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A simple method for preparing active human kgf-2d31

A KGF-2D31 and activity technology, applied in the field of genetic engineering, can solve the problems of limited cell application range, different detection range, and long time-consuming, and achieve the effect of universal detection method, stable biological activity, and simplified purification time

Active Publication Date: 2020-04-07
ABZYME BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the BaF3 of the transceptor FGFR2lllb has good effect and high sensitivity in detecting KGF-2 (EC506 units / mg), but ATCC has suspended the sale, and there is no commercialized BaF of FGFR2lllb on the market 3 For sale, if you want to use this method, it still takes a long time (3-6 months) and effort to build a stable expression cell line
Some research literature and manufacturers also use epithelial cells such as NRK52E, 4MBr-5, ICE-6, RTE and other epithelial cells to detect KGF-2 activity. The detection range is different, and generally isolated, the range of cell application is limited, and the repeatability is poor

Method used

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  • A simple method for preparing active human kgf-2d31
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  • A simple method for preparing active human kgf-2d31

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Embodiment Construction

[0068] Main Reagents and Instruments

[0069] (1) Main reagents:

[0070] 2×YT medium: 16g Tryptone, 10g Yeast Extract, 5g NaCl, add 900mL ultrapure water to dissolve, add 1N NaOH dropwise, adjust the pH value to 7.0, add ultrapure water to make the medium volume to 1L, and autoclave at 121℃ After 20 minutes of incubation, store at 4°C;

[0071] IPTG: Shanghai Sangong, 1M (Self-prepared, repackaged, stored at -20°C)

[0072] Ampicillin and chloramphenicol: purchased from Shanghai Sangong (repacked after self-preparation, stored at -20°C)

[0073] Strong cation exchange chromatography (SP) medium: SP Bestarose Fast Flow, purchased from Shanghai Bergeron Company

[0074]Bacteria-breaking buffer, washing buffer, and elution buffer: adjust the pH to 6.2 after preparing according to the corresponding formula, filter through a 0.45 μm filter membrane, and store at 4°C for later use;

[0075] DMEM(H): Gibco, powder, add NaHCO before use 3 or HEPES preparation, filtered through a...

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Abstract

The invention belongs to the technical field of gene engineering, and specifically relates to a method for simply and conveniently preparing active human KGF-2D31. Hyper-saline concentration (500mM NaCl) is utilized, so that a majority of expression host impurity proteins cannot be combined with strong cation resins, while the KGF-2D31 is combined with the strong cation resins, high-purity KGF-2D31 proteins can be obtained by needing only one-time cation-exchange chromatography and dialysis. Purifying steps are simplified greatly, purifying time is saved, and protein loss rate is reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for conveniently preparing active human KGF-2D31. Background technique [0002] Keratinocyte growth factor-2 was discovered by Yamasaki et al. from mouse embryos in 1996. Because it is closely related to keratinocyte growth factor, it is named Keratinocyte growth factor-2 (KGF-2, hereinafter referred to as this abbreviation) , also known as fibroblast growth factor-10 (FGF-10). Human keratinocyte growth factor-2 consists of 624 nucleotides, encodes 208 amino acids, and has a theoretical molecular weight of 19.3kDa. KGF-2 is mainly synthesized by mesenchymal cells and can specifically bind to fibroblast growth factor receptor 2Ⅲb (FGFR2Ⅲb) and fibroblast growth factor receptor 1Ⅲb (FGFR1Ⅲb) on the surface of target cells. The two receptors trigger the transduction signal, So as to play a physiological role. [0003] KGF-2 can promote the proliferation and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P21/02C07K14/50C07K1/36C07K1/34C07K1/18C12Q1/02C12R1/19
CPCC07K14/50C12N15/70C12N2503/00G01N33/5008G01N2500/10
Inventor 江永海陆雅臣朱月珍
Owner ABZYME BIOTECH
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