Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain

A technology for amylase inhibitors and storage proteins, which is applied in the production and purification of storage protein-type α-amylase inhibitors in chickpea grains, and can solve the problems of low content, unsuitable protein extraction, separation, development and utilization, and complex processes, etc. question

Active Publication Date: 2015-08-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the content of this type of storage protein α-amylase inhibitor in chickpea grains is very small, the extraction and purification of natural chickpea grain materials is complicated and expensive, and it is not suitable for the extraction, separation, development and utilization of this type of protein

Method used

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  • Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain
  • Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain
  • Production and purification method of storage protein type alpha-amylase inhibitor with Cupin structure function domain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] (1) Construction of prokaryotic expression system and induced expression in bacterial culture:

[0091] a. take the cDNA of seeds in the development of Desi type chickpea as a template, obtain the ORF sequence of coding α-amylase inhibitor protein (it is numbered as Q9SMJ4 in NCBI) by PCR method, and primer sequence is

[0092] F1: 5'-TGTCATCATGGCTAAGCTTCTTG-3' (SEQ ID NO.1) and

[0093] R1: 5'-TTGTTTCCGCTAAAAGGTACATG-3' (SEQ ID NO.2);

[0094]b. Use PCR technology to add restriction enzyme site sequences EcoRI and NotI to the 5' and 3' ends of the ORF sequence encoding the Q9SMJ4 protein (removing the signal peptide sequence of the encoded protein), respectively, to obtain the target gene fragment, and the primer sequence is

[0095] F2:5'-CG GAATTC TTGAGAGATCAACCT-3' (SEQ ID NO.3) and

[0096] R2:5'-TTTTCCTTTT GCGGCCGCC AGCTGCTGCTTTGTT-3' (SEQ ID NO. 4);

[0097] c. Use restriction endonucleases EcoR Ⅰ and Not Ⅰ to digest the target gene fragment obtained in st...

Embodiment 2

[0116] a. 5000rpm, centrifuge at 4°C for 5min to collect the bacterial cells in the bacterial liquid in Step (1) of Example 1 after induction, and discard the supernatant;

[0117] b. Resuspend the bacteria in 10 mL of 0.1M phosphate buffer (pH=8.0) for every 50 mL of cultured bacteria collected;

[0118] c. Place the resuspended bacteria solution on ice, and ultrasonically disrupt until the bacterial solution becomes clear. The ultrasonic conditions are ultrasonic disruption for 3 seconds, cooling for 5 seconds, and a power of 400 watts;

[0119] d. Centrifuge the sonicated bacterial solution at 12000rpm at 4°C for 10min, collect the precipitate, and discard the supernatant;

[0120] e. Wash the precipitate once with 0.1M phosphate buffer (pH=8.0), centrifuge at 12000rpm, 4°C for 10min, and collect the precipitate;

[0121] f. Use 10mL solution (100mM NaH 2 PO 4 , 10mM Tris-HCl, 100mM DTT, 8M urea, pH=8.0) to dissolve the precipitate, and incubate at room temperature for 1...

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Abstract

The invention discloses a production and purification method of a storage protein type alpha-amylase inhibitor with a Cupin structure function domain, comprising the steps of: 1, construction of a rokaryotic expression system and bacterium solution cultivation inducible expression; 2, prokaryotic expression of soluble protein purification; and / or 3, prokaryotic expression of inclusion body protein purification and protein renaturation. At present, multiple repeated experiments test that, the protein obtained through producing the alpha-amylase inhibitor by using the technical process provided by the invention is high in yield and purity, the production process is simple and convenient, the production cycle is short, expensive devices are not needed, and thereby the production and purification method is an excellent bioprocess technology for producing and purifying the alpha-amylase inhibitor.

Description

technical field [0001] The invention belongs to the field of biotechnology and food processing, and relates to a production and purification method of a storage protein type α-amylase inhibitor with a Cupin structural functional domain, in particular to a storage protein type α-amylase inhibitor in chickpea grains production and purification methods. Background technique [0002] Carbohydrates, fats and proteins are the three nutrients that provide most of the energy for the human body, of which carbohydrates provide about 60%-70% of the calories. If carbohydrates are consumed too much, they will be converted into fat accumulation In the body, excessive intake and surplus will lead to obesity, high blood sugar, diabetes and other "rich man's disease". [0003] Chickpeas, whose grains are as sharp as chickpeas, are also called peach beans, sheep's head beans, Nuohuti, etc., and have been cultivated in Xinjiang for more than 2,500 years. Chickpea is a commonly used medicinal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/29C07K14/415C07K1/36C07K1/34C07K1/22
Inventor 麻浩王占奎王爽克玉木·米吉提王泽顾爱星张桦
Owner NANJING AGRICULTURAL UNIVERSITY
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