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A method for preparing LPOR protein crystals

A protein and crystal technology, applied in the field of protein engineering, can solve the problems of unstable light-sensitive protein, loss and other problems

Inactive Publication Date: 2019-01-15
ZHEJIANG SHANCE HEQISHI BIO SCI& TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the application of all these methods, care must be taken to preserve the integrity of biological macromolecules and prevent the loss of biological activity of the extracted substances due to acid, alkali, high temperature, and severe mechanical action.
Although the protein purification technology is very mature, it is still a difficult point in protein purification research for some extremely unstable light-sensitive proteins.

Method used

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  • A method for preparing LPOR protein crystals
  • A method for preparing LPOR protein crystals
  • A method for preparing LPOR protein crystals

Examples

Experimental program
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Effect test

Embodiment 1

[0045] LPOR protein expression.

[0046]The plasmid pEBSRC-TEV-LPOR was transformed into C43 host cells, the plasmid pEBSRC-TEV-LPOR had Amp resistance, and the host C43 had no resistance. The medium used in the experiment was LB, and the plates were inverted and cultured overnight at 37°C. Pick the single clone on the transformation plate and transfer it to a 100mL Erlenmeyer flask containing 100mg / L Amp, culture overnight at 37°C and 220r / min, and transfer it to a 1L Erlenmeyer flask containing antibiotics on the second day with 1% inoculum , 37°C, 220r / min to continue culturing. After culturing for 4 h, IPTG was added with a final concentration of 0.4 mM to induce overnight. After the end of the induction, centrifuge at 6000r / min for 20min to collect the bacteria, discard the supernatant, and suspend the bacteria with buffer I.

Embodiment 2

[0048] Purification of LPOR protein

[0049] Such as figure 1 As shown, the specific steps for the purification of LPOR protein are as follows: the bacterial cell suspension is first crushed by a low-temperature and high-pressure cell crusher, and the crushing conditions are as follows: temperature 4°C, pressure 1000 MPa, crushing three times. The crushed solution was centrifuged at 16000r / min for 60 minutes to remove the cell debris in the precipitate, and the supernatant was the crude enzyme solution containing the target protein.

[0050] Pass the crude enzyme solution through the Ni affinity chromatography column, adsorb the target protein on the Ni column, then use buffer I to equilibrate the purification column, and then use buffer II, buffer III and buffer IV to wash the purification column respectively, and each eluted protein The peaks were collected separately for verification by SDS-PAGE, in which LPOR was eluted by buffer III, and the buffer III eluted protein was...

Embodiment 3

[0058] Crystallization of LPOR proteins

[0059] Use Amicon Ultra-30k ultrafiltration membrane to concentrate protein, and use Nanodrop to measure the concentration to be 30mg / mL. After taking 1 μL of concentrated protein for SDS-PAGE to verify the purity and concentration, use conventional crystallization buffer (2-methyl-2, 4-pentanediol) began to point crystals, and the manipulator was used to screen, and a large number of crystal points were carried out for the crystallization conditions of long crystals;

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Abstract

The invention discloses a method for preparing LPOR protein crystals, in particular comprising LPOR protein expression, LPOR protein purification, TEV enzyme digestion, Ni affinity chromatography, molecular sieve and crystallization process. The invention provides a method for crystallizing an LPOR protein, thereby achieving the purpose of analyzing the structure of the LPOR protein and laying a foundation for the structural analysis of other photosensitive overspeed proteins. At the same time, the method lays a foundation for clarifying and defining the catalytic mechanism of photosensitive ultrafast proteins or engineering them.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a method for preparing LPOR protein crystals. Background technique [0002] LPOR (light-dependent protochlorophyllide oxidoreductase) is a key enzyme in the synthesis of chlorophyll in cyanobacteria, algae and multicellular plants. In the photosynthesis pathway, organisms catalyze the reduction of protochlorophyllide Pchlide with two different reductases: one is light-dependent LPOR (light-dependent NADPH: protochlorophyllide oxidoreductase); the other is light-independent oxidation of Pchlide Reductase DPOR (light-independent Pchlide reductase). LPOR or DPOR, as prochlorophyllate reduction catalyzing enzyme, is the key enzyme of chlorophyll synthesis in phototrophic organisms. These two enzymes are widely found in phototrophic organisms. The encoding and catalytic reaction mechanism are completely different. DPOR is encoded by the chloroplast genome and consists o...

Claims

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Application Information

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IPC IPC(8): C12N9/02
CPCC12N9/0004
Inventor 程奇孙文丽
Owner ZHEJIANG SHANCE HEQISHI BIO SCI& TECH CO LTD
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