Gene mutation type recombined protease K and industrialized production method thereof

A protease and amino acid technology, which is applied in the field of gene mutant recombinant proteinase K and its industrial production, and can solve problems such as industrial production of undiscovered gene mutant recombinant proteinase K.

Active Publication Date: 2012-12-26
GPROAN BIOTECH (SUZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, no report on the industrial production of gene mutant recombinant p...

Method used

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  • Gene mutation type recombined protease K and industrialized production method thereof
  • Gene mutation type recombined protease K and industrialized production method thereof
  • Gene mutation type recombined protease K and industrialized production method thereof

Examples

Experimental program
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Embodiment approach

[0059] Examples of such eukaryotic cells include, but are not limited to, yeast cells. According to a preferred embodiment of the present invention, the eukaryotic cells are yeast cells. According to yet another preferred embodiment of the present invention, the yeast cells are Pichia cells, Candida cells, Hansenula polymorpha cells, Torulopsis cells, Schizosaccharomyces cells and Kluyveromyces cells.

[0060] According to an especially preferred embodiment of the present invention, the yeast cells are Pichia pastoris cells. Due to more in-depth research on the fermentation conditions of Pichia pastoris, and the cost of the Pichia pastoris cell is the lowest in the fermentation preparation, which can meet the technical requirements and the needs of product marketization, the Pichia pastoris cell is considered to be a genetic mutation. The most preferred transformed cells for the production of recombinant proteinase K.

[0061] According to a preferred embodiment of the pres...

Embodiment 1

[0083] Example 1 Construction of Proteinase K Expression Vectors pPIC9K-ProK and pPIC9K-mutProK

[0084] Replace the coding sequence of wild-type proteinase K (SEQ ID NO.3) with the DNA sequence composed of Pichia pastoris preferred codons, add the SnaB I restriction site TACGTA at the 5' end, and add the hexamerization site in sequence at the 3' end The histidine tag CACCATCACCACCATCAC (SEQ ID NO.4), the stop codon TAA and the NotI restriction site GCGGCCGC were synthesized by chemical methods (SEQ ID NO.5). Replace the SnaB I restriction site TACGTA added at the 5' end with the EcoR I restriction site GAATTC, and further introduce corresponding mutations (such as figure 1 shown), and the obtained sequence (SEQ ID NO.6-9) was synthesized by chemical method (BioBasic Inc., a Canadian gene synthesis technology service company). The above chemically synthesized sequences have been cloned into the pUC57 plasmid (BioBasic Inc., a Canadian gene synthesis technology service company...

Embodiment 2

[0087] Sequencing results showed that the target fragments were correctly inserted into the above expression vectors. Example 2 Transformation of Proteinase K Expression Vectors pPIC9K-ProK and pPIC9K-mutProK

[0088] The recombinant plasmid constructed in Example 1 was digested with the restriction endonuclease Sal I to make it linearized, and then dissolved to a concentration of 1 μg / μL with TE buffer (pH 8.0), and 20 μL of the dissolved plasmid was mixed with GS115 yeast Competent cells were mixed evenly, transferred to an ice-precooled electroporation cuvette (0.1 cm gap between poles), and kept in an ice bath for 5 min. Using the method of electric shock transformation, the above-mentioned expression vector was transformed into GS115 yeast competent cells by electric shock using the built-in Pichia pastoris transformation parameters of the electric transformation instrument.

[0089] After the electric shock, quickly add 1mL of ice-cooled 1M sorbitol solution to the elec...

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Abstract

The invention relates to a gene mutation type recombined protease K and an industrialized production method thereof. Specifically, the invention relates to the gene mutation type recombined protease K which is obtained by performing gene reformation and protein engineering and has an enzymatic activity being more than two times of the enzymatic activity of the same natural protein, and further relates to the industrialized production method and a technical process for utilizing yeast cells to large-scale culture expressing foreign proteins and prepare the gene mutation type recombined protease K.

Description

technical field [0001] The invention relates to a gene mutant recombinant proteinase K and its industrial production method. More specifically, the present invention relates to a genetically modified and protein-engineered recombinant protease K whose enzyme activity is more than twice that of the same natural protease; The industrial production method and technological process for preparing the gene mutant recombinant proteinase K. Background technique [0002] Proteinase K is a serine protease that was first discovered in extracts of Tritirachium album limber in 1974 (Ebeling W et al., Eur J Biochem. 1974 Aug 15;47(1):91-7 ). Proteinase K is named for its ability to digest native keratin, which is rich in disulfide bonds, and C. albicans limberii can grow on keratin as the sole carbon / nitrogen source. [0003] The biggest feature of proteinase K is that it has a broad-spectrum and high-efficiency cutting ability for natural proteins, and it is the most active protease a...

Claims

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Application Information

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IPC IPC(8): C12N9/60C12N15/57C12N15/63C12N15/81C12N1/19C12R1/725C12R1/84C12R1/72C12R1/78C12R1/88C12R1/645
Inventor 安海谦鲁刚伟冉波任玉珍
Owner GPROAN BIOTECH (SUZHOU) INC
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