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Production method and application of penaeus chinensis COMT (Catechol-O-methyltransferase) recombinant protein

A Chinese prawn and recombinant protein technology, which is applied to the production method of Chinese prawn COMT recombinant protein and its application field, can solve the problems of modification to reduce enzyme activity, limit industrial production, and high production costs, achieve high enzyme activity, overcome expression and The activity is greatly reduced, avoiding the effect of post-treatment

Active Publication Date: 2014-02-26
山东辰泰药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the production of Chinese prawn COMT recombinant protein is only reported by us in 2006 and 2010. This method is to express recombinant COMT by using the Escherichia coli prokaryotic expression system. The lack of post-translational modification of the recombinant COMT will reduce the enzyme activity, and the recombinant COMT needs to be purified. COMT must go through cumbersome steps such as bacterial cell centrifugal recovery, fragmentation, and affinity chromatography. The production cost is high and the efficiency is low, which limits its industrial production.

Method used

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  • Production method and application of penaeus chinensis COMT (Catechol-O-methyltransferase) recombinant protein
  • Production method and application of penaeus chinensis COMT (Catechol-O-methyltransferase) recombinant protein
  • Production method and application of penaeus chinensis COMT (Catechol-O-methyltransferase) recombinant protein

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Experimental program
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Effect test

Embodiment 1

[0031] The expression and purification of COMT recombinase mainly include the following steps:

[0032] i. Express : 3 × 10 per shrimp 6 The amount of cfu bacteria, the mixed Vibrio anguillarum ( Vibrio anguillarum ) and Staphylococcus aureus ( Staphylococcus aureus ) live bacteria were injected into the bottom of the telomere of prawns, and after 24 hours of aerated feeding, the total RNA of induced prawn hemocytes was extracted and reverse-transcribed into SMART cDNA as a template, and the synthesis containing Eco R I and not The forward and reverse primers of the I endonuclease site, the primer sequence is:

[0033] COMT ExF: 5'-TACTCA GAATTC ATGTCTTCTCTGAAGAGTTAC-3'

[0034] COMT ExR: 5’-TACTCA GCGGCCGC TCA ATG ATG ATG ATG ATG ATG

[0035] TTTTTTAAAACAGAGAGACAAGC-3'

[0036] Through polymerase chain reaction (PCR), the COMT ORF of 666bp was amplified, and it was cloned into pPIC9K plasmid (Invitrogen), and the recombinant plas...

Embodiment 2

[0041] The enzymatic activity of the purified COMT protein was determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The main steps include:

[0042] ⅰ. Preparation of reaction system: Design ①, ②, ③ three groups of 1 mL reaction solution, except 100 mM phosphate buffer (pH7.4), 5 mM MgCl 2 , 200 μM DHBAc, 200 μM SAM (added later), ① group without COMT recombinant protein, as a control; ② group with COMT recombinant protein; ③ group with COMT recombinant protein and internal standard - vanillic acid.

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Abstract

The invention belongs to the field of gene product development and application and relates to a production method and application of a penaeus chinensis COMT (Catechol-O-methyltransferase) recombinant protein. The technical scheme is as follows: the production method of the penaeus chinensis COMT recombinant protein comprises the following steps of (1) constructing a comt / pPIC9K recombinant expression vector; (2) obtaining a Pichia pastoris expression strain comt / pPIC9K / KM71 with a large transformant copy number; (3) carrying out fermentation culture and inducible expression on transformants; and (4) purifying COMT. The COMT prepared from eukaryon-Pichia pastoris by using the method has translated modification so as to be more natural in structure and higher in enzymatic activity; the COMT is directly purified by using a yeast liquid, so that the complex protokaryotic thallus recycling and smashing steps are omitted, the production cost is reduced, and the purification efficiency is increased.

Description

technical field [0001] The invention belongs to the field of gene product development and application, and relates to a production method and application of Chinese prawn COMT recombinant protein. Background technique [0002] Catechol-oxygen-methyltransferase (Catechol- O -methyltransferase, referred to as COMT, E.C.2.1.1.6), is a methyltransferase first discovered in the human body in 1958, mainly metabolizing catechol-type compounds, and its reaction mechanism is as follows figure 1 As shown, COMT enzyme (E.C.2.1.1.6) in the presence of divalent magnesium ions (Mg ++ ) in the presence of catalyzed the transfer of a methyl group of S-adenosylmethionine (SAM) to a hydroxyl group of the substrate DHBAc molecule to form the reaction mechanism of methylated products vanillic acid (VA) and isovanillic acid (IVA). [0003] In vivo, COMT has a wide range of substrates, including catecholamines, dopamine, epinephrine, dihydroxybenzoic acid (DHBAc), etc. When tested in vitro, th...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/81
CPCC12N9/1011C12N15/815C12Y201/01006
Inventor 李殿香王金星赵小凡王蕾齐梅
Owner 山东辰泰药业有限公司
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