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Method for preparing N-terminated acetylated thymosin alpha 1 and special engineering bacteria therefor

A technology of thymosin and engineering bacteria, which is applied in the field of preparing N-terminal acetylated thymosin α1, can solve the problem that a single subunit has no catalytic acetyl transfer activity, and achieve the effect of broad application prospects

Inactive Publication Date: 2009-08-05
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Homologous sequences of the catalytic subunits Ard1p, Nat3p and Mak3p of the above three enzymes also exist in higher eukaryotes, but the individual subunits do not have the activity of catalyzing acetyl transfer

Method used

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  • Method for preparing N-terminated acetylated thymosin alpha 1 and special engineering bacteria therefor
  • Method for preparing N-terminated acetylated thymosin alpha 1 and special engineering bacteria therefor
  • Method for preparing N-terminated acetylated thymosin alpha 1 and special engineering bacteria therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Expression and acetylation modification identification of fusion protein between thymosin α1 (Tα1) and L12, S18

[0073] 1. Expression of fusion protein of Tα1 and L12 and identification of acetylation modification

[0074] 1. Construction of engineering strain I expressing fusion protein of Tα1 and L12

[0075] The acquisition of the fusion gene: using the Escherichia coli genome as a template, using L1 and L2 as primers to PCR amplify the sequence of the gene rplL encoding ribosomal protein L12, and the product is about 370bp; using the above-mentioned 370bp PCR product as a template, using Ta2 and L2 as primers Primers were used for PCR amplification to obtain a product of about 320bp; then the product of 320bp was used as a template, and Ta1 and L2 were used as primers for PCR amplification to obtain a product of about 370bp, which was the fusion coding gene of the fusion protein of Tα1 and L12, and sequenced , and its nucleotide sequence is shown in seq...

Embodiment 2

[0113] Example 2. Screening of genes related to acetylation modification of Tα1

[0114] There are three known N-terminal acetyltransferases in Escherichia coli, namely RimI, RimJ, and RimL. There are also many genes of unknown function in the E. coli genome, among which there may also be unknown N-terminal acetyltransferases. The inventors selected five genes (yjaB, yjhQ, yjgM, yhhY, yiiD) presumed to be acetyltransferases from the genome. These 8 genes were knocked out by Red recombination technology, and different enzyme gene deletion mutants were constructed, in which Tα1 was expressed, separated and purified, and acetylated modified forms were identified by mass spectrometry. When the rimJ gene was knocked out, it was found that the fusion protein A2 had no acetylation modification, and it was deduced that this enzyme might be involved in the acetylation modification of Tα1. Now, the related experiments of rimJ gene knockout are described in detail as follows:

[0115]...

Embodiment 3

[0160] Example 3, Preparation of Thymosin Tα1 modified by N-terminal acetylation

[0161] The aforementioned studies have confirmed that rimJ is the key enzyme for the acetylation modification of Tα1. By co-expressing the Tα1 fusion protein with the rimJ gene, all the Tα1 fusion protein can be acetylated. In order to obtain the complete structure of Tα1, the fusion protein needs to be cleaved. The cleavage method can adopt enzymatic cleavage method, chemical cleavage method, and intein-mediated cleavage method. This example describes the use of intein cleavage to obtain N-terminal acetylated thymosin Tα1.

[0162] 1. Construction of engineering bacteria

[0163] (1) Construction of Tα1 and Spl DnaX intein fusion protein expression vector pET-AS

[0164] The gene sequence encoding the Spl DnaX intein is shown as sequence 9 in the sequence table, which was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Use restriction endonucleases EcoR I and Xho ...

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Abstract

The invention discloses a method for preparing N-terminal acetylation extrasin alpha1, and special engineering bacteria thereof. The engineering bacteria is obtained in such a way that encoding genes of the extrasin alpha1 and encoding genes of N-terminal acetyl transferase are introduced to host strains. The N-terminal acetyl transferase is the protein of the following a or b: a) protein is composed of amino acid sequences shown by a sequence 7 in a sequence table; and b) protein is derived from the a) and is provided with the N-terminal acetyl transferase through the replacement and / or deletion and / or adding of one or a plurality of amino acid residue of the amino acid sequences shown by the sequence 7 in the sequence table. Talpha1 obtained through the preparation of the engineering bacteria is all N-terminal acetylized. The invention overcomes the defects of no acetylization or only partial N-terminal acetylization in a traditional gene project technology, thoroughly realizes N-terminal acetylization Talpha1 through a gene engineering technology, and has strong practical purposes.

Description

technical field [0001] The invention relates to a method for preparing N-terminal acetylated thymosin α1 and special engineering bacteria thereof. Background technique [0002] Acetylation is a widespread protein modification method that exists in prokaryotes, archaea, and eukaryotes. About 80-90% of mammalian cytoplasmic proteins, 50% of yeast proteins, and a small amount of prokaryotic or archaeal proteins will Acetylation occurs (Polevoda B, Sherman F. The diversity of acetylated proteins. Genome Biology, 2002, 3: 1-6). [0003] Acetylation modification has effects on the activity and stability of some proteins, such as enzyme activity, enzyme stability, DNA binding, protein-protein interaction, receptor recognition, etc. In some cases, lack of acetylation results in decreased protein thermal stability or changes in other kinetic parameters, or inefficient complex assembly. The acetylation modification of proteins in yeast cells is related to cell growth, utilization of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/00C12P21/02C12R1/19
Inventor 方宏清张旭戴红梅李树龙陈惠鹏
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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