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Pichia pastoris culture medium for screening G-CSF(15-75) polypeptide

A medium and yeast technology, applied in the field of Pichia pastoris medium, can solve the problem of indistinguishability between recombinantly expressed proteins and medium proteins, and achieve the effect of eliminating expression and purification and eliminating influence

Active Publication Date: 2015-10-07
SHANDONG NEWTIME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The BMGY / BMMY medium system can efficiently express most recombinant proteins. The main components of BMGY and BMMY medium include yeast extract, peptone, amino acid-free yeast nitrogen source, etc., rich in amino acids, nucleotides, sugars, Vitamins and polypeptides provide carbon and nitrogen sources for the growth of yeast, but it contains a variety of protein components, especially in electrophoresis at about 6.5KD, the electrophoresis band is obvious, and the recombinant expression protein and the medium protein cannot be distinguished

Method used

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  • Pichia pastoris culture medium for screening G-CSF(15-75) polypeptide
  • Pichia pastoris culture medium for screening G-CSF(15-75) polypeptide
  • Pichia pastoris culture medium for screening G-CSF(15-75) polypeptide

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1, Recombinant G-CSF (15-75) Polypeptide Pichia Pichia Strain Seed Culture and Recombinant Protein Induced Expression

[0030] Pick pPIC9K-GS115 and pPIC9K-G-CSF(15-75)-GS115 monoclonals from the YPD plate and inoculate them into 5ml YPD medium containing 0.5mg / ml G418, cultivate them at 30°C and 220rpm for 7 hours, follow the 10 Inoculate the seed medium in No. 1-9 culture medium with % inoculum amount, cultivate overnight at 30°C, 220rpm, 6000rpm, collect the bacteria by centrifugation for 5min, inoculate into the induction medium in No. 1-9 medium, 28°C , induced expression at 220rpm, added 1% methanol at intervals of 24 hours, induced for 72 hours, stopped the culture, took the bacterial solution and centrifuged, collected the supernatant, precipitated with TCA, and identified the protein expression by tricine-SDS-PAGE electrophoresis. Protein expression is shown in Table 1 and figure 1 , figure 2 . pPIC9K-GS115 is used as a blank control. There should b...

Embodiment 2W

[0059] Embodiment 2Western Blot identification recombinant protein expression

[0060] Centrifuge the Pichia pastoris induced to express the recombinant G-CSF (15-75) polypeptide, collect the supernatant, precipitate with TCA, redissolve in 0.2M NaOH, add reducing buffer, and perform Tricine-SDS-PAGE electrophoresis. Transfer the electrophoresis gel to cellulose acetate membrane, wash with TTBS 3 times, 10 minutes each time, block with 10% fetal bovine serum, 37°C, 2 hours, add rabbit anti-human G-CSF polyclonal antibody (1:2000), 37°C , 2 hours, washed 3 times with TTBS, 10 minutes each time. Add HRP-labeled goat anti-rabbit polyclonal antibody (1:20000), at 37°C for 2 hours, and develop color with DAB. For color results, see image 3 , the target protein was expressed in medium 4-8.

[0061] Table 1. Expression of G-CSF polypeptide in 9 different media

[0062]

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Abstract

The invention discloses a pichia pastoris culture medium for screening and recombining G-CSF polypeptide (15-75) expressions. The culture medium does not contain yeast powder and peptone, contains microelements, and can be used for screening the G-CSF polypeptide (15-75) with the molecular weight being 6.5KD, and influences of the yeast powder and the peptone on G-CSF polypeptide expression authentication are avoided.

Description

technical field [0001] The invention belongs to the field of biological fermentation, and in particular relates to a Pichia pastoris culture medium for screening recombinant G-CSF (15-75) polypeptide expression. Background technique [0002] The main function of granulocyte stimulating factor (granulocyte colony stimulating factor, G-CSF) is to stimulate the proliferation and differentiation of neutrophil lineage hematopoietic cells, increase the number of neutrophils in peripheral blood, and activate the function of neutrophils. Recombinant human granulocyte-stimulating factor (rhG-CSF) was first launched in the United States in 1991, and has been widely used in clinical prevention and treatment of neutropenia and its complications (such as fever, infection, etc.) caused by multiple factors. G-CSF mainly acts on the differentiation, proliferation and activation of neutrophil hematopoietic cells. Injecting G-CSF into tumor patients after chemotherapy can increase the level ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N1/16C12P21/02C12R1/84
Inventor 张贵民金彩艳朱中松
Owner SHANDONG NEWTIME PHARMA
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