Production and purification method of a storage protein type alpha-amylase inhibitor with cupin structural functional domain
A technology for amylase inhibitors and protein storage, applied in biochemical equipment and methods, botany equipment and methods, plant peptides, etc., can solve the problems of high cost, inappropriate protein extraction, separation, development and utilization, and low content
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Embodiment 1
[0090] (1) Construction of prokaryotic expression system and induced expression by 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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