WO2005116220A1 - Production de proteines recombinantes au moyen de prosequences heterologues - Google Patents

Production de proteines recombinantes au moyen de prosequences heterologues Download PDF

Info

Publication number
WO2005116220A1
WO2005116220A1 PCT/GB2005/002151 GB2005002151W WO2005116220A1 WO 2005116220 A1 WO2005116220 A1 WO 2005116220A1 GB 2005002151 W GB2005002151 W GB 2005002151W WO 2005116220 A1 WO2005116220 A1 WO 2005116220A1
Authority
WO
WIPO (PCT)
Prior art keywords
prosequence
nucleic acid
heterologous
polypeptide
recombinant polypeptide
Prior art date
Application number
PCT/GB2005/002151
Other languages
English (en)
Inventor
Monique Gangloff
Nicholas Gay
Original Assignee
Cambridge University Technical Services Limited
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 Cambridge University Technical Services Limited filed Critical Cambridge University Technical Services Limited
Publication of WO2005116220A1 publication Critical patent/WO2005116220A1/fr

Links

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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • This invention relates to methods and means for the production of recombinant proteins.
  • human MD-2 which plays a key role in septic shock and inflammation and is an important pharmaceutical target, tends to form inactive disulfide-linked protein aggregates when it is produced in mammalian cells (Re and Strominger (2001) J Biol. Chem. 276(40) 37692-9).
  • the present inventors have discovered that the yields of polypeptide from recombinant expression systems are ' increased if the polypeptide is expressed with a heterologous pro-sequence.
  • One aspect of the invention provides method of producing a recombinant polypeptide comprise; expressing in a host cell a nucleic acid encoding a fusion polypeptide which comprises said recombinant polypeptide and a heterologous prosequence.
  • the heterologous prosequence may be removed from the fusion polypeptide, for example by proteolysis to produce the recombinant polypeptide.
  • a prosequence (often referred to as a propeptide or prodomain) is an amino acid sequence which is present in a newly synthesized polypeptide chain but is not present in the mature polypeptide. After folding, the immature polypeptide chain is subjected to proteolysis to remove the prosequence and generate the mature polypeptide.
  • the term ⁇ prosequence f as used herein does not include the mature coding sequence of the polypeptide from which the prosequence is derived.
  • the prosequence is heterologous to the recombinant polypeptide i.e. is not naturally associated with the recombinant polypeptide which is produced.
  • a prosequence may be unstructured and display little or no secondary or tertiary structure. Unstructured pro-sequences may, for example, be sensitive to proteases, such as trypsin.
  • the prosequence of a polypeptide may be determined using any convenient technique. For example, the prosequence may be determined by comparing the sequences of the immature and mature polypeptide. In other embodiments, the immature polypeptide may be contacted with trypsin protease and the trypsin-sensitive region determined.
  • Suitable prosequences may be found in eukaryotic polypeptides which have a mature form which is generated by proteolysis following expression.
  • a prosequence may, for example include a cytokine prosequence.
  • the prosequence may be a mammalian cytokine prosequence, for example an NGF cytokine prosequence.
  • NGF cytokines include neurotrophin 1 ( ⁇ -NGF), neurotrophin 2 (BDNF) , neurotrophin 3 (NTF3) , neurotrophin 5 (NTF5) , neurotrophin 6 ⁇ (NTF6A) , neurotrophin 6 ⁇ (NTF6B) and neurotrophin 6 ⁇ (NTF6G) .
  • Suitable NGF cytokines are shown in Table 1.
  • the heterologous prosequence may exclude the hNGF- ⁇ (neurotropin 1) preprosequence .
  • the prosequence may be an insect cytokine prosequence, for example a Drosophila cytokine prosequence such as a Spatzle prosequence.
  • Spatzle cytokines include Spatzle 1 (U05850.1, GI: 458315, and figure 2) and Spatzle 2 to 6 as shown in figures 3, 5, 7, 9 and 11 respectively.
  • Spatzle isoforms and variants are shown in Table 3 and described in more detail in Parker et al (2001) PROTEINS: Structure, Function, and Genetics 45:71-80. Examples of prosequences within Spatzle cytokines are shown in table 5.
  • a prosequence suitable for use in accordance with the invention may comprise the prosequence of a eukaryotic polypeptide described herein or may be an isoform, variant, fragment, mutant or allele thereof.
  • Suitable isoforms, mutants, variants, fragments or alleles of a prosequence retain the biological activity of the wild type sequence in promoting folding and expression.
  • a mutant, variant or derivative may have one or more of addition, insertion, deletion or substitution of one or more amino acids in the polypeptide sequence. Such alterations may be caused by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid.
  • a prosequence which is an amino acid sequence variant, allele, or mutant of a wild-type prosequence may comprise an amino acid sequence which shares greater than 20% sequence identity with the wild-type prosequence, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 55%, greater than 65%, greater than 70%, greater than about 80%, greater than 90% or greater than 95%.
  • the sequence may share greater than 20% similarity with the wild-type prosequence, greater than 30% similarity, greater than 40% similarity, greater than 50% similarity, greater than 60% similarity, greater than 70% similarity, greater than 80% similarity or greater than 90% similarity. Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) .
  • Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J. Mol . Biol . 215: 405- 410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol . 147: 195-197), or the TBLASTN program, of Altschul et al.
  • Similarity allows for "conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or ethionine for another, or the substitution of one polar residue for another, such as arginine for lysine, gluta ic for aspartic acid, or glutamine for asparagine .
  • Suitable host cells may include prokaryotic cells, in particular bacteria such as E. coli, and eukaryotic cells, including mammalian cells such as CHO and CHO- derived cell lines (Lee cells) , HeLa, COS, and HEK293 cells, amphibian cells such as Xenopus oocytes, insect cells such as Trichoplusia ni, Sf9 and Sf21 and yeast cells.
  • Recombinant polypeptides suitable for expression using the methods described herein may include any polypeptide of interest.
  • the recombinant polypeptide is a polypeptide which is not naturally expressed with a prosequence i.e. a polypeptide which, in its natural environment, does not require the presence of a prosequence, in order to be expressed and folded into an active form.
  • the present methods are especially suitable for the production of polypeptides which are only produced in host cells at low levels using standard methods. Examples of such polypeptides include glycosylation deficient mutant polypeptides i.e. polypeptides which lack glycosylation sites and thus remain unglycosylated after expression.
  • Preferred recombinant polypeptides may be mammalian, in particular human polypeptides.
  • suitable recombinant polypeptides may include MD-2 polypeptides shown in Table 2.
  • a method of producing an MD-2 polypeptide may comprise; expressing in a host cell a nucleic acid encoding a fusion polypeptide which comprises said MD-2 polypeptide and a heterologous prosequence, and; removing the heterologous prosequence from the fusion polypeptide to produce the MD-2 polypeptide .
  • the MD-2 polypeptide is human MD-2 and the heterologous prosequence is a Spatzle prosequence, for example a Spatzle 1 prosequence.
  • a MD-2 polypeptide may comprise or consist of the sequence of human MD-2 (AAH20690) or another MD-2 polypeptide shown in Table 2 or may be an isoform, variant, fragment, mutant or allele thereof.
  • Suitable isoforms, mutants, variants, fragments or alleles of human MD-2 retain the biological activity of the wild type sequence in binding Toll-like receptor 4 (TLR4) and modulating inflammatory responses.
  • a mutant, variant or derivative may have one or more of addition, insertion, deletion or substitution of one or more amino acids in the polypeptide sequence. Such alterations may be caused by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid.
  • a MD-2 polypeptide e which is an amino acid sequence variant, allele, or mutant of the human MD-2 sequence may comprise an amino acid sequence which shares greater than 30% sequence identity with the human MD-2 sequence, greater than 35%, greater than about 40%, greater than 45%, greater than 55%, greater than 65%, greater than 70%, greater than about 80%, greater than 90% or greater than 95%.
  • the sequence may share greater than 30% similarity with the human MD-2 sequence, greater than 40% similarity, greater than 50% similarity, greater than 60% similarity, greater than 70% similarity, greater than 80% similarity or greater than 90% similarity.
  • the fusion polypeptide may be coupled to an appropriate signal leader peptide to direct secretion of the fusion polypeptide from cell into the culture medium.
  • signal leader peptide may be a sequence which is naturally associated with the prosequence or the recombinant polypeptide or may be heterologous to both prosequence and recombinant polypeptide.
  • the fusion polypeptide may comprise an affinity tag, which may, for example, be useful for purification.
  • An affinity tag is a heterogeneous peptide sequence which forms one member of a specific binding pair. Polypeptides containing the tag may be purified by the binding of the other member of the specific binding pair to the polypeptide, for example in an affinity column.
  • the tag sequence may form an epitope which is bound by an antibody molecule.
  • Suitable affinity tags include for example, glutathione-S- transferase, (GST), maltose binding domain (MBD) , MRGS(H) 6 DYKDDDDK (FLAGTM), T7-, S- (KETAAAKFERQHMDS) , poly-Arg (R 5 _ 6 ) , poly-His (H 2 _ 10 ) , poly-Cys (C 4 ) poly-Phe (Fu) poly-Asp (D 5 - ⁇ 6 ) , Strept-tag II (WSHPQFEK), c-myc (EQKLISEEDL) , Influenza-HA tag (Murray, P. J.
  • a poly-His tag such as MRGS (H) 6 may be used.
  • the method may further comprise isolating and/or purifying the recombinant polypeptide, after production. This may be achieved using any convenient method known in the art.
  • purification may be performed using an affinity tag as described above.
  • an affinity tag may be used to isolate and/or purify the fusion protein prior to removal of the prosequence, or to isolate and/or purify the recombinant polypeptide after removal of the prosequence .
  • the fusion protein encoded by the nucleic acid may comprise one or more protease recognition sites to allow separation of the recombinant polypeptide from the prosequence and/or affinity tag after expression.
