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Preparation of SP Ion Exchange Media and Its Application in the Purification of Recombinant Human Lactoferrin

Author: BaiZuo
Tutor: MaRunYu
School: Beijing University of Chemical Technology
Course: Microbial and Biochemical Pharmacy
Keywords: Ion exchange medium Allyl activation Ligand coupling Recombinant human lactoferrin Antibacterial activity
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 80
Quote: 1
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Abstract


Lactoferrin (Lactoferrin, LF) is a high-value therapeutic proteins, with higher levels in human milk, but human milk sources are limited, severely limiting its commercial application. With the rapid progress of transgenic technology, LF has been in recombinant bovine mammary gland bioreactor was highly expressed. In order to effectively utilize the recombinant protein, the paper first imported SP ion exchange chromatography media medium is established mammary gland bioreactor expressed recombinant human lactoferrin purified scale production processes, separation and purification from milk pretreatment ( Milk pretreatment), ion exchange chromatography (Ion Exchange Chromatography, IEC), ultrafiltration desalted (Ultrafiltration desalting), lyophilized (Freeze drying) and other steps. Batch processing capacity of 200L recombinant human lactoferrin milk, craft product obtained about 600g of pure recombinant human lactoferrin, electrophoresis and HPLC purity was greater than 95%. However, because of the high cost of imported process uses ion-exchange medium, the production cost is high, limiting the application of recombinant lactoferrin. In order to reduce production costs purification process, the paper made fast flow agarose gel matrix synthesis of different ligand density SP ion exchange media, and successfully applied to recombinant human lactoferrin purification. The activation step in the agarose gel, adding the amount of allyl glycidyl ether investigated, NaBH4 dosage, the amount of anhydrous Na2SO4, reaction time, reaction temperature and concentration of NaOH solution such factors as the initial density of the medium allyl. Then Na2S2O5 sulfonic acid group is coupled to allyl ligand-activated Sepharose, using orthogonal experiment investigated Na2S2O5 adding amount of coupling buffer pH, reaction temperature ligand density of the medium affected. Found impact factor ligand density medium order of pH gt; Na2S2O5 dosage gt; reaction temperature. Controlled by the reaction process optimization can be achieved SP ion exchange medium ligand density control and got high ligand density (0.24mmol/mL) of SP ion exchange media. Finally, this paper examines the development of the SP ion exchange medium reproducibility of preparation, the use of the media during adsorption. The results show that the synthesis of self-stable medium, different reuse good reproducibility between batches, static, dynamic adsorption performance is good, the density of different ligand SP ion-exchange medium may be used to purify the recombinant human lactoferrin . Wherein the high ligand density (0.24 mmol / mL) of the ion-exchange medium containing milk of up to 50 mL / mL medium, rHLF yield 86.5%, purity of 98.5%. Circular dichroism results show that the purified rHLF structure consistent with natural human lactoferrin. Purified recombinant human lactoferrin in iron binding and release activity similar to natural human lactoferrin concentration of 5g / L for the growth of E. coli rHLF inhibited.

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