Reagent for extracting yeast protein

A yeast protein and reagent technology, applied in the field of protein extraction reagents, can solve the problems of complex process, narrow application, low efficiency, etc., and achieve the effect of simple preparation, convenient operation, and simple extraction steps

Active Publication Date: 2016-06-15
WUXI APPTEC SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because yeast cells have very thick cell walls, their disruption requires expensive equipment for mechanical disruption or cell wall dissolving enzyme treatment, etc., and the process is complex, which limits the application of the Pichia pastoris expression system for the expression of insoluble proteins
Although there are yeast lysis methods and commercial reagents reported in the literature at present, the application is narrow, the efficiency is low, and the cost of amplification processing is high, and the effect is not ideal

Method used

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  • Reagent for extracting yeast protein
  • Reagent for extracting yeast protein

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 GPCR membrane protein expression and purification in Pichia pastoris

[0026] In this example, for the expression of GPCR membrane protein in Pichia pastoris, Pichia pastoris X33 and pPICZa expression system (EasySelect TM Pichia Expression Kit, K1740-0, available from invitrogen life technologies).

[0027] 1. Expression plasmid construction

[0028] 1. According to the CDS sequence of the GPCR membrane protein, the gene synthesis company (Shanghai Jierui Bioengineering Co., Ltd.) carried out codon optimization and gene synthesis, and connected it to the pPICZa vector (purchased from invitrogenlifetechnologies);

[0029] 2. The midiprep kit (EndoFreePlasmidezFiterMidiprepKit, Biomiga) was used to prepare the plasmid to obtain the plasmid pPICZa-GPCR.

[0030] 2. Pichia pastoris transformation

[0031] 1. 10 μg of the extracted plasmid pPICZa-GPCR was digested with Sac1 (purchased from Fermentas) and kept at 37° C. for 2 hours. The digested plasmid was rec...

Embodiment 2

[0076] A reagent for extracting total protein from Pichia pastoris, comprising: solution A and solution B;

[0077] Wherein, solution A is 0.1M KOH solution or 0.1M NaOH solution.

[0078] Solution B contained 100 mM Tris-HCl, 0.1% TritonX-100 by volume, 5 mM EDTA, 0.1 g / 100 mL of SDS, 2 mM β-mercaptoethanol and 1 M urea.

[0079] In addition, the above reagents can also be used to extract the total protein of Pichia pastoris, and the steps are as follows:

[0080] 1) Centrifuge the cultured yeast at 2500g for 3 minutes to collect the yeast;

[0081] 2) Add 0.5mlddH per gram of yeast wet weight 2 O resuspended thallus, after obtaining the thallus liquid, add above-mentioned solution A (the volume ratio of the addition amount of solution A and thallus liquid is 1.1:1), after 50 ℃ of water bath incubations for 20 minutes, centrifuge, remove supernatant, collect yeast;

[0082] 3) Add 2ml of the above solution B per gram of yeast wet weight, shake on a shaker at room temperatu...

Embodiment 3

[0084] A reagent for extracting total protein from Pichia pastoris, comprising: solution A and solution B;

[0085] Wherein, solution A is 1M KOH solution or 1M NaOH solution.

[0086] Solution B contained 200 mM Tris-HCl, 3% TritonX-100 by volume, 40 mM EDTA, 2 g / 100 mL of SDS, 200 mM β-mercaptoethanol and 10 M urea.

[0087] In addition, the above reagents can also be used to extract the total protein of Pichia pastoris, and the steps are as follows:

[0088] 1) Centrifuge the cultured yeast at 3500g for 8 minutes to collect the yeast;

[0089] 2) Add 0.8mlddH per gram of yeast wet weight 2 O resuspended thallus, after obtaining the thallus liquid, add the above-mentioned solution A (the volume ratio of the addition amount of solution A to the thallus liquid is 1.2:1), after incubating in a water bath at 70° C. for 5 minutes, centrifuge, remove the supernatant, and collect the yeast;

[0090] 3) Add 3ml of the above solution B according to the wet weight of each gram of y...

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Abstract

The invention discloses a reagent for extracting yeast protein. The reagent contains a solution A and a solution B, wherein the solution A is an alkali solution; and the solution B contains 20mM to 200mM of Tris-HCl, TritonX-100 with the volume concentration of 0.1% to 3%, 5mM to 40mM of EDTA, 0.1g / 100mL to 2g / 100mL of SDS, 2mM to 200mM of beta-mercaptoethanol and 1M to 10M of urea. According to the reagent disclosed by the invention, the preparation is simple; and when in use, protein extracting steps are simple, the operation is convenient, expensive experimental instruments, wall breaking enzymes and the like are not required, the cracking efficiency is equivalent to that of the classic glass bead breaking method, and a large amount of samples can be treated.

Description

technical field [0001] The invention relates to a protein extraction reagent, in particular to a yeast protein extraction reagent. Background technique [0002] The Pichia pastoris expression system is a eukaryotic expression system developed in the past ten years. It is currently one of the most successful exogenous protein expression systems. It has obvious advantages in product processing, exocytosis, post-translational modification and glycosylation modification, and has been widely used in the field of scientific research and pharmaceutical industry to express foreign proteins. [0003] At present, the expression system of Pichia pastoris is mostly used for extracellular protein expression, while membrane proteins such as GPCR and other hydrophobic proteins can only be expressed intracellularly. Because yeast cells have very thick cell walls, their disruption requires expensive instruments for mechanical disruption or cell wall dissolving enzyme treatment, etc., and th...

Claims

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Application Information

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IPC IPC(8): C07K1/14
Inventor 赵永浩周延庆王鹏张楠
Owner WUXI APPTEC SUZHOU
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