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Process for preparing serine-rich protein employing cysteine synthase (cysk) gene

A cysteine ​​and serine technology, which is applied in the field of protein preparation, can solve the problems of a lot of time, no results, and difficulty in improving the yield of heterologous proteins.

Inactive Publication Date: 2004-08-18
KOREA ADVANCED INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is still a problem that it is difficult to increase the yield of heterologous protein preparation when using the current conventional method, because a large amount of time is required after induction of expression
Many efforts have been made to overcome this problem, but no satisfactory results have been reported

Method used

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  • Process for preparing serine-rich protein employing cysteine synthase (cysk) gene
  • Process for preparing serine-rich protein employing cysteine synthase (cysk) gene
  • Process for preparing serine-rich protein employing cysteine synthase (cysk) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Measuring Physiological Changes of Obesin-Producing Bacteria Using Two-dimensional Electrophoresis

[0034] According to a known method, changes in protein levels before and after overproduction of human obesin in E. coli BL21(DE3)(pEDOb5) were compared using two-dimensional electrophoresis (Hochstrasser et al., Anal. Biochem., 173: 424- 5, 1988; Han et al., J.Bacteriol., 183:301-8, 2001): namely, after E.coliBL21(DE3)(pEDOb5) was pre-cultured, the expression of obesin was induced and cultured at a high concentration . Remove the culture broth before and after induction of expression. Each culture broth was centrifuged at 6,000 rpm for 5 minutes at 4°C, and the precipitate was washed with 500 μl of low-salt buffer (KCl 3mM, KH 2 PO 4 1.5mM, NaCl 68mM, NaH 2 PO 4 9mM) wash. Then, the product was suspended in 200 µl of TE buffer (Tris-HCl 10 mM, EDTA 1 mM). The suspension was sonicated with a sonicator and centrifuged at 12000 rpm for 10 min at 4°C. T...

Embodiment 2

[0038] Embodiment 2: the preparation of the recombinant plasmid that introduces cysK gene

[0039] The preparation of the recombinant plasmid pAC104CysK expressing CysK protein is as follows: First, the E.coliBL21 (DE3) chromosome is used as a template for polymerase chain reaction (PCR), primer 1:

[0040] 5'-gc gaattc atgagtaagatttttgaagataa-3' (SEQ ID NO: 1) and primers

[0041] 2: 5'-gc gaattc tatatactgttgcaattctttctc-3' (SEQ ID NO: 2). At 95°C, perform the first denaturation for 5 minutes; the second denaturation at 95°C for 50 seconds, anneal at 55°C for 1 minute, and extend at 72°C for 1 minute and 30 seconds, repeating 30 cycles; finally at 72°C °C extension for 5 minutes. In this way, the cysK gene digested by the restriction endonuclease EcoRI was obtained, and the obtained fragment was inserted into the plasmid p10499A (Park et al., FEMS Microbiol. Lett., 214:217-22, 2002) having the gntT104 promoter , which was digested with the same restriction enzymes to f...

Embodiment 3

[0042] Example 3: Preparation of recombinant plasmids introducing IL-12p40 gene

[0043] The recombinant plasmid pEDIL-12p40 was prepared as follows to express IL-12p40 (interleukin 12β chain) protein. Use plasmid pUC18 / p40 containing human interleukin β chain gene as template, and primer 3: 5'-ggctagc attaat gatatgggaactgaagaaagat-3' (SEQ ID NO: 3) and primer 4: 5' gcc ggatcc ttattaactgcagggcacaga-3' (SEQ ID NO: 4) was subjected to PCR by the same method as in Example 2 to obtain IL-12p40 gene. The gene was digested with restriction enzymes AdeI and BamHI. The obtained fragment was inserted into the obesin expression vector (Jeong and Lee, Appl. Environ. Microbiol., 65:3027-32, 1999) to constitute pEDIL-12p40, wherein the vector had been digested with restriction enzymes NdeI and BamHI ( image 3 ).

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Abstract

The present invention relates to a process for preparing a foreign protein comprising culturing a bacterium containing the cysteine synthase (cysK) gene and a gene encoding the foreign protein. The present invention comprises the steps of culturing a bacterium transformed with an expression vector containing a gene encoding a serine-rich foreign protein and an expression vector containing the cysK gene, or a bacterium transformed with an expression vector containing the cysK gene and a gene encoding a serine-rich foreign protein and isolating the foreign protein therefrom. The present invention is expected to be widely used to increase the production yield of a serine-rich foreign protein.

Description

technical field [0001] The invention relates to a preparation method of serine-rich protein, which comprises culturing bacteria containing cysteine ​​synthetase (cysK) gene and heterologous protein gene. More specifically, the present invention relates to a method for preparing a serine-rich protein, which comprises culturing a bacterium containing both a serine-rich heterologous protein gene and a cysteine ​​synthase (cysK) gene, and isolating a bacterium rich in Serine-containing heterologous proteins. Background technique [0002] E.coli is a commonly used strain for the synthesis and preparation of heterologous proteins. This strain is used to produce proteins such as interferon, interleukin 2, colony-stimulating factor, growth hormone, insulin-like growth factor and human serum albumin through recombinant technology. . In order to efficiently prepare heterologous proteins in E.coli, plasmid vectors expressing heterologous proteins, suitable culture conditions, conditi...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/54C07K14/575C12N1/21C12N15/70C12P21/02C12R1/19
CPCC12P21/02C07K14/5759C12N15/70C07K14/5434C07K14/575
Inventor 李相烨韩美正
Owner KOREA ADVANCED INST OF SCI & TECH
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