Recombinant therapeutic glycine n-acyltransferase

a technology of acyltransferase and glycine, which is applied in the direction of transferases, peptide/protein ingredients, metabolic disorders, etc., can solve the problems of affecting the glycine conjugation capacity of humans, and affecting the glycine conjugation capacity. , to achieve the effect of enhancing detoxification, enhancing detoxification in mammals, and treating and/or preventing metabolic disorders

Inactive Publication Date: 2013-08-29
NORTH WEST UNIV (ZA)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The activated compounds generated by phase I detoxification are often more reactive and toxic than the original metabolites, and are further processed by phase II detoxification systems.
In urea cycle disorders, nitrogen accumulates in the form of ammonia resulting in hyperammonemia which ultimately causes irreversible brain damage, coma and / or death.
Assays on liver samples have however shown that there is great variability in the glycine conjugation capacity in humans.
To date, no system for the bacterial expression and purification of an enzymatically active recombinant GLYAT has been reported.
A disadvantage associated with the lack of a system for expression of an enzymatically active recombinant GLYAT is that there is no commercially viable product currently available for directly improving the capacity of the glycine-conjugation detoxification system, particularly in the case of patients with metabolic disorders.

Method used

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  • Recombinant therapeutic glycine n-acyltransferase

Examples

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example 1

Recombinant Bovine GLYAT

[0059]Recombinant bovine GLYAT was cloned into a set of three modified pColdIII (pColdIII-E, pColdIII-A and pColdIII-EH) expression vectors encoding C-terminal histidine tags.

[0060]In order to clone the coding sequence into the expression vectors, the sequence is amplified through polymerase chain reaction (PCR) using primers containing Ndel and Xhol restriction enzyme sites to facilitate directional cloning. The PCR reaction mixtures contained 1X Takara ExTaq buffer, 10 nmol of each dNTP, 25 pmol of each primer, approximately 50 ng of template DNA and 2 units of Takara ExTaq polymerase, in a final volume of 50 μl. Thermal cycling conditions were 94 degrees Celsius for 1 min, then 30 cycles of 94 degrees Celsius for 30 seconds, 70 degrees Celsius for 30 seconds, and 72 degrees Celsius for 1 minute, followed by a final extension at 72 degrees Celsius for 10 minutes.

[0061]After transforming E. coli with an expression plasmid containing a recombinant GLYAT codin...

example 2

Recombinant Human GLYAT

[0068]The nucleotide sequence encoding the human GLYAT sequence was synthesised and cloned into the pET32 expression vector.

[0069]The pET32 expression vector enables the expression of human GLYAT with an N-terminal hexahistidine tag and an N-terminal Trx-tag, which respectively facilitates the purification and correct folding of the enzyme.

[0070]The expression vector encoding human GLYAT was transformed into Origami expression cells. The cells were also transformed with the pGro7 vector from Takara, which resulted in co-expression of the GroES and GroEL chaperone proteins. Chaperone proteins aid in the correct folding of proteins and increase the yield of soluble recombinant enzymes.

[0071]The Origami cells containing the plasmids for expression of recombinant human GLYAT and the chaperone proteins were grown in liquid culture. It was found that the optimal expression of soluble GLYAT occurs in the absence of IPTG (Isopropyl (β-D-1-thiogalactopyranoside), thus ...

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Abstract

This invention relates to a method of producing a recombinant enzyme, more particularly, this invention relates to a method of producing water soluble enzymatically active recombinant glycine N-acyltransferase (GLYAT (E.G. 2.1.3.13)), including the steps of providing a suitable expression host; preparing a vector including a gene for expressing GLYAT in the expression host to form an expression piasmid; transforming the host with the expression piasmid to form an expression system; expressing the GLYAT gene in the expression system; and separating the expressed GLYAT from the expression system.

Description

INTRODUCTION AND BACKGROUND TO THE INVENTION[0001]This invention relates to a method of producing a recombinant enzyme. More particularly, this invention relates to a method of producing water soluble enzymatically active recombinant glycine N-acyltransferase (GLYAT (E.C. 2.3.1.13)).[0002]Detoxification of toxic metabolites by the human body is an essential physiological process. The detoxification process decreases the toxicity of several endogenous metabolites, such as steroid hormones, and exogenous toxins, which could include compounds in food or industrial chemicals.[0003]The detoxification process is divided into three main phases. Phase I detoxification activates metabolites by adding functional groups. The activated compounds generated by phase I detoxification are often more reactive and toxic than the original metabolites, and are further processed by phase II detoxification systems. In phase II detoxification, a range of conjugation reactions serve to make the activated c...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/10
CPCC12Y203/01013C12N9/1029A61P3/00A61P39/02A61P43/00A61K38/45C12N9/10C12N15/52C12N15/63C12P21/02
Inventor MIENIE, LODEWYK JACOBUSVAN DIJK, ALBERDINA AIKEBADENHORST, CHRISTOFFEL PETRUS STEPHANUSVAN DER SLUIS, RENCIA
Owner NORTH WEST UNIV (ZA)
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