  • the protease recognition site may, for example, be positioned between the recombinant polypeptide and the affinity tag or heterologous prosequence such that contacting the fusion protein with a site-specific protease cleaves the fusion protein at the recognition site to release the recombinant protein from the tag or prosequence .
  • Suitable protease recognition sites are known in the art, including Factor Xa, thrombin, rennin, TEV protease and enterokinase recognition sites.
  • the protease recognition site is a tobacco etch virus (TEV) protease recognition site have the sequence Glu-X-X-Tyr-X-Gln/Ser.
  • TSV tobacco etch virus
  • Other suitable protease recognition sites are described in Richter et al J. Biol. Chem. (2002) 277: 43888-43894.
  • the heterologous prosequence may be removed from the fusion polypeptide by contacting the fusion polypeptide with a site- specific protease which cleaves a site located between the prosequence and the recombinant polypeptide.
  • an affinity tag may be removed by contacting the recombinant polypeptide with a site-specific protease which cleaves a site located between the affinity tag and the recombinant polypeptide
  • heterologous sequences may also be removed the fusion polypeptide using site-specific proteases, to generate the recombinant polypeptide .
  • a method of producing a recombinant polypeptide may comprise; expressing in a host cell a nucleic acid encoding a fusion polypeptide which comprises an recombinant polypeptide, a site specific protease recognition site, a heterologous prosequence and an affinity tag, wherein the site-specific protease recognition site is located between the recombinant polypeptide and the heterologous prosequence and affinity tag, purifying the expressed fusion polypeptide using said affinity tag, and; contacting the fusion polypeptide with a site-specific protease to remove the heterologous prosequence and the affinity tag and produce the recombinant polypeptide.
  • the recombinant polypeptide is an MD-2 polypeptide such as human MD-2 and the heterologous prosequence is a Spatzle prosequence as described above.
  • the site-specific protease may, for example, be a TEV protease which cleaves the site-specific protease recognition site Glu-X-X-Tyr-X- Gln/Ser; factor Xa, which cleaves the sequence Ile-Glu-Gly-Arg; thrombin, which cleaves the sequence Gly-Arg; or enterokinase, which cleaves the sequence Asp-Asp-Asp-Asp-Lys .
  • TEV protease which cleaves the site-specific protease recognition site Glu-X-X-Tyr-X- Gln/Ser
  • factor Xa which cleaves the sequence Ile-Glu-Gly-Arg
  • thrombin which cleaves the sequence Gly-Arg
  • enterokinase which cleaves the sequence Asp-Asp-Asp-Asp-Lys .
  • a non-specific protease may be used to remove the prosequence.
  • the prosequence may be sensitive to proteolysis with a serine protease such as trypsin, while the recombinant polypeptide is resistant to such treatment. Proteolysis with the protease therefore degrades the prosequence leaving the intact recombinant polypeptide.
  • a method as described herein may comprise removing the heterologous prosequence from the fusion polypeptide by contacting the fusion polypeptide with a non-specific protease which digests the prosequence to produce the recombinant polypeptide.
  • the fusion polypeptide may be contacted with the protease prior to isolation/purification, for example by adding a non-specific protease such as trypsin to the medium containing the host cells, or after isolation and/or purification of the fusion polypeptide from the host cells.
  • a non-specific protease such as trypsin
  • the recombinant polypeptide may be isolated and/or purified further.
  • Techniques for the purification of recombinant polypeptides are well known in the art and include, for example HPLC, FPLC or affinity chromatography.
  • Recombinant polypeptide produced using the present methods may be investigated further, for example its biochemical properties and/or its activity may be determined. Methods and means of protein analysis are well known in the art.
  • a therapeutic polypeptide may be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) which comprises the recombinant polypeptide, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents .
  • Methods of the invention may therefore comprise the step of formulating the recombinant polypeptide with a pharmaceutically acceptable carrier, adjuvant or excipient.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be . included, as required.
  • Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the recombinant polypeptide with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention further encompasses nucleic acids, vectors and cells suitable for use in methods of producing recombinant polypeptide as described above.
  • a nucleic acid construct may comprise a nucleotide sequence which encodes an heterologous pro-sequence, the nucleotide sequence further comprising one or more restriction endonuclease sites (i.e. a cloning site), which suitable for insertion of a nucleotide coding sequence capable of expressing a recombinant (i.e. a heterologous) polypeptide fused to said heterologous pro-sequence.
  • restriction endonuclease sites i.e. a cloning site
  • the nucleic acid construct may further comprise a nucleotide coding sequence encoding a recombinant polypeptide for expression as part of said, fusion polypeptide, the nucleotide coding sequence being inserted in the cloning site.
  • the invention thus encompasses an isolated nucleic acid comprising a nucleotide sequence which encodes a fusion protein in which a recombinant polypeptide is fused to a heterologous pro-sequence.
