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Using methanol yeast to produce human kallikrein - 1

A kallikrein and methanol yeast technology, applied in the direction of enzymes, enzymes, hydrolytic enzymes, etc., can solve the problems of low yield, complicated purification process, and low effective yield

Inactive Publication Date: 2007-12-26
上海万兴生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Comprehensive current hK 1 research, all r-hK 1 The preparation of engineering strains has the disadvantages of low expression yield, inhomogeneous products, complicated purification process and low effective yield.

Method used

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  • Using methanol yeast to produce human kallikrein - 1
  • Using methanol yeast to produce human kallikrein - 1
  • Using methanol yeast to produce human kallikrein - 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] k 1 Construction of engineered strains

[0049] 1. hK 1 Gene acquisition and expression vector pPICZa-hK 1 Construct

[0050] Refer to the relevant hK published in GeneBank 1 Complete gene sequence data, designed and synthesized (Shanghai Sangong) can amplify mature hK 1 Two primers of the gene Kal5 (5'-primer, SEQ-3) Kal3 (3'-primer, SEQ-4):

[0051] Kal5 (AA07624):

[0052] 5`-cat ctc gag aaa aga att gtg gga ggc tgg gag tgt gag-3`

[0053] Kal3 (AA07625):

[0054] 5`-cat gcg gcc gc t tag gag ttc tcc gct atg gtg tcc tc-3`

[0055] hK was obtained by PCR using the human kidney cDNA library (P / N: 7202, L / N: P0260602) from Panomics as a template 1 Gene. Primer Kal5 will be in hK 1 Add a DNA restriction enzyme XhoI site (CTCGAG) to the 5'-end of the gene, and the codon AAA AGA corresponding to the recognition sequence Lys-Arg of Kex2 protease, which will ensure that the inserted hK 1 When the gene is secreted and expressed, the α-signal peptide can be succe...

Embodiment 2

[0079] wxya 1 Fermentation of engineered bacteria

[0080] 1. Seed solution preparation

[0081] Take the working seed glycerol cryopreservation tube, after thawing, take 1ml and inoculate it into 500ml YPD medium (1% yeast powder, 2% peptone, 2% glucose), and cultivate in a shaker at 30°C and 300rpm for 30 hours to OD 600 If the value is 6.0±1.0, if the microscopic examination is normal, the seed solution can be used for inoculation. Preparation of basal salt medium BSM for fermentation 1 (K 2 SO 4 60.7 g, MgSO 4 24.2 g, CaSO 4 2H 2 O 3.9 g, H 3 PO 4 89ml, KOH 13.8g, PTM1 14ml, glycerol 400g, foam enemy 2ml, take 10L as an example, take the preparation of 1 liter of trace element medium PTM1 as an example, the content of each component is: CuSO 4 ·5H 2 O 6.0 g, NaI 0.008 g, MnSO 4 3.0 g, NaMoO 4 0.2 g, H 3 BO 3 0.02 g, ZnSO 4 20.0 g, CoCl 2 0.5 g, FeSO 4 ·7H 2 O 65.0 g, biotin 0.2 g, H 2 SO 4 5 ml, add water to make it 1 liter) and carry out solid...

Embodiment 3

[0088] wxya 1 protein purification

[0089] The purification process consists of three steps: hydrophobicity, anion exchange and gel filtration chromatography, among which Phenyl-Sepharose FF is selected for hydrophobic chromatography, Q-Sepharose FF is used for anion exchange medium, and Superdex75 is used for gel filtration chromatography.

[0090] The specific process is as follows:

[0091] 1. Pretreatment of fermentation broth

[0092] Add 0.2M PB, pH 6.0 and 3.0M (NH 4 ) 2 SO 4Solution is respectively 20mM and 1.0M to its final concentration in the fermented liquid, adjusts the pH value to 6.0, leaves standstill 0.45 micron membrane filtration after 1 hour and contains 20mMPB, 1.0M (H 4 ) 2 SO 4 , pH6.0 treated fermentation liquid (sample liquid), just can carry out the chromatographic process.

[0093] 2. Phenyl-Sepharose FF hydrophobic chromatography

[0094] Phenyl Sepharose FF chromatographic column on the fermented liquid after above-mentioned treatment, co...

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Abstract

This invention relates to a method for producing recombinant human kallikrein-1 (r-HK1) with Pichia pastoris. This invention also describes molecular weight, isoelectric point, and sugar content and glycosyl modification of r-HK1. It is proven by in vivo and in vitro activity experiments that r-HK1 can be used to treat and prevent cerebral infraction.

Description

1. Technical field [0001] The present invention adopts the means of genetic engineering to obtain a new type of recombinant protein——recombinant Human Kallikrein-1 (Recombinant HumanKallikrein-1 expressed by Pichia pastoris) on a large scale through the Pichia pastoris expression system , r-hK 1 ), especially for r-hK 1 The study of molecular properties, as well as the determination and test of in vivo and in vitro activities, showed that r-hK 1 It is expected to become a drug for the treatment of cerebral infarction. 2. Background technology [0002] As early as 1926, Frey et al. obtained a component from human urine that could cause a drop in blood pressure, which was called kalaitalin at the time. However, later researchers found that the protease component was also widely distributed in plasma, Pancreas, kidney, salivary glands, intestines, pancreatic juice, especially the highest content in the pancreas. In 1930, Kraut H. of Greece named the component extracted from...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/00C12N9/50C07K1/14
Inventor 黄秀东陈佩新王俊陈耀国袁靖宇王书生潘学工曹之舫
Owner 上海万兴生物制药有限公司
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