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Method for preparing cyanobacterial phytochrome fluorescent marker with orange-red fluorescence

A technology of phytochrome and cyanobacteria, which is applied in the direction of bacterial peptides, chemical instruments and methods, and peptides containing affinity tags, etc., can solve the problems of unstable expression and difficulty in obtaining qualified products with stable quality, so as to increase production, Effects of improving strain stability and screening efficiency, and reducing the number of genes

Inactive Publication Date: 2018-05-18
GUANGZHOU TEBSUN BIO TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of fusion proteins is often limited by the design of the fusion gene and the fermentation process, and cannot be expressed stably, making it difficult to obtain qualified products with stable quality

Method used

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  • Method for preparing cyanobacterial phytochrome fluorescent marker with orange-red fluorescence
  • Method for preparing cyanobacterial phytochrome fluorescent marker with orange-red fluorescence
  • Method for preparing cyanobacterial phytochrome fluorescent marker with orange-red fluorescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Sequence Selection of Cyanobacterial Phytochrome GAF Domain

[0034] The cyanobacterial phytochrome gene sequence alr5272 (algal species PCC7120) has two GAF domains with high homology. The GAF domain gene sequence selected in this program is the first domain gene sequence, which is hereinafter referred to as the gaf gene sequence. The amino acid sequence of the expressed protein is as follows:

[0035] ESLDLETILYQTAKDLRQCLKCDRILIYQIKSDNNGAIVAESTILPNVSLLGKHFRDPCFTGKYKERQGRCCLEIIEDIYAAGVKPCQRDFLASMQVRANIVVPIALKSDLWGLLIAQYCDEPHQWQQIEIDLLKQLATQLGIAIQNTK (SEQ ID NO: 1).

[0036] Selection of transgenic plasmid combinations

[0037] This protocol uses the Duet series double plasmid combination, the sa::gaf fusion gene is inserted into the multiple cloning site 1 of pET Duet; the phycoerythrin synthase plasmid uses pACYC Duet-ho1-pebS.

[0038] The fusion design of streptavidin sa sequence, gaf sequence and 6his-taq affinity tag sequence was carried out, and the gene seque...

Embodiment 2

[0073] Get the monoclonal bacterial classification that the fermentation of embodiment 1 uses, carry out fermentation as follows.

[0074] 1) Preparation of primary seed solution: Take 20 μL of qualified monoclonal bacterial seed solution, insert it into 25 mL of LB medium, shake overnight at 37°C to a saturated concentration;

[0075] 2) Prepare the secondary seed solution: transfer 5mL of the primary seed solution into 1.5L LB medium, culture with medium-speed shaking at 37°C for 3-4h, OD 600 Control below 0.5;

[0076] 3) Inoculation: transfer 1.5L secondary seed solution to a 200L fermenter, medium 150L, ​​fermentation medium formula: peptone 1%, yeast powder 0.5%, sodium chloride 1%, glycerin 0.4%, blood red 0.004% element, the pH value was adjusted to 7.2 with NaOH solution;

[0077] 4) Cultivation: 37°C, 300rpm medium speed cultivation to the early growth stage, the duration is about 1h;

[0078] 5) Cool down: set the tank temperature to 20°C, reduce the rotation spe...

Embodiment 3

[0082] Get the monoclonal bacterial classification that the fermentation of embodiment 1 uses, carry out fermentation as follows.

[0083] 1) Preparation of primary seed solution: Take 20 μL of qualified monoclonal bacterial seed solution, insert it into 25 mL of LB medium, shake overnight at 37°C to a saturated concentration;

[0084] 2) Prepare the secondary seed solution: transfer 5mL of the primary seed solution into 1.5L LB medium, culture with medium-speed shaking at 37°C for 3-4h, OD 600 Control below 0.5;

[0085] 3) Inoculation: transfer 1.5L of secondary seed solution to a 200L fermenter, 150L of culture medium, fermentation medium formula: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 0.4% glycerin, pH value NaOH solution adjusted to 7.2;

[0086] 4) Cultivation: 37°C, 300rpm medium speed cultivation to the early growth stage, the duration is about 1h;

[0087] 5) Cool down: set the tank temperature to 20°C, reduce the rotation speed to 150rpm, and culture a...

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Abstract

The invention discloses a method for preparing a cyanobacterial phytochrome fluorescent marker with orange-red fluorescence. Fusion protein comprises a first GAF structural domain of cyanobacterial phytochrome protein alr5272 and streptavidin. A fusion protein sequence of the fluorescent marker is easily and stably expressed in microorganisms, and the defects that acquirement of high-purity natural phycobiliprotein and cyanobacterial phytochrome is difficult, the preparation cost is high, chemical modifiers are used and the polymerization form of natural phycobiliprotein is unstable are avoided. According to an expression method of the fluorescent marker, participation of phycobiliprotein lyase is not required, the number of transformed genes is reduced, and the stability and screening efficiency of bacterial strains are improved. At the same time, the yield of fusion proteins is greatly increased by optimizing a fermentation culture medium and fermentation conditions.

Description

technical field [0001] The invention relates to the field of fusion protein expression, in particular to a method for preparing a phytochrome fluorescent marker. Background technique [0002] Fluorescent probes (or fluorescent markers) are important basic raw materials for fluorescent immunoassay technology, which are mainly obtained by combining the fluorescent substrate with the biotin-streptavidin system through chemical modification, which can further amplify the fluorescent signal and improve Sensitivity of immunoassay. [0003] Natural phycobiliprotein is one of the most common fluorescent substrates. Its natural structure is generally hexamer, and its subunits are composed of apoprotein and phycobilichrome, which are catalyzed by lyase. Phycobilichromes are formed from heme catalyzed by heme oxidase (HO1) and various biliverdin reductases. Common phycobilichromes include phycoerythrin (PEB), phycourobilin (PUB), Cholin (PCB) and Phycopurinoid (PVB). By combining di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC07K14/195C07K2319/20C12N15/70
Inventor 宋建勋任虎夏坤付卫雷佟顺刚
Owner GUANGZHOU TEBSUN BIO TECH DEV
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