  • the recombinant polypeptide may comprise or consist of an MD-2 polypeptide.
  • the nucleic acid construct may thus further comprise a nucleotide coding sequence encoding an MD-2 polypeptide for expression as part of said fusion polypeptide, the nucleotide coding sequence being inserted in the cloning site.
  • the invention thus encompasses an isolated nucleic acid comprising a nucleotide sequence which encodes a fusion protein in which an MD-2 polypeptide is fused to a heterologous pro-sequence.
  • an affinity tag may be conveniently used in the purification of expressed polypeptides.
  • a nucleic acid construct may further comprise a nucleotide coding sequence encoding an affinity tag which allows purification of the fusion protein or recombinant polypeptide.
  • the recombinant polypeptide may be isolated from heterologous peptide sequences such as the affinity tag and the prosequence using site-specific proteases, as described above.
  • a nucleic acid construct may further comprise a nucleotide sequence encoding one or more site specific protease recognition sites (i.e. cleavable linkers) which allow the liberation of the recombinant polypeptide from the fusion polypeptide and/or affinity tag after expression.
  • the recombinant polypeptide may be fused to the heterogologous pro-sequence via a cleavable linker. Suitable linkers may be cleaved by a site-specific endoprotease such as thrombin, factor Xa, TEV protease, enterokinase or rennin
  • the nucleotide sequence encoding the fusion polypeptide may be operably linked to a heterologous regulatory sequence. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well known in the art.
  • a heterologous regulatory sequence may be an inducible promoter.
  • Such a promoter may induce expression in response to a stimulus. This allows control of expression, for example, to allow optimal cell growth before fusion polypeptide production is induced.
  • inducible promoters will be known to those skilled in the art.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on” or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases in the presence of the relevant stimulus by an amount effective to cause production of polypeptide. Thus an inducible (or “switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which causes little or no accumulation of polypeptide. Upon application of the stimulus, which may for example, be an increase in environmental stress, expression of polypeptide is increased (or switched on) .
  • the heterologous regulatory sequence may be activated by a heterologous transcription factor, such as GAL4 or T7 polymerase.
  • a heterologous transcription factor such as GAL4 or T7 polymerase.
  • GAL4 transcription factor may be expressed using a promoter and may drive expression of a fusion polypeptide coding sequence which is operably linked to the GAL4 promoter.
  • T7 polymerase may be expressed using a promoter promoter and may drive expression of a coding sequence which is operably linked to a T7 promoter.
  • heterologous and recombinant are used to indicate that the sequence of nucleotides in question has been introduced into a nucleic acid construct or a host cell using genetic engineering or recombinant means, i.e. by human intervention and is not naturally found in such a construct or cell.
  • a sequence which is heterologous (i.e. exogenous or foreign) to another nucleotide sequence or host cell is not associated with that sequence or cell in nature.
  • Nucleic acid sequences and constructs as described above may be comprised within a vector.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • a vector may comprise a selectable marker to facilitate selection of the transgenes under an appropriate promoter.
  • Methods of the invention may comprise the step of introducing a nucleic acid construct or vector into a host cell.
  • nucleic acid to be inserted should be assembled within a construct or vector which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the constructor vector into the cell.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome- mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage .
  • transformation technology will be determined by its efficiency to transform the particular host cells employed as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into host cells is not essential to or a limitation of the invention.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying cells containing the nucleic acid of interest, as is well known in the art.
  • a method of producing a recombinant polypeptide as described herein may comprise; introducing a nucleic acid encoding a fusion polypeptide comprising a recombinant polypeptide and a heterologous prosequence into a host cell, and; expressing said nucleic acid to produce the fusion polypeptide .
  • heterologous prosequence may be removed from the fusion protein, for example by proteolysis, to produce the recombinant polypeptide.
  • the recombinant polypeptide may be isolated and/or purified from the host cell and the heterologous prosequence.
  • Nucleic acid may be expressed by culturing the host cells (which may include cells actually transformed, although more likely the cells will be descendants of the transformed cells) under conditions for expression of the nucleic acid, so that the encoded polypeptide is produced.
  • a host cell comprising a vector or nucleic acid construct as described herein, and a method of producing a host cell as described herein may comprise introducing a nucleic acid as described herein into the host cell. Suitable host cells are described in more detail above.
