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Method and system for protein purification

A protein-binding and recombinant protein technology, applied in the field of protein purification, can solve the problems of inconvenient protein purification process, enzyme loss, enzyme activity destruction, etc.

Inactive Publication Date: 2010-11-17
SIMPSON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] These existing protein purification systems have certain disadvantages
Its protein purification process is inconvenient and laborious
Columns for purification are expensive
Limitations of these systems for protein purification include the inability to isolate recombinant proteins in some cases, such as samples containing EDTA, and the relatively large size of the currently used protein tags compared to the target protein of this invention
[0007] There are still some problems in the use of enzyme products in water feeding, such as (a) the rapid loss of enzymes after the feed is added to water, and (b) the enzyme activity will be destroyed when the feed making temperature is higher than 100°C

Method used

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  • Method and system for protein purification
  • Method and system for protein purification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Protein Purification

[0085] SBP-tagged recombinant protein construction and expression

[0086] The SBP gene is amplified by PCR and fused to the N-terminal of the target protein, which contains an endoprotease recognition site between the SBP and target protein genes. The fusion protein was replicated in the Pichia pastoris expression vector pPICZαA under the control of the AOX1 promoter and transformed into Pichia pastoris GS115 for expression. Pichia pastoris transformants carrying the SBP-target protein gene were cultured in BMGY medium for 24 hours. The cells were obtained by centrifugation and resuspension in BMMY containing 0.5% methanol. 0.5% methanol (0.5% v / v) was added every 24 hours to induce the expression of SBP-tagged recombinant protein. After 5 days of induction, the cells were removed by centrifugation and the cell-free broth was collected for subsequent purification. For subsequent purification methods, please refer to figure 1 and th...

Embodiment 2

[0093] The comparison of embodiment 2 thermal stability

[0094] The SBP gene was amplified by PCR and fused to the N-terminus of the target enzyme. The lipase gene from R. oryzae, the xylanase gene from unpurified rumen fungal culture medium and the phytase gene from E. coli were all duplicated and fused to SBP. The fusion proteins such as SBP-lipase, SBP-xylanase and SBP-phytase are expressed by the above method and used in the present invention.

[0095] Thermostability of lipases from different sources

[0096] The lipase activity unit (U) is defined as when the sample releases 1 μmole of p-Nitrophenol per minute in 0.3 mM 4-Nitrophenyl palmitate (4-Nitrophenylpalmitate) at 30 ° C, pH Under the condition of value 7.0. SBP-lipase (52500 U / g) and F-AP15 lipase (32430 U / g, R. oryzae lipase purchased from Amano Enzyme Inc.) were mixed with 15% or 20% water content of fermented soybean flour to reach 100 U per gram. 100U / g lipase mix from different sources was weighed and p...

Embodiment 3

[0121] Example 3 Binding test of starch binding protein-enhanced green fluorescent protein (hereinafter referred to as SBP-eGFP) bound to different types of SBP matrices.

[0122] Binding assay of SBP-eGFP bound to different types of resins

[0123] 2 mg of unwashed branched starch, amylose resin (purchased from Bio-Rad Laboratories Inc., Hercules, California, U.S.), dextrin resin (purchased from GE Healthcare., Waukesha, U.S.) and sephadex (a kind of glucan Sugar commercial product, purchased from Sigma., Saint Louis, Missouri, U.S) was stirred together with SBP-eGFP in binding buffer (50 mM NaOAc, pH 5.5), its concentration was 0.3 mg / mL, and the overall volume was 200 μL. After stirring and incubation at 25°C for 3 hours, the samples were centrifuged. The suspension (unbound protein) and resin pellets (bound protein) were boiled and used for SDS-PAGE. The results of this binding test are shown in Figure 4A to Figure 4C .

[0124] Binding assay of SBP-eGFP bound to algi...

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Abstract

The present invention provides a method and a system for protein purification using a starch binding protein (SBP)-tagged recombinant protein. SBP-binding matrixes are also disclosed in the invention to recover the SBP-tagged recombinant protein. Thus, purifying a target protein is re-usable, convenient and low cost by the present invention.

Description

technical field [0001] The present invention relates to a method and system for protein purification, more particularly, the present invention relates to a method and system for using starch-binding protein. Background technique [0002] Production of proteins by expression in microbial systems has become a major source of high-priced, pharmaceutically useful proteins. Purification and recovery of recombinant proteins is an important consideration in designing fermentation processes. While traditional methods of protein purification can be used to isolate products, improved methods include the use of recombinant proteins. The recombinant protein can be purified by affinity column chromatography, and the desired component of the recombinant protein is purified by its covalent attachment to the polypeptide, which is attached to an affinity matrix (affinity medium). [0003] There are certain systems for separating proteins by the principle of affinity column chromatography. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C12N9/20C12N9/42C12N9/16
CPCC07K1/32C07K2319/20C07K1/22
Inventor 张大慈许嘉钦
Owner SIMPSON BIOTECH CO LTD
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