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Yeast Protein Extraction Reagent

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

Active Publication Date: 2021-02-26
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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  • Yeast Protein Extraction Reagent
  • Yeast Protein Extraction Reagent

Examples

Experimental program
Comparison scheme
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.) was used for codon optimization and gene synthesis, and was connected to the pPICZa vector (purchased from invitrogen lifetechnologies);

[0029] 2. The midiprep kit for endotoxin removal (EndoFree Plasmid ezFiter Midiprep Kit, 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....

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 0.5ml ddH 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 temperat...

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 0.8ml ddH 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 ...

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Abstract

The invention discloses a reagent for extracting yeast protein, which comprises: solution A and solution B; wherein, solution A is an alkaline solution; Triton X-100, 5-40mM EDTA, 0.1g / 100mL-2g / 100mL of SDS, 2-200mM β-mercaptoethanol and 1-10M urea. The reagent of the invention is simple to prepare, and when used, the protein extraction step is simple, the operation is convenient, no need to use expensive experimental instruments and wall-breaking enzymes, etc., the cleavage efficiency is equivalent to the classic glass bead breaking method, and a large number of samples can be processed.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14
Inventor 赵永浩周延庆王鹏张楠
Owner WUXI APPTEC SUZHOU
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