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Fermentation and one-step purifying method for obtaining aromatic isoprenyl transferase

A technology of isopentenyl and transferase, applied in the biological field, can solve problems such as sedimentation, inactive inclusion bodies, protein aggregation, etc., and achieve the effects of simplified purification steps, improved activity, and simple process

Active Publication Date: 2019-05-17
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the E. coli expression system has some disadvantages, for example, high levels of protein expression and inappropriate expression conditions will lead to protein aggregation and sedimentation, resulting in the formation of inactive inclusion bodies

Method used

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  • Fermentation and one-step purifying method for obtaining aromatic isoprenyl transferase
  • Fermentation and one-step purifying method for obtaining aromatic isoprenyl transferase
  • Fermentation and one-step purifying method for obtaining aromatic isoprenyl transferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: A fermentation and one-step purification method for obtaining aromatic prenyltransferase

[0039] Construct the expression vector:

[0040] Get the fusion tag MBP gene:

[0041] Search the MBP sequence on NCBI, and design two pairs of primers according to the sequence:

[0042] F1:5'-CATGCCATGGGCCAAAATTGAAGAAGGTA-3';

[0043] R1: 5'- CCCAAGCTT GGGGTACCTCAGCTGCCCGCATTAGTCT-3';

[0044] F2: 5'-GGAATTCCATATGGCCAAAATTGAAGAAGGTA-3';

[0045] R2: 5'-GGGGTACCTCAGCTGCCCGCATTAGTCTGC-3';

[0046] Using the pMBP plasmid as a template, use primers F1 and R1 for PCR amplification. The PCR amplification system is: 1 μL pMBP plasmid, add 1 μL of 10 μM primers, add 12.5 μL PCR Mix, add ddH 2 O to make up to 25 μL. The amplification parameters were denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 100 s, extension at 72°C for 2 min, and 30 cycles. Fragment 1 was obtained after purification by a PCR purificat...

Embodiment 2

[0059] Example 2: Obtaining soluble aromatic prenyltransferase pure enzyme MBP-NovQ protein

[0060] Pick a single colony of positive Escherichia coli BL21(DE3)-pET28a-MBP-NovQ, inoculate it in LB liquid medium, fill a 250mL shake flask with a liquid volume of 50mL, and cultivate it at 37°C and 200rpm for 18h to obtain activated Escherichia coli BL21(DE3 )-pET28a-MBP-NovQ bacterial solution;

[0061] Absorb 500 μL of the activated E. coli BL21(DE3)-pET28a-MBP-NovQ bacteria solution and inoculate it into a new LB medium. The liquid volume in a 250mL shaker flask is 50mL. Shake at 37°C and 200rpm until the OD value of the bacteria solution is 0.6- 0.8, to obtain Escherichia coli BL21(DE3)-pET28a-MBP-NovQ induction pre-bacteria liquid;

[0062] Add IPTG at a final concentration of 0.5 mM to the above-mentioned pre-induction bacterial solution, culture at 22° C. with shaking at 180 rpm for 3-4 hours. The fermentation broth was centrifuged at 6000 g for 5 min to remove the cultur...

Embodiment 3

[0065] Example 3: Obtaining soluble aromatic prenyltransferase pure enzyme NovQ protein

[0066] (1) Pick a single colony of positive Escherichia coli Rosetta(DE3)-pETDuet-1-MBP-NovQ, inoculate it in LB liquid medium, fill a 250mL shake flask with 50mL, shake at 37°C and 220rpm for 16h, and obtain the activated Escherichia coli Rosetta(DE3)-pETDuet-1-MBP-NovQ bacterial liquid;

[0067] (2) Inoculate 500 μL of the activated Escherichia coli Rosetta(DE3)-pETDuet-1-MBP-NovQ bacterial solution into a new LB medium, fill a 250mL shaker flask with 50mL, shake at 37°C and 220rpm until the bacterial solution is reached The OD value is 0.6-0.8, and the pre-induction bacterial liquid of Escherichia coli Rosetta(DE3)-pETDuet-1-MBP-NovQ is obtained;

[0068] (3) Add IPTG with a final concentration of 0.3mM to the above-mentioned pre-induction bacterial solution, shake and culture at 20°C and 180rpm for 4h. The fermentation broth was centrifuged at 6000g for 5min to remove the culture me...

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Abstract

The invention discloses a fermentation and one-step purifying method for obtaining aromatic isoprenyl transferase. The method comprises the steps that a large amount of soluble isoprenyl transferase NovQ can be obtained in cytosol of cells, high-activity proteins can be obtained through one-step purification, and the obtaining efficiency of proteins is improved. According to the method, zymoprotein with higher purity is obtained under the premise that an enzymatic result is not damaged by using the coexpression of a fusion label and the NovQ protein. Compared with other aromatic isoprenyl transferase acquisition methods, the yield is high, the enzymatic purifying and extracting method is simple, and the method has wide application prospects in in vivo and in vitro generating process of terpene.

Description

technical field [0001] The present invention relates to a fermentation and one-step purification method for obtaining aromatic prenyl transferase, in particular to a method for soluble expression of aromatic prenyl transferase NovQ in Escherichia coli and its application in the production of vitamin K2 , belonging to the field of biotechnology. Background technique [0002] Soluble aromatic prenyltransferase (soluble aromatic prenyltransferase) is a key enzyme in many biosynthetic processes, catalyzing the prenylation of aromatic substrates to form multi-level metabolites. Soluble prenyltransferase has a wide range of substrates, such as 4-hydroxyphenylpyruvate, resveratrol and p-coumaric acid can be prenylated to produce many physiologically and pharmacologically active products. [0003] In recent years, there have been many research reports on ABBA soluble prenyltransferases derived from actinomycetes at home and abroad. They have similar (N / D)DxxD structures and have be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/70C12N15/54C12N9/10C12P7/66
Inventor 郑之明倪文枫赵根海刘会王鹏王丽王晗孙小雯吴荷芳杨强方志伟唐恒芳
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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