| Streptavidin (STV) is one of the most stable proteins known in nature, which can exist in the condition of high temperature, extreme pH, denaturing agent and others while still maintaining its activity. This stable STV can be specifically bounded with biotin to form a Biotin-streptavidin binding system (BSA), which has been widely used in the field of bioscience for the advantages of high sensitivity,strong specificity and signal amplification.Consequently, it is the very prerequisite to get the high yield and purity of STV to play its role effectively. However, there are many defects in the natural STV, such as long expression time, low expression level as well as its tedious and high-cost purification method and so on. Therefore,it is very important to establish a rapid and efficient method for the separation and purification of STV. At present, high performance hydrophobic interaction chromatography (HPHIC) is widely applied in the refolding and purification of proteins because of its mild condition, high speed, high resolution and good repeatability.In this work, in order to overcome the defects of long time of expression and low yield existing in the process of natural STV expression, we firstly constructed the core STV according to the sequence of STV and got an efficient expression in E.coli successfully.The expressed STV which exist in the form of inclusion body was dissolved in 8.0 mol/L urea after washing. Then,a new method to prepare STV was established by refolding and separating of STV using four stationary phases with different ligands in HPHIC mode.This article includes four sections as following:1. Literature reviewSTV has an extreme stability and can combine with biotins to form the BSA system which has the properties of high affinity, good stability and strong signal amplification and is widely applied in the many biotechnologies, for instance, luminescence immunoassay and enzyme linked immune-sorbent assay. Therefore,this chapter will review the structure and function,important use as well as the preparing methods of STV.2. High expression and recovery of streptavidin in Escherichia coliAccording to the constructed STV engineering bacteria, we discussed the effects of culture conditions and recovery conditions of fermentation products. We did also optimize expression condition of STV and the result shows that the STV expression amount can reach up to 43.6% when use 2YT as the medium and glycerol as carbon source with a culture temperature of 37 ?, induction time of 3h and a IPTG concentration of 0.6 mmol/L. We screened the washing liquid and found that the purity of the purified STV reached up to 68.7% when adding sodium deoxycholate (DOC) in Tris-HCl buffer.3. The synthesis of stationary phase modified with benzyl amine and refolding with simultaneous purification of STVA new HPHIC stationary phase with benzyl amine containing a hydrophobic group and electrostatic group as ligand was synthesized, and showed that benzyl has been successfully bonded in silica using elemental analysis and X-ray photoelectron spectroscope. The separation and retention characteristics of the protein were investigated,and it was found that six kinds of standard proteins (Cyt-c; Myo; Lys; a-lact; a-Amy; Ins)can be baseline separated in HPHIC mode. However, the protein was not retained in the weak anion exchange chromatography (WAX) mode. The good separation performance of protein was obtained when pH is 7.0 and the concentration of (NH4)2SO4 is 3.0mol/L. This stationary phase was uesd to separate egg white, and compared with phenyl,phenylethylamine, phenylalanine and tryptophan column, and found that different ligands showed different separation properties. This stationary phase was used to separate and purify OVA in egg white, Cyt-c in pig heart and STV in inclusion body, and the obtained quality of recovery was 93.7%, 97.2% and 30.1% while the purity was 92.1%, 96.4% and 93.3%, respectively.4. The refolding with simultaneous purification of STV using high hydrophobic interaction chromatographyFour kinds of stationary phases with ligand of benzyl amine, phenylethylamine,phenylalanine and tryptophan, respectively, were used to refold and purify STV in HPHIC mode. It was found that the refolding efficiency and the purity of ligand with tryptophan were much higher than the others. We further optimized the chromatographic condition of the stationary phase with tryptophan to refold and purify STV,and the result showed the purity of STV reached 95.3 % and the mass recovery reached 48.7% when the mobile phase pH was 7.5 and the urea concentration was 3 mol/L. Circular dichroism spectrum and biological tests can both verify that these three stationary phases are able to refold and purify STV. RPLC and Cs-930 dual wavelength thin layer chromatogram scanning was used to precisely analyze the obtained purity of STV, and the results were consistent. |
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