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

A technology of Escherichia coli and endotoxin, applied in the direction of microorganisms, animal/human proteins, microorganism-based methods, etc., can solve the problem of low production of hGH

Inactive Publication Date: 2004-04-07
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 CE.(oli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein
  • Modified i CE.(oli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein
  • Modified i CE.(oli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 to 10

[0117]Examples 1 to 10 describe the construction of a vector containing a gene encoding an MST / hGH fusion protein according to the present invention, wherein MST represents a modified E. coli endotoxin II signal peptide. The STII gene or STII SD sequence in the plasmid pT14SSH obtained in Step 3 of Preliminary Example 3 was modified according to site-directed mutagenesis (Papworth C. et al., Strategy 9:3 (1996)) by using a PCR was carried out with sense primers of the nucleotide sequence and antisense primers with nucleotide sequences complementary to the sense primers.

Embodiment 1

[0120] Example 1: Construction of a vector containing the gene encoding the MST1 / hGH fusion protein

[0121] (step 1)

[0122] The vector pT14SSH obtained in Preliminary Example 3 was subjected to PCR using the following complementary primers S4 and AS4 designed to replace the fourth codon (ATT) of STII with the Thr codon (ACA).

[0123] Sense primer S4 (SEQ ID NO: 23)

[0124] 5'-GGTGTTTTATGAAAAAGACAATCGCATTTCTTC-3'

[0125] Antisense primer AS4 (SEQ ID NO: 24)

[0126] 5'-GAAGAAATGCGATTGTCTTTTTCATAAAACACC-3'

[0127] The vector thus obtained was cut with XbaI and MluI to obtain a 0.1 kb XbaI / MluI fragment, which was inserted into the XbaI / MluI portion of vector pT14SSH to obtain vector pT14SSH-4T. The vector pT14SSH-4T contains a gene encoding a modified STII / hGH fusion protein in which the fourth amino acid of STII is replaced by Thr.

[0128] (step 2)

[0129] PCR was performed on the vector pT14SSH-4T using the following complementary primers S5 and AS5, which were ...

Embodiment 2

[0144] Example 2: Construction of a vector containing the gene encoding the MST2 / hGH fusion protein

[0145] The process of step 2 in Example 1 was repeated except that the following complementary primers S7 and AS7 were used, which were designed to replace the 20th and 22nd codons of STII (AAT and TAT) with Val and Gln codons (GTT and CAA), respectively. ) to obtain the vector pT14SSH-4T20V22Q.

[0146] Sense primer S7 (SEQ ID NO: 29)

[0147] 5'-GTTTTTCTATTGCTACAGTTGCCCAAGCGTTCCAACCATTCCC-3'

[0148] Antisense primer AS7 (SEQ ID NO: 30)

[0149] 5'-GGGAATGGTTGGGAACGCTTGGGCAACTGTAGCAATAGAAAAAAC-3'

[0150] Then, the process of step 3 in the example was repeated, except that the vector pT14SSH-4T20V22Q was used to obtain the vector pT14S1SH-4T20V22Q. The vector pT14S1SH-4T20V22Q contains the modified STII SD sequence and a gene encoding MST1 / hGH fusion protein, the 4th, 20th and 22nd amino acids of STII of the protein are replaced by Thr, Val and Gln respectively.

[0151...

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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 Patents(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