  • the nucleic acid, vector or nucleic acid construct may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. In other embodiments, the vector or nucleic acid construct may be extra- chromosomal .
  • a host cell may further comprise a heterologous nucleic acid sequence encoding a site-specific protease and/or a non-specific protease, as described above.
  • the expressed protease may be used to liberate the recombinant polypeptide from the fusion polypeptide in situ within the cell or the cell medium.
  • a host cell may contain a nucleic acid sequence encoding a fusion polypeptide as described herein as a result of the introduction of the nucleic acid sequence into an ancestor cell.
  • Another aspect of the invention provides the use of a nucleic acid, vector or cell as described above in a method of producing a recombinant polypeptide as described herein.
  • Control experiments may be performed as appropriate in the methods described herein.
  • the performance of suitable controls is well within the competence and ability of a skilled person in the field.
  • Figure 1 shows (A) Prosequence fusion proteins.
  • B Prosequence co- expression system.
  • C Table of constructs with genes X for the prosequences used and Y for the genes of interest to be tested in cis and in trans expression experiments.
  • Figures 2 to 12 show the amino acid and encoding nucleic acid sequences of Spatzle 1 to 6 respectively.
  • Figure 2 shows the sequences of the Spzl proteins, HL01462 and LD02813 variants.
  • Figure 3 shows the deduced amino acid sequence for the gene CG18318 (spz2) .
  • the putative signal sequence is underlined.
  • the region predicted to adopt a Spz-like cystine-knot fold is underlined and shown in bold. Nucleotides flanking the four predicted introns are shown in bold.
  • Figure 4 shows Genie/Genscan transcript prediction for the gene CG18318 ( spz2) . Nucleotides flanking the four predicted introns are shown in bold.
  • Figure 5 shows the deduced amino acid sequence of the spz3 EST clone LP11516.
  • the putative signal sequence is underlined.
  • the region predicted to adopt a Spz-like cystine-knot fold is underlined and shown in bold.
  • Figure 6 shows the reconstructed nucleotide of the spz3 EST clone LP11516. The nucleotides that flank the four introns are shown in bold.
  • Figure 7 shows deduced amino acid sequence for the gene CG14928 ( spz4) .
  • the putative signal sequence is underlined.
  • the region predicted to adopt a Spz-like cystine-knot fold is underlined and . shown in bold.
  • Figure 8 shows Genie/Genscan transcript prediction for the gene CG14928 ( spz4) .
  • the nucleotides that flank the two predicted introns are shown in bold.
  • Figure 9 shows the deduced amino acid sequence of the spz5 EST clone LD26258.
  • the putative signal sequence is underlined.
  • the region predicted to adopt a Spz-like cystine- knot fold is underlined and shown in bold.
  • Figure 10 shows the reconstructed nucleotide of the spz ⁇ EST clone LD26258. The nucleotides that flank the five introns are shown in bold.
  • Figure 11 shows the deduced amino acid sequence of a spz ⁇ transcript, derived from the 5' EST sequence of the spz ⁇ cDNA clone CK00311 and the sequences of the spz ⁇ cDNA clones LP10725 and LP10866.
  • the putative signal sequence is underlined.
  • the region predicted to adopt a Spz-like cystine-knot fold is underlined and shown in bold.
  • Figure 12 shows the reconstructed nucleotide sequence of the spz ⁇ transcript of figure 11. The nucleotides that flank the three introns are shown in bold.
  • Figure 13 shows partial sequence alignment of Spzl variants, indicating the extent of the prosequence (the last sequence is Spz3 (AAL33883)).
  • the smallest Spzl prosequence in this list is 56 amino acid (AAF98125) .
  • the longest in this list is 265 amino acid (LD02813) .
  • the prosequence of HL01462 is 163 amino acid and marked by a black arrow.
  • Figure 14 shows cis (a) and trans (b) expression systems for MD-2 protein production.
  • Figure 15 shows the results of an Alkaline phosphatase assay for T.ni cells infected with SEAP and Spatzle HL01462 prosequence-SEAP fusion (HL-SEAP) viruses at different multiplicities of infection.
  • SEAP Spatzle HL01462 prosequence-SEAP fusion
  • Figure 16 shows circular dichroism spectroscopy of the purified Spatzle prosequence.
  • Table 1 shows examples of NGF cytokines.
  • Table 2 shows examples of MD2 polypeptides.
  • Table 3 shows examples of Spatzle cytokines.
  • Table 4 shows pairwise percentage identities for the putative Spz homologues and members of the neurotrophin superfamily of known structure. The table was created using Malform. The identities to Spz ⁇ are for the alignment regions 1 - 64 and 97 - 112. Identities > 29% are shown in bold.
  • Table 5 shows examples of prosequences within Spatzle cytokines.
  • cDNAs were amplified by polymerase chain reaction using the following specific primers .
  • the forward primer was (#6335) 5' CCGGAATTCG CCGCCACCAT
  • the forward primer was (#6325) 5' CCGGAATTCG CCGCCACCAT GTCCATGTTG TTCTAC 3' and the reverse primer was (#6326) 5' CATTTGGTTC ATATGCTGCA GGCGCTTGCT CCTGTGAGT 3' .
  • These forward primers all encode a 5' EcoRI restriction site (GAATTC) followed by a Kozak sequence (GCC GCC ACC) , which immediately precedes the starting codon (AUG) .
  • the 3' primers contains a Pstl and a Ndel restriction sites.
