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Modified i(E.coli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein

A heterologous protein, E. coli technology, applied in microorganisms, animal/human proteins, microorganism-based methods, etc., can solve problems such as low hGH production

Inactive Publication Date: 2001-10-17
HANMI SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method yields low hGH and requires the use of the expensive expression inducer IPTG

Method used

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  • Modified i(E.coli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein
  • Modified i(E.coli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein
  • Modified i(E.coli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein

Examples

Experimental program
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Effect test

Embodiment 1

[0064] The following examples are intended to further illustrate the invention without limiting its scope. Preliminary Example 1: Screening of human growth hormone cDNA gene

[0065] (Step 1) Construction of human pituitary cDNA library

[0066] To 1 g of human pituitary gland was added 10 ml of guanidine solution (4M guanidine isothiocyanate, 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, and 5% 2-mercaptoethanol) and homogenized. The homogenate was centrifuged at 10000 rpm for 10 minutes at 6°C. 1 / 10 volume of 2% sarkosyl ether (Sigma, USA) was added to the supernatant, and the mixture was kept at 65°C for 2 minutes. Cesium chloride was added to the resulting solution to a final concentration of 0.1 g / ml, and the mixture was centrifuged at 25,000 rpm for 16 hours on 9 ml of a cushion solution (5.7M CsCl and 0.1 mM EDTA) to obtain an RNA pellet. The precipitate was dissolved in 3ml of suspension (5mM EDTA, 0.5% sarkosyl and 5% mercaptoethanol), and then washed successively with pheno...

Embodiment 2

[0077] In order to confirm that the clone contains the human growth hormone gene, the cloned phage DNA was cut with EcoRI, and then the DNA fragment was subjected to Southern blot using a mixed sequence oligonucleotide probe (Southern, E., Journal of Molecular Biology 98:503 (1975 )). In addition, a 0.65 kb EcoRI fragment containing human growth hormone gene was inserted into the EcoRI site of M13mp18 vector (Pharmacia, USA) to obtain vector M13-hGH. The nucleotide sequence of the human growth hormone gene in the vector M13-hGH was determined by the dideoxy-mediated chain termination method (Sanger, F. et al., Proc. Acad. Sci. USA 74:5463-5467 (1977)). Preliminary Example 2: Preparation of Gene Encoding Mature Human Growth Hormone

[0078] To prepare a cDNA gene encoding mature human growth hormone, the vector M13-hGH obtained in Step 2 of Preliminary Example 1 was subjected to PCR using the following primers S1 and AS1. The sense primer S1 was designed to provide an NdeI re...

Embodiment 3

[0084] figure 1 The procedure for constructing the vector pT-hGH above is shown. Preliminary Example 3: Construction of a Vector Containing a Gene Encoding Escherichia coli Endotoxin II Signal Peptide / hGH Fusion Protein

[0085] (Step 1) Cloning the Escherichia coli endotoxin II signal peptide gene

[0086] In order to prepare the Escherichia coli endotoxin II signal peptide gene, design the following pair of complementary oligonucleotides based on the nucleotide sequence of the Escherichia coli endotoxin II signal peptide, and use a DNA synthesizer (type 380B, Applied Biosystem, USA) synthesis.

[0087] Sense strand oligonucleotide STⅡS1 (SEQ ID NO:8) 5'-TCATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTCTATTGCTACAAATGCCTACGCGT-3'

[0088] Antisense strand oligonucleotide STⅡ AS1 (SEQ ID NO:9)

[0089] 5'-ACGCGTAGGCATTTGTAGCAATAGAAAAAACGAACATAGATGCAAGAAGAAATGCGATATTTCTTTTTCATGA-3'

[0090] The oligonucleotide was designed to have NcoI and BspHI restriction sites upstream of...

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Abstract

A heterologous protein is produced by: (i) culturing a microorganism transformed with an expression vector comprising a gene encoding a modified E. coli enterotoxin II signal peptide fused with the heterologous protein to produce and secrete the heterologous protein to periplasm, the modified E. coli enterotoxin II signal peptide being obtained by replacing at least one of the 2nd, 4th, 5th, 12th, 20th, and 22nd amino acids of E. coli enterotoxin II signal peptide of the following amino acid sequence (SEQ ID NO: 1) with another amino acid, with the proviso that at least one of the 2nd and 4th amino acid of the modified peptide is lysine; and (ii) recovering the heterologous protein from the periplasm.

Description

field of invention [0001] The present invention relates to a modified Escherichia coli endotoxin II signal peptide, a gene encoding the peptide, a vector comprising the gene fused together with a gene encoding a heterologous protein, a microorganism transformed with the vector, and the preparation of The method of said heterologous protein. Background of the invention [0002] Many heterologous proteins have been produced by intracellular or secretory methods using genetically engineered host microorganisms. [0003] In the intracellular approach, genes encoding heterologous proteins are expressed and accumulated in the cytoplasm of the microorganism. Although this method is known to yield higher yields of heterologous protein, the expressed heterologous protein is not in the native active form but is amino-terminally methioninated. In addition, biologically inactive heterologous proteins prepared by this method often form insoluble inclusion bodies, which must be solubili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/245C07K14/61C07K19/00C12N1/21C12N15/31C12N15/62C12N15/70C12P21/02C12R1/19
CPCC12N15/625C07K2319/02C12N15/70C07K2319/75C07K14/61C07K14/245C07K2319/034Y02A50/30C12N15/11C12N15/62
Inventor 权世昌郑圣烨申勋崔宰道崔基斗李宽淳
Owner HANMI SCI CO LTD
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