Method for production of a bioengineered form of tissue plasminogen activator

A technology of plasminogen and activator, applied in biochemical equipment and methods, drug combinations, enzymes, etc., can solve the problem of low incidence of non-cerebral hemorrhage events

Inactive Publication Date: 2008-07-09
阿维斯塔金格兰技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the incidence of ICH and other non-cerebral hemorrhage events will also be lower

Method used

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  • Method for production of a bioengineered form of tissue plasminogen activator
  • Method for production of a bioengineered form of tissue plasminogen activator
  • Method for production of a bioengineered form of tissue plasminogen activator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The DNA sequence encoding tissue plasminogen activator was obtained by de novo synthesis. This approach can be more conducive to better codon optimization for a particular cell line. And, making synthetic DNA the subject of eukaryotic / prokaryotic expression provides isolatable quantities of polypeptides exhibiting the biological properties of naturally occurring t-PA and the biological activities of t-PA in vivo and in vitro.

[0029] The nucleotide sequence (TENECT1) encoding recombinant tissue plasminogen activator is shown in SEQID.No.1. Codons in the DNA sequence encoding tissue plasminogen activator have been altered as part of a codon optimization process to ensure optimized expression of the recombinant protein in mammalian cell lines such as CHO K1 and HEK293, these altered Codons for are highlighted with capital letters. SEQ ID.No.2 represents the codon-optimized nucleotide sequence (TENECT2) encoding tissue-type plasminogen activator

[0030] The paired seq...

Embodiment 2

[0031] Example 2: Confirmation of the authenticity of the de novo synthesized cDNA encoding tissue-type plasminogen activator

[0032] The authenticity of the de novo synthesized cDNA provided by a commercial service provider was confirmed by automated DNA sequencing, and the results obtained are depicted in Figures 2 and 3.

Embodiment 3

[0033] Example 3: Subcloning TENECT and TENECT-Opt cDNAs into pcDNA3.1D / V5-His mammalian cell-specific expression vector

[0034] After verifying the authenticity of the de novo synthesized cDNA molecules (TENECT and TENECT-Opt) by automated DNA sequencing as shown above, TENECT and TENECT-Opt were respectively subcloned into the mammalian cell-specific expression vector pcDNA3.1D / V5-His , to generate constructs for transfection. The methodology employed is detailed below:

[0035] A. Reagents and Enzymes

[0036] 1. QIAGEN Gel Extraction Kit and PCR Purification Kit

[0037] 2. pcDNA 3.1D / V5-His vector DNA (Invitrogen)

[0038] enzyme

supplier

U / μl

10× buffer

1. BamHI

Bangalore Genei

10

Buffer E

2. Xhol

Bangalore Genei

10

Buffer E

3. Hind III

Bangalore Genei

20

Buffer E

4. Xhol

Bangalore Genei

10

Buffer E

5. T4 DNA ligase

Bangalore Genei

40

Ligase...

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PUM

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Abstract

The present invention relates to the recombinant method used for the production of soluble form of human tissue plasminogen activator variant. In this variant the threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine leading to a new glycosylation site. At position 117 of the endogenous tissue plasminogen activator asparagine has been replaced by glutamine, leading to the removal of an N linked glycosylation site. At position 296-299 the amino acids lysine, histidine, arginine, and arginine have been replaced by four alanine amino acids. The invention further relates to the de novo synthesis of the nucleic acid sequence encoding tissue plasminogen activator, transformation of the constructed nucleic acid sequences into competent bacteria and sub-cloning of the same into mammalian expression vectors for the expression of the desired protein. DNA constructs comprising the control elements associated with the gene of interest have been disclosed. The recombinant human tissue plasminogen activator, according to the invention, and the salts and functional derivatives thereof, may comprise the active ingredient of pharmaceutical compositions for treatment of treatment of heart attack and stroke patients. These compositions are yet another aspect of the present invention.

Description

field of invention [0001] The present invention relates to recombinant methods for producing soluble forms of human tissue plasminogen activator variants. In this variant, threonine 103 of endogenous tissue-type plasminogen activator is replaced by asparagine to create a new glycosylation site. The asparagine at position 117 of endogenous tissue-type plasminogen activator is replaced by glutamine, thereby removing the N-linked glycosylation site. At positions 296-299, lysine, histidine, arginine and arginine were replaced by four alanines. [0002] The present invention also relates to the de novo synthesis of the nucleic acid sequence encoding tissue plasminogen activator, transforming the constructed nucleic acid sequence into competent bacteria and subcloning it into a mammalian expression vector to express the desired protein. [0003] DNA constructs comprising control elements that bind to a gene of interest have been disclosed. [0004] The recombinant human tissue-ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72C12N15/67A61K38/00
CPCC12Y304/21069C12N9/6459A61P35/00A61P7/02C12N9/6456C12N15/67A61K38/00
Inventor V·莫拉瓦拉帕特尔
Owner 阿维斯塔金格兰技术有限公司
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