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Pichia pastoris surface co-display system based on cellulosome as well as construction method and application of pichia pastoris surface co-display system

A technology of surface co-display and Pichia pastoris, applied in the fields of application, chemical instruments and methods, botany equipment and methods, etc., can solve the problems of difficult foreign protein display efficiency quantification, low amino acid sequence homology, etc., and achieve improvement The effect of display ability, improvement of display ability, and cost saving of preparation

Inactive Publication Date: 2019-09-13
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Their protein structures have certain similarities, both are a hollow cylinder formed by β chains, but the amino acid sequence homology between mCherry and GFP is very low
[0006] Quantitative analysis of multi-protein co-display systems on the cell surface has always been a difficult problem, which can only be compared through the final catalytic effect of the multi-enzyme system, and it is difficult to quantify the display efficiency of exogenous proteins on the cell surface

Method used

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  • Pichia pastoris surface co-display system based on cellulosome as well as construction method and application of pichia pastoris surface co-display system
  • Pichia pastoris surface co-display system based on cellulosome as well as construction method and application of pichia pastoris surface co-display system
  • Pichia pastoris surface co-display system based on cellulosome as well as construction method and application of pichia pastoris surface co-display system

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Embodiment 1: Design and synthesis of MixA2 gene:

[0081] The genome DNA sequences of C.thermocellum and C.cellulolyticum were searched in the NCBI database respectively. The accession numbers of these two microorganisms in the NCBI database are: CP000568 and NC_011898. The coding DNA sequences of the respective cohesin modules were confirmed in these genomes, and then these coding sequences were electronically spliced ​​by DNAMAN software to form preliminary mini-scaffold protein genes. According to the codon usage frequency database information Codon UsageDatabase, the coding MixA2 was optimized according to the preferred codons of Pichia pastoris. Under the premise of not changing the amino acid sequence, the JCat codon optimization software was used to select the optimal codon, and at the same time, fine-tuning was performed with reference to the secondary structure of the mRNA to make the activation energy as high as possible, so that the mRNA was easier to expres...

Embodiment 2

[0082] Example 2: Construction of recombinant plasmid pPICZαA-egfp-mixa2-sed1:

[0083] Mini-scaffold protein MixA2 gene primer: upstream primer P1: 5'-ggaattc catatg gttcgtataaaagttgacacggtc-3' (with Nde I restriction site); downstream primer P2: 5'-aaggaaaaaa gcggccgc acctctacttggtgt-3' (with NotI restriction site).

[0084] Mutated green fluorescent protein gene egfp primer: upstream primer P3: 5'-ccg gaattc agcaagggcgaagagctgttcac-3' (contains EcoR I restriction site); downstream primer P4: 5'-ggattc catatg aagctttacagttcatccatgccgagagt-3' (with Nde I restriction site).

[0085] Sed1 primer of Saccharomyces cerevisiae cell wall protein gene: upstream primer P5: 5'-aaggaaaaaa gcggccgc caattttccaacagtacatctgcttc-3' (with Not I restriction site); downstream primer P6: 5'-catg tctaga ttataagaataacatagcaacaccagccaaac-3' (containing Xba I restriction site).

[0086] PCR reaction system: 1 μL MixA2 synthetic sequence, 1 μL upstream primer P1, 1 μL downstream primer P2...

Embodiment 3

[0090] Example 3: Transfer of recombinant plasmid pPICZαA-egfp-mixa2-sed1 into Pichia pastoris

[0091] The recombinant plasmid vector pPICZαA-egfp-mixa3-sed1 was linearized by PCR with ultra-fidelity DNA polymerase to improve the linearization effect and increase the transformation efficiency of yeast cells. The sequences of the linearization primers are as follows: upstream primer P7: 5'-gagctcgctcattccaattcctt-3', downstream primer P8: 5'-caatcaagcccaataactgggctg-3'.

[0092] Transform into Pichia pastoris KM71H by electric shock, select a single colony on the YPDS plate of Zeocin antibiotic (100 μg / mL), extract the genome as a template, and perform PCR amplification on the target DNA with specific primers, and identify positive according to the size of the PCR band Turn.

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Abstract

The invention provides a pichia pastoris surface co-display system based on cellulosome as well as a construction method and application of the pichia pastoris surface co-display system and belongs tothe technical fields of molecular biology, food and gene engineering. The system comprises anchoring protein and polyprotein display chassis protein, wherein the anchoring protein is saccharomyces cerevisiae cell wall protein Sed1p; and the polyprotein display chassis protein is artificial mini scaffold protein MixA2 of clostridium cellulosome. The pichia pastoris surface co-display system basedon cellulosome can realize polyprotein co-display on cell surfaces; heterologous protein for fusion expression of dockerin is prepared from recombinant bacteria, purification is not needed, multiple proteins can be displayed on cell surfaces by direct specific binding of dockerin on recombinant protein and cohesion on MixA2, and the preparation cost of recombinant protein can be saved effectively.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology, food and genetic engineering, and in particular relates to a cellosome-based co-display system on the surface of Pichia pastoris and its construction method and application. Background technique [0002] Microbial cell surface display is introduced into microbial host cells by fusing the target protein gene sequence with a specific carrier protein gene sequence. The target protein is expressed and positioned on the microbial cell surface under the guidance of the carrier protein, maintaining a relatively independent spatial conformation and original biological activity. The surface display system has the advantages of high efficiency and safety, and is a good platform for expressing foreign proteins. In recent years, its application has become more and more extensive. Whole-cell catalysts; displaying foreign proteins on the cell surface and discovering substances that interact with th...

Claims

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Application Information

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IPC IPC(8): C07K14/39C12N15/31C12N1/19C12N15/81C12N15/70C12N1/21
CPCC07K14/39C12N15/70C12N15/815
Inventor 李迅王亮亮王月皎李文谦王飞张瑜
Owner NANJING FORESTRY UNIV
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