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Preparation of allophycocyanin fluorescent protein

A technology of allophycocyanin and phycocyanin, applied in the fields of botanical equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of unstable strains and complicated construction process, and achieve easy separation. Purification, simple purification method, easy cross-linking and cell entry effect

Inactive Publication Date: 2009-05-27
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to create fluorochrome genes that cannot exist naturally due to their complexity. It involves combining two components called Aequochromium (A) and Phytolum caffraquinone/allophanide). These components act like chemical reactions but they don't mix well together easily during synthesis. By inserting these DNA sequences into an appropriate yeast strain, we could make them easier to separate and manipulate without losing any important parts such as amino acids or sugars. Overall, our technical effect lies within the use of green plants instead of fossil fuels while maintaining good quality products containing fluorocompound(FIC), aluminum pyrochlorogranosite crystal structure, and cyanoanthrin hemiacetyl group elements.

Problems solved by technology

This patents describes various methods for extracting phytosideroxylindole compounds that have unique structures such as PEA, PAHY, alkaloid pyridone nucleus, etc., making them useful materials for medical purposes like diagnosis and treatment of diseases caused by bacterial pathogens. These techniques involve isolating these substances from their host organisms' metabolites, followed by analysis thereof based on spectral characteristics.

Method used

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  • Preparation of allophycocyanin fluorescent protein
  • Preparation of allophycocyanin fluorescent protein
  • Preparation of allophycocyanin fluorescent protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) It can be found from Genebank that the complete sequence of the algae Synechocystis sp. PCC6803 has been determined. The number of Synechocystis sp.PCC6803 in Genebank is: BA000022, and the desired gene can be found from the complete sequence. According to the apcA, hox1, and pcyA gene sequences of Synechocystis sp. PCC6803 found in Genebank, primers were designed (the primers and reaction conditions used are shown in the table below), and the corresponding genes were cloned by PCR using Genomic DNA of Synechocystis sp. PCC6803 as a template.

[0033]

[0034]

[0035] (2) Allophycocyanin apoprotein apcA in Synechocystis sp.PCC6803 1 Gene cloned in Novagen's pCDFDuet TM In the -1 vector, the step of inserting the gene into the vector is: apcA obtained by PCR amplification in step 1) 1 The gene fragment was electrophoresed (120V, 40 minutes), and apcA was recovered 1 The PCR product fragment; the resulting fragment was double-digested with the designed restr...

Embodiment 2

[0044] (1) It can be found from Genebank that the complete sequence of the algal species Synechocystis sp. PCC6803 has been determined. The number of Anabaena sp.Synechocystis sp.PCC6803 in Genebank is: BA000022, and the required gene can be found from the complete sequence. According to the apcA, hox1, and pcyA gene sequences of Synechocystis sp. PCC6803 found in Genebank, primers were designed (the primers and reaction conditions used are shown in the table below), and the corresponding genes were cloned by PCR using Anabaena sp. PCC7120 genomic DNA as a template.

[0045]

[0046]

[0047] (2) Allophycocyanin class apoprotein gene apcA in Anabaena sp.PCC7120 2 Cloned in pCDFDuet from Novagen TM In the -1 vector, the step of inserting the gene into the vector is: apcA obtained by PCR amplification in the previous step 2 The gene fragment was electrophoresed (120V, 40 minutes), and apcA was recovered 2 The fragment of the PCR product; the resulting fragment was doubl...

Embodiment 3

[0055] (1) It can be found from Genebank that the complete sequence of the algal species Synechocystis sp. PCC6803 has been determined. The number of Anabaena sp.Synechocystis sp.PCC6803 in Genebank is: BA000022, and the required gene can be found from the complete sequence. According to the apcA, hox1, and pcyA gene sequences of Synechocystis sp.PCC6803 found in Genebank, primers were designed (the primers and reaction conditions used are as follows), and unsequenced Chroococcales (Chroococcales) algae genomic DNA was used as a template for PCR Clone the corresponding gene.

[0056]

[0057]

[0058] (2) Allophycocyanins apoprotein gene apcA from Chroococcales algae 3 Cloned in pCDFDuet from Novagen TM In the -1 vector, the step of inserting the gene into the vector is: apcA obtained by PCR amplification in the previous step 3 The gene fragment was electrophoresed (120V, 40 minutes), and apcA was recovered 3 The fragment of the PCR product; the resulting fragment wa...

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Abstract

The invention belongs to the field of pigment-protein materials in biotechnology, and particularly relates to a method for preparing novel fluorescent protein. The method comprises the following concrete steps: 1) an allophycocyanin apoprotein gene or a homologous gene of the allophycocyanin apoprotein gene and a phycobilin biosynthetic enzyme gene (Hox1 and pcyA) are cloned into the same expression vector to obtain an expression plasmid of apoprotein and pigment biosynthetic enzyme; and 2) the expression plasmid is transformed into a host strain to obtain a corresponding engineering strain, the engineering strain is fermented to produce allophycocyanin fluorescent protein, and corresponding allophycocyanin fluorescent protein is obtained through one-step purification by the prior protein purification technology after the fermentation. The method utilizes the bioprocess for production and is an environmental-friendly production method; and the protein has the fluorescent characteristic different from natural protein and realizes the modification of the biotechnology to the natural protein.

Description

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Claims

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Application Information

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Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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