temperature appropriate to the host cell line employed.
  • the inclusion bodies comprising the obesity protein are solubilized in a denaturant at a concentration sufficient to solubilize the protein
  • the inclusion bodies are solubilized in about 6 to 7 M urea at a pH of about 8 to 10.
  • the desired protein concentration is about 0.1 mg/mL to the solubility of the protein in the solution; more preferably 0.1 to 50 mg/mL and most preferably 0.5 mg/mL to 5.0 mg/mL. Under these conditions the protein is denatured.
  • conformations of the molecule may be formed.
  • the present invention provides a key intermediate in the renaturation.
  • the claimed intermediate is prepared by adding about 1 to 100 mM, preferably 1 to 20 mM and most preferably 1 to 10 mM, of a thiol reducing reagent containing a free -SH that is operable in a disulfide interchange, preferably, cysteine, cystamine, BME and the like.
  • a thiol reducing reagent containing a free -SH that is operable in a disulfide interchange preferably, cysteine, cystamine, BME and the like.
  • Preferred thiol reagents are cysteine and cysteamine.
  • the intermediate forms in about 1 minute to 24 hours.
  • the intermediate can be purified by filtration, chromatography or other conventional methodology. Because the intermediate is in the correct tertiary structure (native conformation), the intermediate can be used as a biologically active therapeutic agent and may offer advantages in efficacy or onset of action. However, the intermediate is preferably converted to the biologically active proteins of Formula III.
  • the intermediate is converted to the biologically active protein of the Formula II by reducing the concentration of the thiol reducing reagent and denaturant concentration of the solution to effect disulfide bond formation.
  • the reduction of thiol and denaturant may be carried by
  • the solution is diluted to a protein concentration about 0.05 mg/mL to about 5.0 mg/mL and about 1 to 20 mM thiol and then dialyzed or diafiltered.
  • the buffer used for dialysis or diafiltration is preferably PBS (phosphate buffered saline with about 5 to about 10 mM phosphate and 50 to 500 mM NaCl) at a pH of about 7.0 to 12.0 and more preferably 7.5 to 9.0.
  • Suitable buffers include, but are not limited to, 4- (2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS).
  • HEPES 4- (2-hydroxyethyl)-1-piperazineethane-sulfonic acid
  • TRIS tris(hydroxymethyl)aminomethane
  • the thiol reducing reagent and denaturant concentration is reduced by diafiltration or dialysis against PBS or about 5 to 10 mM TRIS.
  • the conversion from the intermediate to the biologically active conformation is most remarkable -- approaching quantitative conversion.
  • the biologically active protein of the Formula II is produced in high yield.
  • the formation of the intermediate also allows the fold to be conducted at higher protein concentrations than a fold carried out directly from the free -SH.
  • the preferred range includes 0.05 to 5 mg/mL, preferably 0.1 to 3 mg/mL, and most preferably 1.0 to 2 mg/mL.
  • a higher concentration during the fold translates into lower volumes (smaller tanks) and less downstream processing.
  • the fold may be carried out in the absence of glycerol or other agent added to prevent protein aggregation.
  • the ability to fold at large scale in the absence of such agents is significant because such agents, particularly glycerol, must be removed in downstream purification.
  • the present invention further provides an efficient process of preparing proteins of the Formula II:
  • a thiol reducing reagent at a concentration of 1 to 100 mM at a pH from about 7 to about 12;
  • inclusion bodies are solubilized by the addition of about 6 to 8 M urea and about 3 to 7 mM cysteine in a 8 to 12 mM Tris buffer at about pH 8 to 12 and more preferably at a pH of about 8 to 10. Under these conditions the mixed disulfide intermediate forms and is optionally purified by filtration and/or chromatography.
  • the efficiency of the formation of the single intra-chain disulfide from the intermediate is increased by the adding additional thiol reducing reagent prior to dilution, diafiltration or dialysis.
  • thiol is added so that it is in molar excess -- preferably 1 to 6000 fold, more preferably 3 to 6000 fold, excess.
  • thiol preferably cysteine
  • the protein is purified from the reaction mixture by
  • the intermediate of Formula (I) is stable in the presence or absence of denaturant.
  • the intermediate is soluble in PBS suggesting proper tertiary structure
  • the intermediate may be purified by techniques known in the art and including size exclusion, ion exchange, reversed phase chromatography.
  • the dilute protein solution was allowed to stand without mixing at room temperature for 8 hours, and the pH of the solution was raised to 8.68 by addition of solid Tris base to 10 mM.
  • the solution was clarified by centrifugation and analyzed by SDS-PAGE under non-reducing conditions, reverse phase HPLC and ESI-mass spectroscopy. Analysis indicated an overall recovery of 68 % of the protein of which 7 % was covalent dimer yielding a 63 % recovery of monomeric protein and a 32 % loss of protein.
  • Solubilized protein was clarified by
  • the dilute protein solution was allowed to stand without mixing at room temperature for 18 hours.
  • the solution was clarified by centrifugation and analyzed by SDS-PAGE under non-reducing conditions, reverse phase HPLC and ESI-mass spectroscopy. Analysis indicated a recovery of >95 % of the protein of which 16 % was covalent dimer yielding a recovery of monomeric protein of 80 %.
