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Method of high efficiency expression tPA exogenic protein using methanol yeast system and its purification preparation technology of expressed product

A protein and protein technology, applied in recombinant DNA technology, fermentation, DNA/RNA fragments, etc., can solve the problems of research work without large-scale production and high expression

Inactive Publication Date: 2006-04-12
黄秀东 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] This patent describes the construction process of this expression system, and also describes in detail the examples of using this expression system to successfully achieve a large amount of expression of some target proteins (such as rPA, Anx-scUK, mut-hNGF, etc.), because such exogenous There are generally no formal reports of successful overexpression of proteins in methanolic yeast or related research work for large-scale production

Method used

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  • Method of high efficiency expression tPA exogenic protein using methanol yeast system and its purification preparation technology of expressed product
  • Method of high efficiency expression tPA exogenic protein using methanol yeast system and its purification preparation technology of expressed product
  • Method of high efficiency expression tPA exogenic protein using methanol yeast system and its purification preparation technology of expressed product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of expression vector PICZa-DoI

[0053] This vector is transformed on the basis of Invitrogen's commercial secretory vector pPICZαA. The biggest difference between it and pPICZαA is that the domain I of HSA (HSA-DoI) is inserted after the α-signal peptide sequence, and pPICZα series vectors are eliminated. c-myc epitope and 6×His region after the multiple cloning site.

[0054] 1. pPICZαA vector

[0055] See Invitrogen's operating manual pPICZαA, B, and C-----Pichia expression vectors for selection on Zeocin TM and purification of secreted, recombinant proteins (Catalog no. V 195-20).

[0056] 2. Obtaining the domain I (DoI) gene-joint region DNA sequence of HSA and constructing the PICZa-DoI vector. Purchase the commercialized human fetal liver cDNA library (Lot: A604235) from Biochain, and synthesize primers:

[0057] HSA5:

[0058] 5`-cat ctc gag aaa aga gat gca cac aag agt gag gtt gct cat cgg ttt aag-3`

[0059] HSA3:

[0060] 5`-cat gcg gcc...

Embodiment 2

[0078] Expression, assay, purification and preparation of mutant tPA

[0079] 1. Acquisition of mutant tPA gene

[0080] According to the literature report of the study, the whole gene sequence of the tPA active fragment was artificially synthesized, and the 184th and 448th Asn of the original tPA were mutated to Gln during the synthesis, so as to avoid possible occurrence in the tPA protein molecule during yeast secretion and expression. N-glycosylation modification. The synthetic whole gene sequence is shown in SEQ-2, and the corresponding amino acid sequence is shown in SEQ-3.

[0081] 2. Activity determination of expression products

[0082] The construction of expression vectors and the screening of engineered strains all adopt the conventional methods in the Invitrogen manual. The tPA gene fragment was inserted between KpnI and NotI, and the DNA sequence corresponding to the recognition sequence Glu-Asn-Leu-Tyr-Phe-Gln-Ser of rTEV protease was recreated behind the Kpn...

Embodiment 3

[0093] Expression of Human Annexin V-Human Urokinase Active Fragment Fusion Protein (Anx-scUK)

[0094] Two primers were synthesized, and the whole gene of human annexin V (AnnexinV) was obtained from the cDNA library of human placenta tissue by PCR method, and the active fragment of human urokinase (scUK for short) was obtained according to the ProUK amino acid sequence 144-411, a total of 268 For the amino acid sequence, the gene fragment is synthesized by the whole gene synthesis method, and its gene sequence is described in SEQ-4.

[0095] 1. Acquisition of Human Annexin V Gene

[0096] The human annexin V gene is a gene fragment obtained by PCR from the commercialized cDNA library of Biochiain Company (Lot: A700109) through the following two primers AnxP5 and AnxP3, and inserted into the pUC18 vector. Primer AnxP5 introduces a restriction endonuclease KpnI cutting site for the 5'-end of the Annexin V gene, and introduces the enterokinase recognition site Asp-Asp-Asp-Asp-...

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Abstract

A process for configuring the universal expression carrier PICZa-DoI of methanol yeast in disclosed. It can be used to produce the exogenous recombinant protein in the form of fusion protein which can not be highly expressed by conventional method. The mutant human tissue plsminogen activator (rPA), human annexin V-urokinase action fragment fusion protein (Ank-scUK) and mutant human nerve growth factor (mut-hNGF) has been successfully expressed by it. Said fusion protein can be used to prepared high-purity medical protein through direct capturing by chelating sepharose medium from the fermented liquid, desalting, enzyme (or chemical) severing, and chromatography for purifying.

Description

technical field [0001] The invention relates to a method for producing a kind of recombinant protein in the way of fusion, secretion and expression by adopting a methanol yeast expression vector PICZa-DoI. In particular, using this system, the recombinant human tissue plasminogen activator mutant (rPA), human annexin V-urokinase active fragment fusion protein (Anx-scUK) and human nerve growth factor have been successfully expressed in methanolic yeast. (hNGF) and its mutants, etc. Background technique [0002] The yeast expression system has many advantages over the expression systems of Escherichia coli (E.coli) and mammalian cells (such as CHO, etc.), such as convenient cultivation, high yield, relatively simple purification, and low production cost. However, actual studies have found that many foreign proteins are difficult to achieve high expression in yeast expression systems, and some have little or no expression at all. We can retrieve many research reports on succe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C12N15/62
Inventor 黄秀东李树刚于廷和
Owner 黄秀东
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