Expression system for producing recombination tryptophanase and preparation method and application of recombination tryptophanase

A technology of tryptophanase and expression system, applied in peptide preparation methods, biochemical equipment and methods, botany equipment and methods, etc., can solve problems such as labor-intensive and time-consuming, protein loss, and low tryptophanase activity , to achieve the effect of reducing production cost and improving recovery rate

Inactive Publication Date: 2004-05-19
WUHAN UNIV
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Problems solved by technology

However, the separation and purification of this expressed protein is difficult, the recovery rate is only 20%, and the tryptophanase activity produced by this expression vector is low, many proteins are lost in the separation and purification, and it is laborious and time-consuming, especially the preparation of a large amount of color This is especially true for the amino acid enzyme protein

Method used

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  • Expression system for producing recombination tryptophanase and preparation method and application of recombination tryptophanase
  • Expression system for producing recombination tryptophanase and preparation method and application of recombination tryptophanase
  • Expression system for producing recombination tryptophanase and preparation method and application of recombination tryptophanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0034] Example: 1. Construction of tryptophanase protein expression vector

[0035] PCR amplification of tryptophanase gene

[0036] Use a sterilized pipette tip to pick a single colony of E.coli TG1 and suspend it as a template for cloning genes, and float it in 20 μL of lysate (the composition of the lysate is: 0.5 volumes of NP-40 and Tween-20, respectively, dissolved in 99 volumes of double Distilled water, mix evenly, each concentration is 0.5%), add 20 μL of water, fully shake to disperse, incubate at 100C for 15 min, centrifuge (15000 rpm, 10 min) to take 10 μL of supernatant for PCR reaction with a total volume of 100 μL. The PCR reaction system is 100 μL, and the sample addition list is as follows:

[0037] 10×buffer: 10μL

[0038] Primer 1: 1 μL

[0039] Primer 2: 1 μL

[0040] 4dNTPs: 2 μL

[0041] Sterilized ddH 2 O: 75 μL

[0042] Template 10μL

[0043] Taq DNA polymerase: 1 μL

[0044] Pre-denaturation at 95°C for 5min

[0045] Cyclic conditions: denatu...

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Abstract

An expression carrier for preparing the recombinant tryptophanase is pEVT (CCTCC M202038). The process for preparing said expression carrier and the process for purifying bacterial spawn and preparing tryptophanase protein are disclosed. As the tryptophanase fusion protein expressed by said recombinant carrier contains 6 continuous histidines, it is easy to bind the Ni-affinity fixed histidine-protain chromatographic column and the histidine does not affect the activity of target protein, so resulting in high recovery rate and purify of target protein (more than 60% and more than 95%) and low cost.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, in particular, the invention relates to an expression system for producing recombinant tryptophanase, and also relates to a preparation method of recombinant tryptophanase and its application in tryptophan production. Background technique: [0002] L-tryptophan, an aromatic amino acid, is an essential amino acid in humans and animals, and is very important for the nutritional supply and growth promotion of humans and animals. It is widely used in the pharmaceutical and food industries, and is the third largest added amino acid in animal feed after threonine and lysine. [0003] The research and production of tryptophan have gone through more than 20 years. There are various production methods, mainly including proteolytic extraction, chemical synthesis, microbial fermentation and enzymatic transformation. As far as the current situation is concerned, no matter in terms of high yield or high eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/36C12N1/21C12N9/88C12N15/60C12N15/70C12P13/22
Inventor 王业富龚睿
Owner WUHAN UNIV
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