| TEV protease(tobacco etch virus protease) is a 27 KD protein catalytic area encoded in NIa protein of plaque tobacco virus.Because it can specifically recognize the specific sequencecontaining 6 amino acids(ENLYFQ) and cut the protein containing the sequence having higher enzyme activity and enzyme digestion conditions with forbearance. Therefore it plays an important role in the field of recombinant proteins.Our laboratory had cloned TEV fragments successfully by using the method of overlapping PCR based on e. coli preference codon and TEV gene sequence, At present the study of TEV enzyme mainly concentrate in improving the expression and activity of TEV enzymethrough different methods, and the TEV enzyme immobilization, For the TEV enzyme activity detection, mostly means are by semi-quantitative analysis, namely is analyzed by analysis software on the size of the stripe by SDS-PAGE after enzyme cutting protein.This experiment build different recombinant carrier such as p ET28b-MBP-his- TEV?p ET28b-MBP-his-ACP-TEV?p ET28b-MBP-ybb R-his-TEV successfully and succeed to express and purify the target protein, found that the protein groups ACP and the flexible short peptide ybb R had a promoting effect for TEV enzyme activity,and short peptide ybb R has an evident promoting effect; ACP and ybb R were known as be available to participate in immobilization,so the analysis of the accurate enzyme activity will provide technical support for the immobilization of TEV enzyme,for this reason, this experiment builds a series of recombinant carrier such as p BAD24, p BAD24-?, p BAD24-his-MBP-?, p BAD24- his-MBP2-?, after conversion in DH5?, preliminary finding it can form ?-complementary to produce beta- galactose glucoside enzyme, chromogenic reaction of beta- galactose glucoside enzyme can indirectly research TEV enzyme activity, it will lay the foundation on how to measure the enzyme activity of TEV enzyme with spectrophotometry by quantitative. |
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