Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide

An acetylase, recombinant engineering bacteria technology, applied in biochemical equipment and methods, chemical instruments and methods, botanical equipment and methods, etc., can solve problems such as inability to acetylate Tβ4, and achieve the effect of broad application prospects.

Active Publication Date: 2012-07-04
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

Our latest results found that RimJ can catalyze the acetylation of Tα1 but not Tβ4 (Fang, H., Zhang, X., Shen, L., Si, X., Ren, Y., Dai, H. , Li, S., Zhou, C., Chen, H., 2009. RimJ is responsible for N α -acetylation of thymosin α1 in Escherichia coli. Appl. Microbiol. Biotechnol. 84, 99-104)

Method used

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  • Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide
  • Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide
  • Method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1, Escherichia coli host construction expressing the acetylase of halophilic archaea

[0048] 1. Design and synthesis of the acetylase ssArd1 gene of halophilic archaea

[0049] According to the amino acid sequence reported in the literature (Dale T.Mackay, Catherine H.Botting, Garry L.Taylor and Malcolm F.White. An acetylase with relaxed specificity catalysesprotein N-terminal acetylation in Sulfolobus solfataricus. Molecular Microbiology, 2007, 64: 1540-1548 ) and the partial tropism of Escherichia coli gene codons, the following gene sequence was designed and sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for synthesis. The synthetic ssArd1 gene sequence has NdeI and HindIII restriction sites added on both sides respectively, and the specific sequence is shown in sequence 1 (the 4th-501st nucleotide sequence at the 5' end of sequence 1 in the sequence listing is the coding sequence) , the encoded amino acid sequence is shown in Sequence ...

Embodiment 2

[0121] Example 2, Preparation of recombinant N-terminal acetylated Tα1

[0122] Tα1 has a small molecular weight and is difficult to express directly in cells, and needs to be fused with other protein genes. 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.

[0123] 1. Construction of engineering bacteria I expressing fusion protein of Tα1 and intein

[0124] The gene sequence encoding the Spl DnaX intein is shown as sequence 3 in the sequence listing, and was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0125]Use restriction endonucleases EcoR I and Xho I to double-digest the gene encoding the Spl DnaX intein, then use restriction endonucleases EcoR I and Xho I to double-digest the pET22b v...

Embodiment 3

[0167] Example 3, Preparation of recombinant N-terminal acetylated Tβ4

[0168] Tβ4 has a small molecular weight and is difficult to express directly in cells, and needs to be fused with other protein genes. To obtain the complete structure of Tβ4, 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 Tβ4.

[0169] 1. Construction of engineering bacteria II expressing fusion protein of Tβ4 and intein

[0170] Preparation of human thymosin β4 with plasmid pET-B2 containing Tβ4 gene (Si Xinxi, Dai Hongmei, Fang Hongqing, Chen Huipeng. Chemical cleavage of recombinant fusion protein. Biotechnology Communications, 2009, 20(5), 677-679) (Military Medicine of the Chinese People's Liberation Army Institute of Bioengineering, Academy of Sciences) as a template, PCR amplified Tβ with prime...

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Abstract

The invention discloses a method and special engineering bacteria for producing N-terminal acetyl protein or polypeptide. The recombinant engineering bacteria capable of expressing transferase of archaebacteria on chromosomes are obtained by integrating the expression box of the transferase of archaebacteria onto the chromosomes of a host; the expression box for expressing the transferase of archaebacteria comprises a promoter and an archaebacteria transferase gene connected with the downstream of the promoter; and the nucleotide sequence of the archaebacteria transferase gene is represented by the sequence No.1 in a sequence list, and the amino acid sequence of the archaebacteria transferase gene is represented by a sequence No.2 in a sequence table. The engineering bacteria of the invention can directly produce complete N-terminal acetyl protein or polypeptide, such as thymosin extrasin alpha 1 and thymosin extrasin beta 4, the drawback of incompetence of acetylation or partial acetylation in a conventional genetic engineering technique is overcome, the production of N-terminal acetyl thymosin extrasin by the genetic engineering technique is realized completely, and the method and the special engineering bacteria are very practical.

Description

technical field [0001] The invention relates to a method for preparing N-terminal acetylated polypeptide, especially thymosin and special engineering bacteria. 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, u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/10C12N15/54C12N15/62C12N15/70C07K14/575C07K19/00C12R1/19
Inventor 方宏清戴红梅孙旭任元涛司信喜李树龙陈惠鹏周长林
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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