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Method for quantitatively detecting target protein yield of cell-free protein synthesis system and method for screening zymoprotein with high catalytic activity

A technology for protein synthesis and quantitative detection, applied in the field of molecular biology and synthetic biology, can solve the time-consuming and labor-intensive problems of expression and purification

Pending Publication Date: 2022-02-08
林影 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Screening enzyme proteins with high catalytic activity from enzyme homologues from different species is a common method to improve the yield and production efficiency of the target product in the biosynthetic pathway, but this process is often time-consuming because it usually requires the expression and purification of multiple proteins strenuous

Method used

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  • Method for quantitatively detecting target protein yield of cell-free protein synthesis system and method for screening zymoprotein with high catalytic activity
  • Method for quantitatively detecting target protein yield of cell-free protein synthesis system and method for screening zymoprotein with high catalytic activity
  • Method for quantitatively detecting target protein yield of cell-free protein synthesis system and method for screening zymoprotein with high catalytic activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1 expresses Hrbks-GFP 11 Construction of fusion protein plasmid vector and Hrbks-GFP 11 Establishment of the standard curve between fluorescence intensity value and protein concentration of fusion protein under different incubation time conditions

[0094] (1) Connect the GFP11 gene to the C-terminus of the human (Homo sapiens) ribokinase HRBKS gene (NCBI: NP_071411.1) (see reference "Cabantous, Stéphanie, Waldo, et al. In vivo and in vitroprotein solubility assays using split GFP .[J].Nature Methods,2006." GFP 11M3 OPT , referred to as GFP11 in the embodiments, its nucleotide sequence is shown in SEQ ID NO.1: 5'-CGTGACCACATGGTCCTTCATGAGTACGTAAATGCTGCTGGGATTACA-3'), and in order to ensure the active expression of the HRBKS gene, a section is inserted between the HRBKS gene and the GFP11 gene Linker sequence (nucleotide sequence shown in SEQ ID NO.2: 5'-GATGGAGGGTCTGGTGGCGGATCAACAAGT-3') to obtain the complete HRBKS-GFP11 gene sequence (SEQ ID NO.4). Entru...

Embodiment 2

[0109] Example 2Hrbks-GFP 11 , Hprs-GFP 11 with Cnampt-GFP 11 The establishment of the standard curve between the fluorescence intensity value and the protein concentration of the fusion protein under the condition of the same incubation time

[0110] (1) Referring to step (1) plasmid pET28a-Hrbks-GFP in Example 1 11 The method of construction is to construct the human (Homo sapiens) ribose phosphate pyrophosphate kinase HPRS gene (Uniprot: P60891) and the nicotinamide phosphoribosyltransferase CNAMPT gene (GenBank : RYF34637.1) plasmid vector pET28a-Hprs-GFP 11 with pET28a-Cnampt-GFP 11 .

[0111] (2) Plasmid pET28a-Hprs-GFP 11 with pET28a-Cnampt-GFP 11 Transform into BL21 (DE3) competent cells respectively, and carry out positive transformant selection on LBK kanamycin resistance plate.

[0112] (3) Select the correct transformants identified in step (2) and refer to the literature (Li L, Liao Y, Luo Y, et al. Improved Efficiency of the Desulfurization of Oil Sulfur ...

Embodiment 3

[0115] Example 3 Using the CFPS system to express a variety of fusion proteins and detect the fluorescence intensity values ​​under the same time conditions as the luminescence liquid incubation

[0116] (1) Referring to step (1) plasmid pET28a-Hrbks-GFP in Example 1 11 The construction method, the ribose kinase ERBKS gene (Uniprot: P0A9J6) derived from Escherichia coli, the ribose phosphate pyrophosphate kinase PPRS gene (NCBI: ABO08552.1) derived from Pyrobaculum calidifontis (NCBI: ABO08552.1), Plasmids carrying the human (Homo sapiens) nicotinamide phosphoribosyltransferase HNAMPT gene (Uniprot: P43490) and the Thermus ruber (Meiothermus ruber)-derived nicotinamide phosphoribosyltransferase MNAMPT gene (NCBI: ADD29592.1) Vector pET28a-Erbks-GFP 11 , pET28a-Pprs-GFP 11 , pET28a-Hnampt-GFP 11 with pET28a-Mnampt-GFP 11 .

[0117] (2) with pET28a-Erbks-GFP 11 , pET28a-Pprs-GFP 11 , pET28a-Hnampt-GFP 11 , pET28a-Mnampt-GFP 11 With the pET28a-Hrbks-GFP in embodiment 1 s...

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Abstract

The invention discloses a method for quantitatively detecting the target protein yield of a cell-free protein synthesis system and screening zymoprotein with high catalytic activity. According to the method for quantitatively detecting the yield of the target protein in the CFPS system, the expression quantity of the target protein in the CFPS system can be rapidly analyzed by segmenting the intensity value of fluorescence emitted by spontaneous binding of fluorescent protein, and rapid detection of the expression quantity of the target protein in the CFPS system is facilitated; meanwhile, based on the method, the enzyme homologues with high catalytic activity can be quickly screened by calculating the ratio of the quantity of products obtained by catalysis of each enzyme protein to the fluorescence intensity value after spontaneous binding of segmented fluorescent protein, and a conventional large intestine transformation-expression-purification process is not needed, so that the time and the material cost are greatly saved, the screening speed is increased, and moreover, the screening leakage risk of the high-catalytic-activity protein caused by too low protein expression quantity is avoided, and rapid and accurate screening of the high-catalytic-activity zymoprotein is facilitated.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and synthetic biology, in particular to a method for quantitatively detecting the yield of a target protein in a cell-free protein synthesis system and screening enzyme proteins with high catalytic activity. Background technique [0002] Segmentation of fluorescent protein is a technique for the modification of fluorescent protein: the complete fluorescent protein is divided into two or more polypeptides at a specific position. These polypeptide chains will not produce fluorescence when they exist alone, but they will spontaneously assemble when they exist together. Forms an intact fluorescent protein and fluoresces. By fusing and expressing the fragmented short-chain fluorescent protein polypeptide with the target protein and detecting its assembly with another fluorescent protein polypeptide, this technology has been successfully applied to the analysis of protein soluble expression i...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64C07K19/00C12N15/70C12N1/21C12N15/54C12R1/19
CPCG01N33/68G01N21/6486C12N15/70C12N9/1077C12N9/1211C12Y207/01015C12Y207/01018C12Y204/02012C07K2319/60
Inventor 林影袁清焱梁书利
Owner 林影
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