Method for producing heme binding protein

A technology for binding protein and hemoglobin, which is applied in the field of hemoglobin-binding protein production to achieve the effect of reducing production cost and saving energy

Pending Publication Date: 2022-04-12
HANGZHOU RUIFENG BIOTECH LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for utilizing transgenic soybeans to produce hemopexin (hemoglobin / myoglobin) for the deficiencies in the current production methods of hemoglobin / myoglobin. and sustainable production of hemopexin without regard to heme supply

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  • Method for producing heme binding protein
  • Method for producing heme binding protein
  • Method for producing heme binding protein

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1, the synthesis of hemoglobin / myoglobin gene

[0032] Through the NCBI gene database, directly search the hemoglobin / myoglobin gene sequence from different sources. For example, enter Soybean leghemoglobin to search for several soybean hemoglobin genes, randomly select the gene LBC2 (GeneID: 100527379); artificially synthesize the soybean (Glycine max) hemoglobin gene LGH according to the plant codon preference, the amino acid is shown in SEQ ID NO: 1, The nucleotide sequence is shown in SEQ ID NO:3.

[0033] Enter bovine myoglobin to search for a bovine myoglobin gene MB (Gene ID: 280695). The bovine (Bostaurus) myoglobin gene BMG is artificially synthesized according to the plant codon preference, the amino acid sequence is shown in SEQ ID NO:2, and the nucleotide sequence is shown in SEQ ID NO:4.

Embodiment 2

[0034] Embodiment 2, construction soybean genetic transformation vector

[0035] Theoretically, for the 2 kinds of hemoglobin / myoglobin genes in Example 1 of the present invention, a promoter that can initiate high gene expression in soybean seeds is added at the 5' end, and a terminator is added at the 3' end to obtain The expression box for expressing hemoglobin / myoglobin, and further, other elements for enhancing expression can be added to the expression box for hemoglobin / myoglobin. Then construct a plant genetic transformation vector containing a selectable marker. The plant genetic transformation vectors expressing different hemoglobin / myoglobin in soybean seeds mainly differ only in the gene encoding hemoglobin / myoglobin.

[0036] 1. Binary T-DNA transformation vector GLLGH

[0037] Taking the soybean hemoglobin LGH gene whose nucleotide sequence was synthesized in Example 1 as shown in SEQ ID NO:3 as an example, the Arabidopsis pAtUbi10 promoter (478-1741bp in SEQ ID...

Embodiment 3

[0054] Embodiment 3, the acquisition of transgenic soybean

[0055] The soybean transformation process mainly refers to Paz's Agrobacterium-mediated half-seed transformation method (Paz MM, MartinezJC, Kalvig AB, et al. (2006), Improved cotyledonary node method using analternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Reports, 25(3):206-213). details as follows:

[0056] ①Sterilization of soybean seeds: Select high-quality soybean seeds (variety: Wandou 28), put them in a desiccator, and treat them with chlorine gas for 16 hours to sterilize. Chlorine gas was generated by slowly adding 8 mL of concentrated hydrochloric acid to a beaker containing 100 mL of 30% (v / v) aqueous sodium hypochlorite in a desiccator.

[0057] ② Seed germination: Soak the sterilized soybean seeds in step ① in sterile distilled water at room temperature for 16 hours to obtain germinated soybean seeds.

[0058]3. Preparation of Agroba...

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Abstract

The invention discloses a method for producing a heme-binding protein, which is characterized by comprising the following steps: transferring an expression vector containing a heme-binding protein gene expression cassette into a soybean genome and expressing to obtain soybean seeds rich in heme-binding protein, and separating and extracting to obtain the heme-binding protein. The invention provides a method for producing hemoglobin/myoglobin by using transgenic soybean seeds. The hemoglobin/myoglobin produced by the method can be used as a coloring additive and a flavor regulator of food; the invention discloses a promoter and a regulatory sequence which can be highly expressed in soybean seeds. The content of hemoglobin/myoglobin in the soybean seeds of a high-expression strain reaches 0.1% or above of the weight of the seeds; according to the hemoglobin/myoglobin produced from the transgenic soybeans, the production cost is reduced, energy is saved compared with a traditional microbial fermentation method, and the method is a more environment-friendly production mode.

Description

(1) Technical field [0001] The invention relates to the production of hemopexin, in particular to a method for producing hemopexin by utilizing transgenic soybean expressing hemopexin. (2) Background technology [0002] Hemoglobin / myoglobin is a common hemoglobin protein, including animal myoglobin and plant hemoglobin. With the rise of the vegetable meat industry, as an important coloring and flavor additive in vegetable meat, the demand for hemoglobin / myoglobin is increasing. A method for mass production of hemoglobin / myoglobin is urgently needed in real production. [0003] Livestock blood can be used as a source of hemoglobin, but the extraction process is cumbersome and there is a risk of pathogen contamination, so it is not suitable for large-scale industrial production. [0004] Microbial fermentation is also an important way to prepare hemoglobin. For example, Chinese patent application 2016800292580.4 and Chinese patent application 202110607028.1 disclose the prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/805C07K14/415C07K14/47C12N15/12C12N15/113C12N15/29C12N15/84C12N15/65C12N5/10A01H5/10A01H6/54
Inventor 郑挺赵宇沈志成
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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