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Growth hormone and serum albumni recombinant fusion protein

A serum albumin, growth hormone technology, applied in the direction of serum albumin, growth hormone, albumin peptide, etc., can solve the problem of inactivity

Inactive Publication Date: 2004-07-28
NOVOZYMES BIOPHARMA DK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Activity assays show that the conjugate maintains full activity in vitro, possibly even increased activity, but is inactive in vivo

Method used

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  • Growth hormone and serum albumni recombinant fusion protein
  • Growth hormone and serum albumni recombinant fusion protein
  • Growth hormone and serum albumni recombinant fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Cloning of hGH cDNA.

[0088] The cDNA of hGH was obtained from the human pituitary gland cDNA library by the method of PCR amplification (catalogue number is HL1097V, (Clontech Laboratories, Inc). Two oligonucleotide primers suitable for hGH cDNA amplification: HGH1 and HGH2 use Biological system 380B (Applied Biosystems380B) oligonucleotide synthesizer synthesis.

[0089] HGH1: 5′-CCCAAGAATTCCCTTATCCAGGC-3′

[0090] HGH2: 5′-GGGAAGCTTAGAAGCCACAGGATCCCTCCACAG-3′

[0091] HGH1 and HGH2 differ from the corresponding part of the hGH cDNA sequence by two and three nucleotides, respectively (Fig. 1, Martial et al., 1979), thus, after PCR amplification, an EcoRI site was introduced at the 5'-end of the cDNA , a BamHI site was introduced at the 3′-end. In addition, there is a Hind III site in HGH2 immediately downstream of the hGH sequence.

[0092] Carrying out PCR amplification with Perkin-ELmer-Cetus Thermal Cycler 9600 amplification instrument and Perkin-El...

Embodiment 2

[0094] Example 2: Expression of hGH cDNA

[0095] The polylinker sequence of the phagemid pBluescribe(+) (Stratagene) was replaced by an oligonucleotide linker formed by annealing two 75-mer oligonucleotides between the EcoRI and Hind III sites to form pBST ( +) (Figure 3). The new polylinker contains a unique NotI site (see Figure 3 for the full sequence of the polylinker region).

[0096] The NotI HSA expression cassette of pAYE 309 (EP 431 880) containing the PRB1 promoter, DNA encoding the HSA / MFα-1 hybrid leader sequence, DNA encoding HSA, and the ADH1 terminator was transformed into pBST(+) to form pHA1 (Figure 4). The coding sequence of HSA was removed from the plasmid by digestion with Hind III, and re-ligated to form pHA2 (Fig. 4).

[0097] Cloning of the hGH cDNA described in Example 1 provided the hGH coding region lacking the original hGH sequence and the first 8 base pairs (bp) of the mature hGH sequence. To construct an expression plasmid that secretes hGH in...

Embodiment 3

[0103] Embodiment 3: Cloning and expression of HSA-hGH fusion protein

[0104] To fuse HSA cDNA to the 5'-end of hGH cDNA, the pHA1 Hind III-Bsu 36I fragment (containing most of the HSA cDNA) and the EcoR1-Hind III fragment of pHGH1 were ligated by two oligonucleotides HGH7 and HGH8:

[0105] HGH7: 5′-TTAGGCTTATTCCCAAC-3′

[0106] HGH8: 5′-AATTGTTGGGAATAAGCC-3′

[0107] The Hind III fragment thus formed was cloned into pHA2 digested with Hind III to form pHGH10 (Fig. 5), and the expression cassette of this plasmid NotI was cloned into pSAC35 digested with Notl to form pHGH16 (Fig. 5).

[0108] pHGH16 was used for transformation into Saccharomyces cerevisiae DB1, and the culture supernatant was analyzed by the method in Example 2. A main band with a molecular weight of about 88KD was observed, corresponding to the combined size of HA and hGH. Western blotting with anti-HSA and anti-hGH antisera (Sigma) confirmed the presence of both components in the fusion protein.

[0109...

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Abstract

The fusion protein may be expressed from a polynucleotide having a promoter element and / or a transcription termination sequence, in each case operatively associated with a polynucleotide encoding the fusion protein. The yeast may be S. cerevisiae, S. pombe, Pichia (Hansenula) sp. or Kluyveromyces sp.

Description

[0001] The application of the present invention is a divisional application of the invention application with the application number 96199466.5, the application date is 96.12.19, and the invention name is "recombinant fusion protein of growth hormone and serum albumin". field of invention [0002] The present invention relates to recombinant fusion proteins, growth hormone (GH), serum albumin and protein products in yeast. Background technique [0003] Human serum albumin (HSA) is a protein of 585 amino acids that is an important component of osmolarity in serum and acts as a carrier of endogenous and exogenous ligands. Currently, HSA used clinically is extracted from human plasma. The production of recombinant HA (rHA) in microorganisms has been disclosed in EP 330 451 and EP 361 991 . [0004] The role of albumin as a carrier molecule and its inert nature are essential features for its use as a stabilizer and carrier f...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K31/00A61K38/00A61K38/27A61P5/00A61P5/06A61P43/00C07K14/61C07K14/765C07K19/00C12N1/19C12P21/02C12R1/865
CPCC07K2319/00C07K14/61A61K38/27C07K14/765A61P43/00A61P5/00A61P5/06C12N15/62
Inventor 戴维·J·巴兰斯
Owner NOVOZYMES BIOPHARMA DK AS
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