  • Protein of SEQ ID NO: 14 wherein Xaa at position 28 is Gln and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies).
  • the granules were isolated by a standard procedure using differential centrifugation. These granules were solubilized in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine at a protein concentration of 0.1 mg/mL. Renaturation of the protein solution was initiated by dialysis against PBS to remove excess denaturant and cysteine. The solution was clarified by centrifugation and analyzed by SDS-PAGE under non- reducing conditions, reverse phase HPLC and ESI-mass
  • Protein of SEQ ID NO:11 wherein Xaa at position 28 is Gln with a Met-Asp N-terminal extension was produced as granules (inclusion bodies). The granules were isolated by a standard procedure using differential centrifugation.
  • granules constituted the starting material for further purification.
  • the granules were suspended in buffer A (8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine) and found to be soluble in this buffer at high concentrations (up to 40 mg protein/mL). Solubilized protein was clarified either by centrifugation or by filtration. The protein migrated as a doublet band on nonreducing SDS-PAGE gels and as a single band on reducing SDS-PAGE gels. This is due to the presence of some protein with an internal disulfide bond and some protein with the cysteine residues present as mixed
  • the protein was initially purified by DEAE anion exchange chromatography in the presence of buffer A. Protein bound to the DEAE resin was eluted with a NaCl gradient to 0.250 mM.
  • Nonreducing SDS-PAGE analysis of fractions indicated that most of the contaminating proteins were present in the leading edge of the main Ob peak.
  • Conservative pooling of the DEAE fractions resulted in relatively pure Ob protein for renaturation.
  • Refolding of the protein was initiated by dilution of the protein to 0.1 mg/mL in PBS. The example was repeated by dilution into buffer A and removing the denaturant and thiol by dialysis into PBS. The protein remained soluble after the dialysis and migrated as a single band on nonreducing SDS-PAGE. Reduction of the protein resulted in a single band with slightly slower mobility on SDS-PAGE indicating that the disulfide bond was completely formed during the renaturation process.
  • a protein of SEQ ID NO: 12 was prepared in a manner analogous to Example 1.
  • a protein of SEQ ID NO:13 may be prepared in a manner analogous to Example 1.
  • the mixed disulfide between Ob and cysteine is stable even after removal of denaturant.
  • the mixed disulfide Ob protein is soluble in PBS suggesting proper secondary structure formation. Addition of excess thiol regent stimulates disulfide exchange and gradual removal of the thiol regent favors formation of the Ob molecule with the internal disulfide bond.
  • Protein of Formula (III) (SEQ ID NO:1) wherein Xaa at position 28 is Gln and Trp at position 100 is replaced with Glu and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies). The granules were isolated by a standard procedure using differential
  • the inclusion bodies were solubilized in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine; and the mixed disulfide protein was purified by anion exchange chromatography. Purified protein was
  • Renaturation of the solubilized protein was initiated by dilution of the protein to 0.1 mg/mL with 8 M urea, 10 mM Tris at pH 8.0 and dialysis initiated against PBS. After 24 hours, a 1 mL sample was eluted over a C18 HPLC at 1
  • disulfide intermediate of the present invention prior to conversion to the disulfide containing protein.
  • Protein of Formula (III) (SEQ ID NO:1) wherein Xaa at position 28 is Gln and Trp at position 100 is replaced with Gln and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies). The granules were isolated by a standard procedure using differential
  • Renaturation of the protein was initiated by dilution of the protein to 0.1 mg/mL and dialysis against PBS to remove excess denaturant and cysteine.
  • Protein of Formula (III) (SEQ ID NO:1) wherein Xaa at position 28 is Gln and Trp at position 100 is replaced with Ala and having a Met-Arg N-terminus extension was produced as granules (inclusion bodies). The granules were isolated by a standard procedure using differential
  • N-terminal Met-Arg dipeptide is removed by the using dipeptidylaminopeptidase (dDAP) by techniques
  • Example 9 Protein of Formula (III) (SEQ ID NO:1) wherein Xaa at position 28 is Gln and Trp at position 100 is replaced with Ala and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies). The granules were isolated by a standard procedure using differential
  • the urea solubilized mixed disulfide intermediate was purified by anion exchange chromatography on a Big Bead Q- Sepharose column (Pharmacia Fine Chemicals) in 7 M urea, 10 mM Tris, 5 mM cysteine at approximately pH 8. The product is eluted by a linear gradient in NaCl. Additional cysteine is added to the anion exchange purified intermediate, and the pH adjusted to approximately pH 9. The intermediate is then diluted to approximately 2 mg/mL with 7 M urea.
  • the protein is administered in a dose between about 1 and 1000 ⁇ g/kg.
  • a preferred dose is from about 10 to 100 ⁇ g/kg of active compound.
  • a typical daily dose for an adult human is from about 0.5 to 100 mg.
  • compounds of the Formula (I) can be administered in a single daily dose or in multiple doses per day.
  • the treatment regime may require administration over extended periods of time.
  • the amount per administered dose or the total amount administered will be determined by the

