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Preparation method of low-temperature-resistant and low-salt cyanobacteria phycobilisosome sample

A low-temperature-resistant, cyanobacterial algae technology is applied in the field of preparation of cyanobacterial phycobilisome samples, which can solve the problem of poor contrast of protein samples, and it is difficult to study the interaction between phycobilisomes and photosystem complexes, as well as the mechanism and structural resolution of energy transfer. rate limits etc.

Active Publication Date: 2022-08-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high-salt normal temperature conditions required for the complete phycobilisomes purified in vitro also greatly limit the analysis of the structure of phycobilisomes and the research on the interaction and energy transfer mechanism of phycobilisomes and photosystem complexes.
On the one hand, high phosphate conditions make it difficult to obtain crystallization of phycobilisomes by crystallography, and it is impossible to use crystallographic methods to analyze the three-dimensional structure of phycobilisomes; on the other hand, high phosphate conditions make it difficult to use cryo-TEM When analyzing the three-dimensional structure of phycobilisomes, the contrast of protein samples will be very poor, and various other solutions need to be used to solve the contrast problem, but the resolution of the corresponding resolved structure will be greatly limited; on the other hand , high-salt and low-temperature conditions do not take advantage of the stabilization of photosystem protein complexes, making it difficult for us to study the interaction between phycobilisomes and photosystem complexes and the mechanism of energy transfer in vitro

Method used

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  • Preparation method of low-temperature-resistant and low-salt cyanobacteria phycobilisosome sample
  • Preparation method of low-temperature-resistant and low-salt cyanobacteria phycobilisosome sample
  • Preparation method of low-temperature-resistant and low-salt cyanobacteria phycobilisosome sample

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1. Preparation of low-salt and low-temperature cyanobacterial phycobilisome samples

[0025] 1. Prepare buffer A (0.9 M sodium phosphate buffer pH 8.0, 1 liter contains 300.41 g Na 2 HPO 4 •12H 2 O, 9.55 g NaH 2 PO 4 • 2H 2 O and 10 mM EDTA) and buffer B (0.75 M potassium phosphate buffer pH 8.0, 1 liter contains 160.9 g K 2 HPO 4 , 6.12 g KH 2 PO 4 Buffer A and 10 mM EDTA), buffer A and buffer B are the storage solutions of buffer C, and buffer A and buffer B are mixed in equal volumes to obtain buffer C;

[0026]2. Add 0.0075% (w / v, g / mL) 1,5-glutaraldehyde (GA) and 10 nM BS3 to buffer C to make buffer D;

[0027] 3. Sterilize the solid sucrose, and use buffer D to prepare 15% (w / v, g / mL) and 45% (w / v, g / mL) sucrose solutions;

[0028] 4. Take 1 g of thylakoid membrane-free cyanobacteria cinerea Gloeobacter violaceus PCC 7421, ground and crushed in liquid nitrogen, and the following steps should be protected from light as much as possible;

[002...

Embodiment 2

[0033] Example 2. Preparation of low-salt and low-temperature cyanobacterial phycobilisome samples

[0034] The preparation steps are the same as those in Example 1, except that 0.015% (w / v) 1,5-glutaraldehyde (GA) and 500 nM BS3 cross-linking agents were added in step 2, and a complete low-temperature and low-salt-resistant cross-linking agent was also obtained. phycobilisome samples.

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Abstract

The invention discloses a preparation method of a low-temperature-resistant and low-salt cyanobacteria phycobilisosome sample. Compared with a conventional cyanobacteria phycobilisome in-vitro purification method, the cyanobacteria phycobilisome in-vitro purification method disclosed by the invention comprises a special treatment step: two cross-linking agents, namely 1, 5-glutaraldehyde (GA) with the final concentration of 0.0075-0.015% (w / v) and BS3 with the final concentration of 10-500 nM, are added into a phosphate buffer solution subjected to sucrose gradient purification. The obtained phycobilisome sample can maintain the stable structure within 48 hours under the low-temperature and low-salt conditions of 4 DEG C and 10 mM phosphate, and cannot be depolymerized. The low-temperature-resistant and low-salt phycobilisome provides possibilities for analyzing the structure of the phycobilisome by a cryoelectron microscope, researching the interaction between the phycobilisome and a photosystem complex in vitro, an energy transfer mechanism and the like.

Description

technical field [0001] The invention belongs to the field of microbial protein sample preparation, in particular to a method for preparing a low-temperature and low-salt-resistant cyanobacterial phycobilisome sample, which provides more possibilities for the application of cyanobacterial phycobilisomes in other biochemical fields. Background technique [0002] Photosynthesis is one of the most important biochemical reactions on earth, and the first step of photosynthesis is the efficient capture and transfer of light energy. The cyanobacterial phycobilisome is such a large light-harvesting antenna protein complex, which is located on the side of the inner thylakoid membrane of cyanobacterial cells facing the matrix, and can capture and transmit light energy to the photosystem with an efficiency of no less than 98%. reaction center. Cyanobacteria phycobilisomes are structurally composed of phycobiliproteins and connexins. Phycobiliproteins in different oligomeric forms, coup...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34G01N23/20008G01N23/04
CPCG01N1/34G01N23/20008G01N23/04
Inventor 郑正高王宏蕊董春霞赵进东
Owner PEKING UNIV
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