  • the forward primer was (#6705) 5' GTGAGCTCTC TGCAGCATAT GCATCACCAT CACCATCACG ATTACGAAAA CCTGTATTTT CAGGGCGCTA GCGAAGCTCA GAAGCAG 3' and the reverse primer was (#6328) 5' AAGGAAAAAA GCGGCCGCCT AATTTGAATT AGGTTG 3' .
  • the forward primer #6705 contained a Nhel site followed by a sequence encoding a hexahistidine-tag (His-tag) then a TEV cleavage site and a Nhel restriction site in frame with the sequence of mature MD-2.
  • His-tag hexahistidine-tag
  • the forward primer was (#6329) 5' TAGCTAGCTA GCACCACGCC AGAACCTTGT 3' and the reverse primer was (#6330) 5' AAGGAAAAAA GCGGCCGCCT AGTTCATTGA GCCCTCGTG 3' .
  • the prosequences were digested with EcoRI and Ndel, whereas the His-tagged TEV-cleavable MD-2 gene was digested with Ndel and Notl.
  • the pFastBac-1 containing the prosequence of HL01462, the His-tag, the TEV cleavage site and wild type MD-2 was the first construct generated.
  • the pFastBac-1 construct was checked by DNA-sequencing and subsequently used for the cloning of all the other constructs, in which either the prosequence or the gene of interest was changed (Fig.lA).
  • the C-terminal Flag-tagged prosequences for co-expression experiments were generated by PCR with forward primer #6335 and reverse primer ( # 6943 ) 5 ' TCGACAAGCT TGTCGTCATC GTCTTTGTAG TCAGCCTGCA GAGAGCTCAC 3 ' using HL01462 as a DNA template . Digestion was carried out on the PCR product and on pFastBac-1 with EcoRI and Hindl ll
  • the forward PCR primer was (#6331) 5' TAGCTAGCTA GCGAAAGCTG GGAGCCCTGC 3 ' and the reverse primer was ( # 6332 ) 5 ' CATTTGGTTC ATATGCTGCA GAGAGCTCAC ATCCGTGGG 3 ' .
  • the forward PCR primer was ( # 6329 ) 5 ' TAGCTAGCTA GCACCACGCC AGAACCTTGT 3 ' and the reverse primer was ( # 6330 ) 5' AAGGAAAAAA GCGGCCGCTC AGTTCATTGA GCCCTCGTG 3' .
  • the forward PCR primer was ( #8797 ) 5' TAGCTAGCTA GCATCATCCC AGTTGAGGAG GAG 3 ' and the reverse primer (#8798 ) 5 ' AAGGAAAAAA GCGGCCGCCT ATGTCTGCTC GAAGCGGCCG GC 3 ' .
  • Forward primers contain a Nhel cleavage site and reverse primers a Notl site, that allows cloning in the corresponding sites of the pFastBac-1 containing the prosequence of HL01462 , the His-tag, the TEV cleavage site (as described for MD-2 ) .
  • the Bac-to-BacTM system (InvitrogenTM) was used following the manufacturer's instruction. Briefly, the recombinant pFast-Bacl transfer plasmid was introduced into DHlOBac E. coli cells, which allow recombination into the baculovirus DNA (bacmid) . The recombinant bacmid was extracted from the bacterial strain and the recombination event was checked by polymerase chain reaction using the M13 primers. Spodoptera frugiperda (Sf9) cells were transfected with recombinant bacmid using CellfectinTM (InvitrogenTM) .
  • Sf9 Spodoptera frugiperda
  • His-tagged protein was purified on Ni-NTA resin in native conditions following the manufacturer's instructions (Qiagen).
  • Recombinant His-tagged proteins were produced in the baculovirus system and purified using Ni-NTA affinity chromatography.
  • constructs were subcloned into pFastBacl (Invitrogen) and sequenced.
  • Recombinant baculoviruses were generated using the Bac-to-Bac system (Invitrogen) .
  • Viruses were amplified by infection of Sf9 suspension cultures with 2% fetal bovine serum and protein expression in the supernatant was confirmed by immunoblot using an anti-His antibody (Amersham) .
  • T.ni cells were infected at different MOIs and harvested 1, 2, 3 and 4 days post infection.
  • protein containing supernatants were concentrated on a Centramate tangential flow system (Pall Filtron) with buffer exchange to binding buffer (150 mM NaCl, 20 mM TrisHCl pH 7.0, 20mM imidazole) . His-tagged proteins were purified on Qiagen Superflow NiNTA agarose and gel filtration on Superdex 75 or 200 columns (Pharmacia) .
  • the products of digestion were purified by cobalt affinity chromatography on a 5ml HiTrap Chelating column prepared following the manufacturer's instructions (Pharmacia) and equilibrated in 150mM NaCl, 20mM Tris- HCl pH7.0, 20mM imidazole.
  • the flow-through contained MD-2 and the His-tagged prosequence was eluted from the column in a linear imidazole gradient (from 20mM to 500mM imidazole) . Both were further purified by gel-filtration on Superdex 75 (Pharmacia) in lOOmM NaCl, 20mM sodium acetate pH 5.0. Only monomeric samples were used in biophysical and functional studies.
  • Vivaspin-20 (Sartorius) .
  • the sample was diluted to 0.3mg/ml in distilled water.
  • the CD spectrum was recorded at 293 K in the range of 190-250 nm using a JASCO J-810 spectropolarimeter in continuous scanning mode.
  • the acquisition parameters were 50 nm/min with a 4 s response and a lnm band width. Results of a buffer control were subtracted.
  • HEK293 cells Mammalian protein production HEK293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 200 U/ml penicillin, and 100 ⁇ g/ml streptomycin. Expression constructs were transfected into HEK293 cells using Lipofectamine 2000 transfection reagents (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293 cells were seeded at 4 x 10 4 cells per well in a 6-well plate.
  • HEK293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 200 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • Expression constructs were transfected into HEK293 cells using Polyfect transfection reagents (Qiagen) according to the manufacturer' s instructions .