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Abstract

L'invention concerne des protéines contre l'obésité, qui régulent le tissu adipeux lorsqu'on les administre à un patient. Elle concerne l'avant-dernier intermédiaire dans la voie de renaturation de la protéine contre l'obésité. Elle concerne, de plus, la préparation dudit intermédiaire, ainsi que son utilisation afin de préparer une protéine biologiquement active contre l'obésité ou un analogue de celle-ci.
EP96921702A 1995-06-22 1996-06-20 Intermediaires de proteines contre l'obesite, leur preparation et leur utilisation Ceased EP0833835A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US45195P 1995-06-22 1995-06-22
US451 1995-06-22
PCT/US1996/010613 WO1997000886A1 (fr) 1995-06-22 1996-06-20 Intermediaires de proteines contre l'obesite, leur preparation et leur utilisation

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EP0833835A1 true EP0833835A1 (fr) 1998-04-08
EP0833835A4 EP0833835A4 (fr) 1999-05-26

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EP (1) EP0833835A4 (fr)
JP (1) JPH11508134A (fr)
AU (1) AU6284896A (fr)
CA (1) CA2224867A1 (fr)
WO (1) WO1997000886A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2217698A1 (fr) * 1996-12-20 1998-06-20 Eli Lilly And Company Proteines contre l'obesite
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
DE10059336A1 (de) 2000-11-29 2002-06-13 Scil Proteins Gmbh Herstellung von rekombinantem BMP-2
EP2219031B1 (fr) 2001-10-22 2013-04-24 Amgen, Inc. Utilisation de Leptine pour traiter la Lipoatrophine humaine et procédé pour déterminer une prédisposition à ce traitement
US20120071401A1 (en) 2009-04-10 2012-03-22 Amylin Pharamaceuticals, Inc. Amylin agonist compounds for estrogen-deficient mammals
CA3138758A1 (fr) 2010-09-28 2012-04-19 Amylin Pharmaceuticals, Llc Leptines hautement solubles

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018227A2 (fr) * 1993-02-04 1994-08-18 Denzyme Aps Procede ameliore de repliement de proteines
WO1996005309A2 (fr) * 1994-08-17 1996-02-22 The Rockefeller University Modulateurs de masse corporelle, proteines et acides nucleiques correspondants, et utilisations a des fins therapeutiques et diagnostiques
WO1996027385A1 (fr) * 1995-03-03 1996-09-12 Eli Lilly And Company Proteines contre l'obesite
WO1996034885A2 (fr) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Fragments peptidiques biologiquement actifs de proteines ob
WO1997006816A1 (fr) * 1995-08-17 1997-02-27 Amgen Inc. Procedes pour diminuer ou maintenir un niveau bas de lipides dans le sang, en utilisant des compositions de proteine ob

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018227A2 (fr) * 1993-02-04 1994-08-18 Denzyme Aps Procede ameliore de repliement de proteines
WO1996005309A2 (fr) * 1994-08-17 1996-02-22 The Rockefeller University Modulateurs de masse corporelle, proteines et acides nucleiques correspondants, et utilisations a des fins therapeutiques et diagnostiques
WO1996027385A1 (fr) * 1995-03-03 1996-09-12 Eli Lilly And Company Proteines contre l'obesite
WO1996034885A2 (fr) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Fragments peptidiques biologiquement actifs de proteines ob
WO1997006816A1 (fr) * 1995-08-17 1997-02-27 Amgen Inc. Procedes pour diminuer ou maintenir un niveau bas de lipides dans le sang, en utilisant des compositions de proteine ob

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MURAKAMI T ET AL: "CLONING OF RAT OBESE CDNA AND ITS EXPRESSION IN OBESE RATS" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 209, no. 3, 26 April 1995, pages 944-952, XP002003621 *
See also references of WO9700886A1 *

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CA2224867A1 (fr) 1997-01-09
WO1997000886A1 (fr) 1997-01-09
EP0833835A4 (fr) 1999-05-26
AU6284896A (en) 1997-01-22

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