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A method for immobilizing human arginase-1 by surface display

A technology of arginase and surface display, which is applied in the field of immobilization of human arginase-1, can solve problems such as ineffective realization of surface display and immobilization of multimeric proteins, so as to improve display efficiency and shorten Process flow, effect of simplified purification steps

Active Publication Date: 2018-02-16
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current ice nucleation protein display system cannot effectively realize the surface display and immobilization of multimeric proteins

Method used

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  • A method for immobilizing human arginase-1 by surface display
  • A method for immobilizing human arginase-1 by surface display
  • A method for immobilizing human arginase-1 by surface display

Examples

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Effect test

Embodiment 1

[0031]The method of the present invention is used to display human arginase-1 in Escherichia coli. First, the constructed recombinant plasmid containing InaK-N / ARG1 was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C. Then, pick a single colony and inoculate it in 100mL LB medium, the final concentration of antibiotic ampicillin is 50μg / mL, shake culture at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, and shake at 37°C Incubate for 8 hours. Then, the cells were collected by centrifugation at 12000 RPM at 4°C, washed twice with pre-cooled PBS to remove the residual medium, and finally resuspended in 10 mL of pre-cooled PBS buffer for later use. Then it was measured by Chinard reaction and converted to the corresponding human arginase-1 enzyme activity. ( image 3 , Figure 4 )

Embodiment 2

[0033] The method of the present invention is used to display human arginase-1 in Escherichia coli. First, the constructed recombinant plasmid containing ssMalE-InaK-N / ARG1 was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C. Then, pick a single colony and inoculate it in 100mL LB medium, the final concentration of antibiotic ampicillin is 50μg / mL, shake culture at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, and shake at 37°C Incubate for 8 hours. Then, the cells were collected by centrifugation at 12000 RPM at 4°C, washed twice with pre-cooled PBS to remove the residual medium, and finally resuspended in 10 mL of pre-cooled PBS buffer for later use. Then it was measured by Chinard reaction and converted to the corresponding human arginase-1 enzyme activity. ( image 3 , Figure 4 )

Embodiment 3

[0035] The method of the present invention is used to display human arginase-1 in Escherichia coli. First, the constructed recombinant plasmid containing ssTorA-InaK-N / ARG1 was transformed into Escherichia coli competent cell Rosetta Blue strain, and cultured overnight at 37°C. Then, pick a single colony and inoculate it in 100mL LB medium, the final concentration of antibiotic ampicillin is 50μg / mL, shake culture at 37°C, the OD value is between 0.5 and 0.6, add IPTG, the final concentration is 1mM, and shake at 37°C Incubate for 8 hours. Then, the cells were collected by centrifugation at 12000 RPM at 4°C, washed twice with pre-cooled PBS to remove the residual medium, and finally resuspended in 10 mL of pre-cooled PBS buffer for later use. Then it was measured by Chinard reaction and converted to the corresponding human arginase-1 enzyme activity. ( image 3 , Figure 4 )

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Abstract

The invention discloses a method for immobilizing human source arginase-1 through surface display. The method comprises the steps of adding signal peptide and charged polypeptide to the amino terminal of a protein cleavage variant (InaK-N) formed on an ice core, fusing human source arginase-1 into the carboxyl terminal, and designing an HA label at the carboxyl terminal; constructing various recombinant plasmids to convert competent escherichia coli cells, so that different genetic engineering strains are obtained; conducting shake-flask culture on the strains; detecting the display efficiency and enzyme activity of human source arginase-1; selecting the strain with the highest enzyme activity for mass culture, conducting efficient L-arginine conversion, and synthesizing L-ornithine. Human source arginase-1 fused in the protein cleavage variant formed on the ice core is effectively displayed on the surface of an escherichia coli cell, so that human source arginase-1 is immobilized. Compared with an original ice core protein display system, the method has the advantage that the display efficiency and enzyme activity of human source arginase-1 are improved remarkably. Compared with a chitin immobilizing method, the method has the advantages that cost is reduced, process is shortened, and purification steps are simplified.

Description

technical field [0001] The present invention relates to the immobilization technology of human arginase-1 (Human Arginase 1), especially the method of realizing immobilization through optimized ice nucleation protein display, which is the immobilization of human arginase I A new idea, a new approach, a new method. Background technique [0002] Human Arginase-1 exists in human liver cells in the form of a trimer, participates in the urea metabolic cycle, and catalyzes the hydrolysis of L-Arg (L-Arginine) to generate L-Orn (L-Ornithine) and urea. Arginase I deficiency can lead to a series of related diseases such as developmental delay, mental retardation, epilepsy and movement disorders. Human arginase-1 can also be used as a drug to treat cancer. [0003] Since most arginases are difficult to achieve high-efficiency soluble expression in Escherichia coli, arginase is extracted from the liver as the main source of arginase, but there is a risk of carrying various viruses. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/70
CPCC12N9/78C12Y305/03001
Inventor 马立新张贞王亚平唐荣兴
Owner HUBEI UNIV
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