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Peptides having affinity for polydimethylsiloxane, and uses thereof

A technology of polydimethylsiloxane and polydimethylsiloxane materials, which is applied to peptides containing affinity tags, peptides containing His tags, peptides containing GST tags, etc., and can solve unknown problems

Inactive Publication Date: 2017-09-26
NAT UNIV KYOTO INST OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, peptides with affinity for polydimethylsiloxane substrates are still unknown

Method used

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  • Peptides having affinity for polydimethylsiloxane, and uses thereof
  • Peptides having affinity for polydimethylsiloxane, and uses thereof
  • Peptides having affinity for polydimethylsiloxane, and uses thereof

Examples

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preparation example Construction

[0064] The peptide having affinity for PDMS of the present invention can be prepared by known genetic engineering technology or chemical synthesis technology. For example, a desired peptide can be produced by inserting a polynucleotide encoding the peptide shown in any one of SEQ ID NOS. 1 to 9 into a vector, and then culturing a transformant into which the vector has been introduced. The peptide having an affinity for PDMS of the present invention can also be obtained by isolating and purifying the peptide from a microorganism capable of producing the peptide. The peptide having an affinity for PDMS of the present invention can also be synthesized by known chemical synthesis techniques based on the amino acid sequence shown in any one of SEQ ID NOS. 1 to 9 or the nucleotide sequence encoding it. Chemical synthesis techniques include peptide synthesis methods such as liquid-phase or solid-phase methods. A more detailed example is the steps described in the example below. In ...

Embodiment 1

[0133] According to the following steps, the parapolydimethylsiloxane (PDMS ) affinity.

[0134] 1. Process

[0135] 1-1. Elongation factor Tu (ELN) expression vector, tryptophanase (TPA) expression vector, outer membrane protein C (OMC) expression vector build

[0136] 1) DNA Purification Kit (Promega) was used to extract the chromosomal DNA of Escherichia coli BL21(DE3) (Novagen).

[0137] 2) Use the chromosomal DNA as a template, and use KOD plus ver.2PCR kit (Toyobo Co., Ltd.) to perform PCR to amplify the ELN gene.

[0138] 3) The amplified ELN gene was mixed with the pET-22 vector (Novagen Company) in a molar ratio of 3:1, and Ligation High (Toyobo Company) equal to the mixed solution was added, and then at the Nde I position The amplified ELN gene was inserted between the dot and the Not I site for cloning.

[0139] 4) Escherichia coli HST08Premium (Takara Bio Company) was transformed with the above vector, and cultured in LB-Amp agar medium overnight.

[014...

Embodiment 2

[0187] Following the steps below, studies were performed to examine whether peptide-fused GSTs immobilized to PDMS substrates maintained the original activity of GSTs.

[0188] 1. Steps

[0189] 1-1. Construction of peptide fusion GST

[0190] In this example, ELV1 peptide-fused GST, TPV1 peptide-fused GST, OCV1 peptide-fused GST, OAT1 peptide-fused GST, and wt-GST prepared in Example 1 were used.

[0191] 1-2. Determination of GST residual activity

[0192] 1. Steps

[0193] 1) Each GST solution of wt-GST, ELV1 peptide-fused GST, TPV1 peptide-fused GST, OCV1 peptide-fused GST, and OAT1 peptide-fused GST was diluted with PPB to prepare 2940 μL of each GST solution, so that after adding 30 μL of 100 mM CDNB, After 30 μL of 100 mM GSH, the GST concentration was 0.5 μg / mL.

[0194] 2) Add 30 μL of 100 mM CDNB and 30 μL of 100 mM GSH, measure the absorbance at 340 nm for 3 minutes at 25 ° C with a spectrophotometer (V-630BIO, JASCO Corporation), and calculate the chang...

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Abstract

The purpose of the present invention is to provide: a polydimethylsiloxane substrate obtained through the bonding of peptides having an affinity for polydimethylsiloxane (PDMS); a method for immobilizing a target protein on the polydimethylsiloxane substrate; peptides having an affinity for PDMS; polynucleotides for coding the peptides having an affinity for PDMS; and vectors and the like that use the polynucleotides. A polydimethylsiloxane substrate obtained through the bonding of peptides having an affinity for polydimethylsiloxane, said peptides comprising (1a) or (1b) below or fragments thereof. (1a) A peptide comprising an amino acid sequence represented by at least one sequence selected from the group consisting of SEQ ID Nos. 1 to 9. (1b) A peptide having an affinity for polydimethylsiloxane, and comprising an amino acid sequence in which one or a plurality of amino acids have been deleted, substituted and / or added in the amino acid sequence of (1a).

Description

technical field [0001] The present invention relates to peptides having an affinity for polydimethylsiloxane and uses of the peptides. Background technique [0002] Techniques for immobilizing proteins, nucleic acids, cells, etc. to substrates for detection, quantification, or analysis of desired substances have been widely used in various fields including clinical examinations, drug development, environmental monitoring, and biochemistry. For example, a technique is known in which a protein such as an enzyme or an antibody is immobilized to a substrate to detect a desired substance based on an enzymatic reaction or an antigen-antibody reaction with the immobilized protein. [0003] Today, these techniques are still an active field of research, and aim to allow high-precision and high-efficiency detection, quantification, analysis, and the like. For example, studies report techniques in which the surface of a substrate is treated by chemical treatment or plasma treatment, d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/08C07K14/00C12N7/08C12N9/00C12N15/00
CPCC07K17/08C07K2319/00C07K2319/23C12N9/88C12Y401/99001C07K2319/21C07K14/245C07K14/4737C07K7/08C08G77/42C07K14/00C12N7/08C12N9/00C12N15/00C08G77/04C12N15/63
Inventor 熊田阳一大槻领子赤井亮太片山淳子的场一隆
Owner NAT UNIV KYOTO INST OF TECH
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