  • HEK293 cells were seeded at 4 * 10 4 cells per well in a 96-well plate. After 48 hours incubation at 37°C in 5% C02, cells were transfected with 0.5 ng/well of pcDNA3-TLR4, 0.5 ng/well wild type pEFIRES-MD-2 or pEFIRES-Spatzle HL01462, LD02813 or rat NGF prosequence-MD-2 fusion constructs, 5ng/well of pLuc, the NF-KB-luciferase reporter plasmid and pRL-TK encoding the constitutively active renilla luciferase, in presence and absence of 0.5 ng/well pcDNA3-CD14.
  • HEK293 cells were treated as before without MD-2 encoding vectors. They were incubated instead for 6h with 10 ng/ml LPS and 2 ⁇ l of serial dilutions of purified monomeric MD-2. NF-KB activities were measured in triplicate by using the Promega Dual Luciferase Reporter assay system.
  • SEAP Secreted alkaline phosphatase
  • T.ni and Sf9 cells maintained in SF900-SFM were infected with SEAP and Spatzle HL01462 prosequence-SEAP fusion viruses at multiplicity of infection (MOI) of 0.1, 1.0 and 10.
  • MOI multiplicity of infection
  • the fusion protein was observed to migrate further compared to the sample in reducing conditions, providing indication of the presence of intra-molecular disulfide bridges.
  • the prosequence does not enhance secretion when expressed in trans
  • human MD-2 was produced both in cis and in trans with Spatzle prosequence, as shown in Figure 14 a and b, respectively.
  • Both Sf9 and T.ni cells were infected by increasing amounts of recombinant baculoviruses . Proteins from cell culture supernatant were separated by 12% SDS-PAGE and analyzed by anti-His and anti-FLAG Western blotting.
  • SEAP is secreted in presence of the prosequence, which modulates its level of production
  • SEAP was produced in absence and presence of the Spatzle HL01462 prosequence fusion. Both Sf9 and T.ni cells were infected by increasing multiplicities of infection (MOIs of 0.1, 1 and 10) of recombinant baculovirus. Proteins from cell culture supernatant were separated by 12% SDS-PAGE and analyzed by anti-His Western blot 2 days post infection. Optimal secretion was observed in Sf9 cells, at a MOI of 10. Wild type SEAP was highly expressed in Sf9 cells (optimal production estimated at ⁇ 1.7 mg/L) , but not in T.ni cells (optimal production estimated at ⁇ 0.05 mg/L).
  • HL-SEAP Spatzle HL01462 prosequence-SEAP fusion
  • SEAP was found to be secreted and active when fused to the Spatzle HL01462 prosequence, as shown by alkaline phosphatase assay (figure 15) .
  • the prosequence enhances secretion of human MD-2
  • Human MD-2 was not secreted efficiently in a baculovirus expression system using the endogenous MD-2 signal as the secretion signal. By fusing mature human MD-2 to the Drosophila Spatzle prosequence, secretion was observed to increase by at least 10-fold. Both Sf9 and T.ni cells were infected by increasing amounts of recombinant baculoviruses. Proteins from cell culture supernatant were separated by 12% SDS-PAGE and analyzed by anti-His Western blot over a period of 3 days post infection. The time course of expression showed that optimal secretion of the protein was obtained in Sf9 cells, at a multiplicity of infection of 10 when culture medium is harvested 2 days post infection. Typical yield after purification is about 2.5mg per litre of cell culture.
  • HEK cells were transiently transfected with wild type MD-2 or with the different prosequence-MD-2 fusions (HL01462, LD02813 and rat nerve growth factor) . Proteins from culture supernatant were separated on non- reducing 12% SDS-PAGE, immobilized on nitrocellulose and blotted using anti-His antibody.
  • the spatzle HL01462 prosequence was found to increase production levels of MD-2, compared to wild-type and mammalian NGF, but did not prevent aggregation of pro-MD-2.
  • Prosequences from two Spatzle isoforms (HL01462 and LD02813 referred to as HL and LD, respectively) and from rat NGF cytokine were assessed for their ability to promote secretion of MD-2 in a baculovirus expression system using two different insect cell lines,
  • the prosequence is unordered
  • Circular dichroism experiment was carried out on purified Spatzle prosequence.
  • the CD spectrum was recorded at 293 K in the range of 190-250 nm using a JASCO J-810 spectropolarimeter in continuous scanning mode.
  • the acquisition parameters were 50 nm/min with a 4 s response and a lnm band width and the results shown in figure 16.
  • the CD spectrum shown in Figure 16 is typical for a random coil.
  • the prosequence -although unordered- is stable in solution and monomeric, as indicated by size-exclusion chromatography.
  • the methods described herein generate high levels of recombinant polypeptides using the prosequence of unrelated proteins such as the one from the Drosophila Spatzle isoforms or mammalian nerve-growth factor beta (NGFb) either in fusion proteins or in co-expression experiments .
  • the prosequence may work as protein chaperone, helping proper folding and subsequent secretion of proteins.
  • human MD-2 does not possess an endogenous prosequence, fusing MD-2 to the Drosophila Spatzle prosequence is shown herein to improved significantly the secretion yield.
  • AAF98132 AAF98132.1 isoform 8.29 [Drosophila melanogaster] spatzle alternatively spliced
  • AAF98131 AAF98131.1 isoform 8.24 [Drosophila melanogaster] spatzle alternatively spliced
  • AAF98129 AAIT98129.1 isoform 8.20 [Drosophila melanogaster] spatzle alternatively spliced
  • AAF98124 AAF98124.1 isoform 11.32 [Drosophila melanogaster] spatzle alternatively spliced
  • AAF98123 AAF98123.1 isoform 11.27 [Drosophila melanogaster]
  • AAQ6 944 AAQ64944.1 spz [Drosophila melanogaster] AAQ64943 AAQ64943.1 spz [Drosophila melanogaster]
  • AAQ64941 AAQ64941.1 spz [Drosophila melanogaster]
  • AAQ6 940 AAQ64940.1 spz [Drosophila melanogaster]

