Physarum polycephalum stress-inducible protein 1 (PSTI1) for improving stress resistance of prokaryote and application of PSTI1

A prokaryotic and stress-resistant technology, which is applied in the field of improving the stress resistance of prokaryotes, and can solve problems such as the undiscovered STI1 protein

Inactive Publication Date: 2012-07-04
深圳市裕德凯机械租赁有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Hernández et al. [25] After analyzing the protein-coding sequences of various bacteria, it was found that the TPR-DP structural protein also exists in prokaryotes, but the STI1 protein has not been found yet

Method used

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  • Physarum polycephalum stress-inducible protein 1 (PSTI1) for improving stress resistance of prokaryote and application of PSTI1
  • Physarum polycephalum stress-inducible protein 1 (PSTI1) for improving stress resistance of prokaryote and application of PSTI1
  • Physarum polycephalum stress-inducible protein 1 (PSTI1) for improving stress resistance of prokaryote and application of PSTI1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Isolation of complete cDNA of PSTI1

[0048] Referring to Daniel et al. [28] The method suspension culture Physcoma polycephalum (p.polycephalum PpII (+ / -)) bacterial strain, this bacterial strain is donated by Institute of Biophysics, University of Regensburg, Germany, and can also be purchased from the American Type Culture Collection (preservation number ATCC NO: 24467), or purchase the strains of Phymophora polycephalum from commercial companies, such as Promega, Invitrogen, etc.

[0049] Take 100 mg (wet weight) of Physomum polycereus microplasma, and use RNeasy Plant Mini Kit (QIAGEN, Germany) to extract total RNA. with GeneRacer TM Kit (Invitrogen, USA) prepared mRNA containing 5'-cap structure into complete cDNA. According to the 3′-cDNA sequence of PSTI1, two generacer TM The downstream PCR primers R1 and R2 required by Kit (see Table 1). Use the primers in Table 1 for GeneRacer TM 5′Primer / R1 and GeneRacer TM 5'Nested Primer / R2, namely th...

Embodiment 2

[0056] Example 2: Recombinant expression of PSTI1 and its functional domains TPR1 and TPR2

[0057] Using the complete cDNA as a template, use the primer pair psti1-F / psti1-R in Table 2 to clone the gene encoding PSTI1. The PCR product was inserted into the BamH I and Sal I restriction sites of the vector pET-32a (+) (Novagen, the U.S.), and the recombinant plasmid pET-psti1 was cloned in E.coliTop10 (Invitrogen, the U.S.):

[0058] Extract the plasmid pET-psti1 and transform it into E.coli Origami TM (DE3) (Invitrogen, USA), positive transformant PSTI1(+) was screened on an LB culture plate containing 50 μg / ml Amp and 30 μg / ml Kan to obtain recombinant plasmid pET-psti1.

[0059] Using pET-psti1 as a template, clone the PSTI1 conserved peptide TPR1 (15- 107aa) and TPR2 (135-233aa) gene fragments tpr1 and tpr2, and recombined into the vector pET-32a(+), to prepare plasmids pET-tpr1 and pET-tpr2.

[0060] Then transform E.coli Origami separately TM (DE3), prepared as transfor...

Embodiment 3

[0064] Embodiment 3, Western Blot test

[0065] Culture PSTI1(+), TPR1(+), TPR2(+) and PSTI1(-) in LB medium to OD 600 =0.5-0.6, add IPTG at a final concentration of 1 mmol / L and continue to culture for 4 hours (30°C) to induce the expression of Trx-fused PSTI1, TPR1 and TPR2.

[0066] Take 1ml of the bacterial liquid and mix it with an equal volume of 2×loading buffer and boil it for 10 minutes, then take the supernatant and perform SDS-PAGE on a 12% polypropylene gel. After the protein in the polypropylene gel was transferred to the nitrocellulose membrane, it was blocked overnight with skimmed milk powder, and detected with mouse anti-Trx Tag polyclonal antibody (ABGENT, USA) and alkaline phosphatase-labeled goat anti-mouse IgG (Proteintech, USA) Trx, Trx-PSTI1, Trx-TPR1 and Trx-TPR2 [use BCIP / NBT (Bromo-Chloro-Indolyl Phosphate / NitroBlue Tetrazolium) as the chromogenic substrate].

[0067] The molecular weight of Trx is 17kD, and the theoretical molecular weights of Trx-...

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Abstract

The invention relates to a physarum polycephalum stress-inducible protein 1 (PSTI1) for improving stress resistance of a prokaryote and application of the PSTI1, and in particular provides a PSTI1 separated from physarum polycephalum of eucaryon and coded genes and application of the product in improvement of stress resistance of the prokaryote. Experiments prove that the over-expressed PSTI1 in the prokaryote can widely improve the salt, osmotic pressure, heavy metal ion, oxidation, oxygen deficiency and pH change stress tolerance of the prokaryote and the PSTI1 can widely affect the stress response function of prokaryotic cells. Therefore, the PSTI1 can be used for converting the prokaryote so as to improve the environmental pollution treatment capacity of the prokaryote such as bacteria and the like, and can also be used for improving the production efficiency of bioengineering bacteria used in the field of biotechnology.

Description

technical field [0001] The present invention relates to a polycephala protein PSTI1 for improving prokaryotic stress resistance ability and its application, specifically providing a PSTI1 protein and coding gene isolated from eukaryotic polycephala, and using the product Use for improving prokaryotic stress resistance. Background technique [0002] STI1 protein (Stress-inducible protein 1) is a human [1] ,mouse [2] Leishmania [3] , soybean [4] and yeast [5] A class of evolutionarily conserved co-molecular chaperones associated with biotic and abiotic stress responses found in . The common feature of STI1 family members is that they contain more than 2 TPR domains (tetratricopeptide repeat domain), each domain usually consists of 3 TPR motifs (34 amino acid repeat sequences, containing helix-turn-helix secondary structure) constitute [6，7] . When cells respond to heat stress, STI1 interacts with heat shock proteins (Heatshock proteins, HSPs) Hsp70 and Hsp90 through it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/31C12N15/63C12N1/21
Inventor 邢苗刘士德张建华陈汗英李明华
Owner 深圳市裕德凯机械租赁有限公司
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