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Process for preparing recombined placental growth factor

A technology of placental growth factor and form, which is applied in the field of preparation of recombinant placental growth factor, and can solve the problems of not describing conditions and experimental details

Inactive Publication Date: 2005-05-25
GEYMONAT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method disclosure only gives a simple list of known applicable techniques without describing the conditions and experimental details necessary to obtain the PLGF protein in the purity and quantity required for pharmaceutical use

Method used

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  • Process for preparing recombined placental growth factor
  • Process for preparing recombined placental growth factor
  • Process for preparing recombined placental growth factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1: fermentation

[0052] The following steps relate to the method of fermentation and induction of a genetically modified microorganism (MOGM) [Bl21(DE3)pLysS PLGF-1] in a fermentation vessel using 1 mM IPTG.

[0053] Material:

[0054] Solution SBM consists of the following:

[0055] Solution A (per 1 liter)

[0056] Bacto Yeast Extract (Difco) 34g

[0057] Ammonium sulfate 2.5g

[0058] Glycerin 100ml

[0059] h 2 O about 900ml

[0060] Solution B (10X) (per 100ml)

[0061] K H 2 PO 4 1.7g

[0062] K 2 HPO 4 -3H 2 O 20g, or

[0063] K 2 HPO 4 15.26g

[0064] h 2 O add appropriate amount to 100ml

[0065] Solutions A and B were autoclaved separately and mixed under aseptic conditions for use. Alternatively, solutions A and B are mixed under aseptic conditions and filtered.

[0066] IPTG 200mM (200X) was prepared by dissolving 5g of pure material in 100ml of distilled water. The solution was filtered through a 0.22 μm filter, subdivi...

Embodiment 2

[0096] Example 2: Extraction and purification of inclusion bodies

[0097] The following steps involve the preparation and refolding of inclusion bodies of PLGF-1. Upon refolding, the PLGF-1 bacterial protein is partially restored to the dimeric form.

[0098] Material:

[0099] Mixer of appropriate capacity

[0100] Lysis solution: 1mM Mg 2 SO 4 +20mM Tris-HCl pH8+1%TritonX100.

[0101] Washing solution: 0.5% triton X100+10mM EDTA pH8.

[0102] BD (denaturing buffer): 8M urea, 50mM Tris pH8, ethylenediamine 20mM.

[0103] dissolve in water.

[0104] Oxidized Glutathione 200x: 100mM, in water

[0105] Reduced Glutathione 200x: 250mM in water.

[0106] Dilution buffer: PEG 4000 (2.4g / l) at a final concentration of 600uM

[0107] 50mM Tris-HCl pH8, 20mM NaCl.

[0108] Antifoaming agent: Antifoam O-10 (non-silicon) Sigma

[0109] Preparation of PLGF-1 inclusion bodies.

[0110] Lysis and wash solutions were equilibrated at room temperature (RT).

[0111] Freeze / thaw...

Embodiment 3

[0122] Example 3: Renaturation of protein

[0123] Inclusion bodies were dissolved in 7ml of denaturing buffer BD (containing urea 8M) and further diluted in BD until OD280 was 0.8. Subsequently, 0.6 volumes of dilution buffer were added in order to bring the final concentration of urea to 5M.

[0124] Thereafter, 1 / 200 reduced glutathione 200× (final concentration 1.25 mM) and 1 / 200 oxidized glutathione 200× (final concentration 0.5 mM) were added. 15 μl of the sample for examination (prerefol) was taken, and the solution was then incubated at 20° C. with stirring for 18-20 hours.

[0125] At the end of the incubation period, the medium was centrifuged at 10.000 x g for 10 minutes at 20° C., filtered against a 0.45 or 0.8 μm filter and a 15 μl sample was taken for inspection (postrefol).

[0126] Results: 15 μl of the solution before and after refolding were analyzed by SDS-PAGE electrophoresis ( Figure 5 ).

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Abstract

Process for extracting and purifying the recombinant Placental Growth Factor (PLGF) expressed in inducible prokaryotic expression systems comprising the following steps: I) fermentation of the bacterial cells, II) extraction and purification of the inclusion bodies, III) renaturation of the expressed protein, IV) ion-exchange chromatography, V) reverse-phase chromatography.

Description

field of invention [0001] The present invention relates to a method for extracting and purifying recombinant placental growth factor from genetically modified cells. Background technique [0002] Placental growth factor (PLGF) is a homodimeric glycoprotein with a similar structure to vascular endothelial growth factor (VEGF). The complete polynucleotide sequence encoding the PLGF protein is described by Maglione and Persico in Italian patent EP-B-550 519 (WO-A-92 / 06194) of September 27, 1990 claiming priority. Different processes of splicing the ARN of PLGF generate three homologous forms of placental growth factor, specifically PLGF-1, PLGF-2 and PLGF-3, with distinct polypeptide sequences, all described in the literature. [0003] The above-mentioned patents also describe methods for the production of PLGF factors, including the use of an inducible prokaryotic expression system characterized by a host cell modified by an expression vector in which the human PLGF gene is i...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K1/18C07K1/20C07K14/475C07K14/515C12N15/12C12P21/02
CPCC07K14/515C07K1/36C07K1/14C12N15/70
Inventor D·玛格里尼M·巴缇斯特E·康缇G·萨尔维亚M·图斯
Owner GEYMONAT
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