Abstract

L'invention concerne des procédés et des moyens de production de protéines recombinantes, ledit procédé comprenant l'expression, dans une cellule hôte, d'un acide nucléique codant un polypeptide de fusion comprenant ledit polypeptide recombinant et une proséquence hétérologue, puis élimination de la proséquence pour produire le polypeptide recombinant. Des proséquences appropriées comprennent des proséquences de cytokine, par exemple, des proséquences Spatzle de la drosophile.
PCT/GB2005/002151 2004-05-28 2005-05-31 Production de proteines recombinantes au moyen de prosequences heterologues WO2005116220A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0412186A GB0412186D0 (en) 2004-05-28 2004-05-28 Production of recombinant protein
GB0412186.9 2004-05-28

Publications (1)

Publication Number Publication Date
WO2005116220A1 true WO2005116220A1 (fr) 2005-12-08

Family

ID=32671395

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2005/002151 WO2005116220A1 (fr) 2004-05-28 2005-05-31 Production de proteines recombinantes au moyen de prosequences heterologues

Country Status (2)

Country Link
GB (1) GB0412186D0 (fr)
WO (1) WO2005116220A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015091144A1 (fr) * 2013-12-20 2015-06-25 F. Hoffmann-La Roche Ag Procédés de production améliorés de polypeptides recombinés
US11584793B2 (en) 2015-06-24 2023-02-21 Hoffmann-La Roche Inc. Anti-transferrin receptor antibodies with tailored affinity
US11603411B2 (en) 2015-10-02 2023-03-14 Hoffmann-La Roche Inc. Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use
CN116179608A (zh) * 2023-01-05 2023-05-30 昆明理工大学 一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用
US11787868B2 (en) 2015-10-02 2023-10-17 Hoffmann-La Roche Inc. Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001817A2 (fr) * 1998-07-06 2000-01-13 Schering Corporation Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000001817A2 (fr) * 1998-07-06 2000-01-13 Schering Corporation Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FORD C F ET AL: "FUSION TAILS FOR THE RECOVERY AND PURIFICATION OF RECOMBINANT PROTEINS", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, US, vol. 2, 1991, pages 95 - 107, XP000979744, ISSN: 1046-5928 *
GIOANNINI T L ET AL: "Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 101, no. 12, 23 March 2004 (2004-03-23), pages 4186 - 4191, XP002323010, ISSN: 0027-8424 *
KÜHNEL BLANKA ET AL: "Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases.", PROTEIN ENGINEERING. OCT 2003, vol. 16, no. 10, October 2003 (2003-10-01), pages 777 - 783, XP002345018, ISSN: 0269-2139 *
MUROI M ET AL: "MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions of Salmonella Lipid A", INFECTION AND IMMUNITY, vol. 70, no. 7, July 2002 (2002-07-01), pages 3546 - 3550, XP002345196 *
VIRIYAKOSOL S ET AL: "MD-2 binds to bacterial lipopolysaccharide", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 276, no. 41, 12 October 2001 (2001-10-12), pages 38044 - 38051, XP002323009, ISSN: 0021-9258 *
WEBER ALEXANDER N R ET AL: "Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling.", NATURE IMMUNOLOGY. AUG 2003, vol. 4, no. 8, August 2003 (2003-08-01), pages 794 - 800, XP002345019, ISSN: 1529-2908 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015091144A1 (fr) * 2013-12-20 2015-06-25 F. Hoffmann-La Roche Ag Procédés de production améliorés de polypeptides recombinés
US10370692B2 (en) 2013-12-20 2019-08-06 Hoffmann-La Roche Inc. Recombinant polypeptide production methods
US11584793B2 (en) 2015-06-24 2023-02-21 Hoffmann-La Roche Inc. Anti-transferrin receptor antibodies with tailored affinity
US11603411B2 (en) 2015-10-02 2023-03-14 Hoffmann-La Roche Inc. Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use
US11787868B2 (en) 2015-10-02 2023-10-17 Hoffmann-La Roche Inc. Bispecific anti-human A-beta/human transferrin receptor antibodies and methods of use
CN116179608A (zh) * 2023-01-05 2023-05-30 昆明理工大学 一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用
CN116179608B (zh) * 2023-01-05 2024-03-01 昆明理工大学 一种dsRNA在免疫缺陷中华蜜蜂模型构建中的应用

Also Published As

Publication number Publication date
GB0412186D0 (en) 2004-06-30

Similar Documents

Publication Publication Date Title
Nilsen et al. Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity
Valenzuela et al. The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases
KR101304958B1 (ko) 트립신 변이체에 의한 인슐린 전구체의 절단
Bányai et al. Amoebapore homologs of Caenorhabditis elegans
JP2004522424A (ja) アポリポタンパク質類似体
US7442526B2 (en) Method for preparing a physiologically active IL-18 polypeptide
JP2009502125A (ja) 高レベルでの、大腸菌におけるリソスタフィンの分泌発現の方法
WO2005116220A1 (fr) Production de proteines recombinantes au moyen de prosequences heterologues
Schád et al. A novel human small subunit of calpains
JP2953477B2 (ja) クロストリジウム・ヒストリティカムからの組換えプロティナーゼ並びに細胞及び細胞群の単離のためのその使用
KR101695684B1 (ko) 신규한 자가저해 단백질 융합체 및 이의 용도
Austin et al. The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags
He et al. High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells
US20020164762A1 (en) Modified interleukin-1beta converting enzyme with increased stability
Rasulova et al. The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity
Servos et al. Catalytically active soluble ecto‐5′‐nucleotidase purified after heterologous expression as a tool for drug screening
Masumoto et al. Overexpression, purification, and characterization of human m-calpain and its active site mutant, m-C105S-calpain, using a baculovirus expression system
KR20200124255A (ko) 돼지 트립신의 변이체
US20150141617A1 (en) Production and purification of active eukaryotic formylglycinegenerating enzyme (fge) variants
EP0759079A1 (fr) ADN CODANT UN PRECURSEUR DE LA CYSTEINE PROTEINASE III APPARENTEE A L'ENZYME DE CONVERSION DE L'INTERLEUKINE-1$g(b) (ICE rel?-III)
Fisher et al. Post‐translational processing and Thr‐206 are required for glycosylasparaginase activity
US7084246B2 (en) Epidermal growth factor agonists
US7662935B2 (en) Processing of peptides and proteins
Thomas et al. Isolation of juvenile hormone esterase and its partial cDNA clone from the beetle, Tenebrio molitor
Hyoung Lee et al. Enhanced production and secretion of streptokinase into extracellular medium in Escherichia coli by removal of 13 N-terminal amino acids

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase