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Method for quantitatively co-expressing multiple proteins in vitro and application thereof

A co-expression and protein technology, applied in the field of quantitative co-expression of multiple proteins in vitro, to achieve the effect of simplification of complex phenomena and simple operation

Active Publication Date: 2020-08-04
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After literature search, no report was found on the quantitative co-expression of multiple proteins in the same reaction system using in vitro cell-free protein synthesis technology

Method used

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  • Method for quantitatively co-expressing multiple proteins in vitro and application thereof
  • Method for quantitatively co-expressing multiple proteins in vitro and application thereof
  • Method for quantitatively co-expressing multiple proteins in vitro and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] DNA Screening and Codon Optimization of Expression Sequences of Different Fluorescent Proteins

[0144] By searching different databases, Blast was performed on the coding sequences of 18 different fluorescent protein genes, and the codons were optimized to make them suitable for Kluyveromyces lactis in vitro cell-free protein synthesis system.

Embodiment 2

[0146] Construction of in vitro cell-free protein synthesis system plasmids containing eukaryotic cell translation regulatory sequences, fluorescent protein coding sequences, and tag (protein expression and purification sequences)

[0147] 2.1 Whole gene synthesis

[0148] The optimized DNA sequence in Table 2 was used for genome synthesis.

[0149] 2.2 Primer design

[0150] Primers were designed with oligo 7.0 software, and the primer sequences are shown in Table 2.

[0151] Table 2 Primer Sequence

[0152]

[0153]

[0154]

[0155] 2.3 Construction of plasmid

[0156] Insert the gene sequence of the target protein to be expressed into the pD2P plasmid (see Figure 11 ), the specific construction process is as follows:

[0157] Using molecular cloning technology, pD2P was used as a template for plasmid construction, and two pairs of primers were used for PCR amplification, and 8.5 μL of the two amplification products were mixed with 1 μL of DpnI and 2 μL of 10...

Embodiment 3

[0160] Expression of different fluorescent proteins by yeast in vitro cell-free protein synthesis system

[0161] Using the method of phi29 DNA polymerase amplification, using the above plasmids as templates, using random seven-base primers to amplify the fragments located between the 5'UTR of the transcription initiation sequence and the 3'UTR of the termination sequence in all plasmids, the amplified The product serves as a DNA template for the synthesis of various fluorescent proteins. Wherein, the transcription start sequence 5'UTR and the termination sequence 3'UTR contain one or more tandem combinations, and the tandem combinations include enhanced translation regulatory elements and protein expression purification tag elements.

[0162] According to the instructions for use, the fluorescent protein DNA template prepared above (the concentration of the template mother solution of different fluorescent proteins is equal) was added to the self-made Kluyveromyces lactis in ...

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Abstract

The invention provides a method for quantitatively co-expressing multiple proteins in vitro, comprising the following steps of: (1) establishing a standard curve, namely establishing a standard curveof the relationship between standard protein mass concentration and a luminous value; (2) constructing a vector containing a target protein gene, and obtaining an in-vitro protein synthesis system; (3) establishing a relation curve between the target protein concentration and the carrier concentration; (4) calculating the concentration or / and concentration ratio of the carrier for quantitatively co-expressing multiple target proteins; and (5) quantitatively co-expressing the target protein. According to the invention, a plurality of proteins can be synthesized by quantitative co-expression inthe same reaction system according to the proportional relation of the template addition amount of the target protein. The invention further provides application of the in-vitro acellular protein synthesis system in the method. The in-vitro acellular protein synthesis system is used for quantitative co-expression of various different proteins.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for quantitatively co-expressing multiple proteins in vitro and its application. Background technique [0002] Fluorescent proteins are widely used in many research fields of biology. Molecular probes and labeling methods based on fluorescent proteins have become important tools for dynamic imaging of living cells or in vivo studies of biological macromolecules or cell functions. Since the green fluorescent protein gene was cloned from jellyfish in 1992, new fluorescent proteins have been cloned from many marine species and new mutants have been transformed, which can specifically "light up" biological molecules or cells, And it shows the activity of biomolecules, which can help us reveal the law and nature of the activities of these molecules or cells. The reported fluorescent protein spectrum is distributed in the entire visible light region. They are widely used in the r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12P21/02C07K14/435
CPCC12N15/815C12N15/81C12P21/02C07K14/43595C07K2319/60G01N33/68G01N33/582C12Q1/6844C12N2800/22C12Q2563/107
Inventor 郭敏李海洋宋娅凯于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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