A kind of affinity adsorbent with yeast flocculin as ligand and its application
A flocculation protein and adsorbent technology, applied in the field of affinity adsorbent, can solve the problems of many side reactions, difficult separation and purification, etc., and achieve the effect of high efficiency
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Embodiment 1
[0025] Example 1 Expression of exogenous N-Lg-Flo1p in recombinant Escherichia coli
[0026] Based on the theory of yeast flocculation, the N-terminus of yeast cell wall flocculation protein was exogenously expressed in Escherichia coli, and used as a ligand to prepare an affinity adsorbent for separating functional sugars such as mannan oligosaccharides. Specific steps are as follows:
[0027] (1) Synthesize the gene encoding N-Lg-FLO1 shown in SEQ ID NO.1, connect the gene between the pET21b (+) carrier NdeI and XhoI, induce expression in Escherichia coli, screen positive clones, and inoculate into In the LB medium containing 50 μg / mL, cultivate at 37°C and 200r / min for 12h, transfer to the LB medium containing ampicillin with 1% inoculum size, and cultivate at 37°C and 200r / min until the logarithmic growth phase ( OD 600 value reached 0.6), and induced with a final concentration of 0.2mmol / L IPTG at 37°C and 200r / min for 8h, the target protein was present in the cell in t...
Embodiment 2
[0034] Example 2 Preparation of Affinity Adsorbent Using Yeast Flocculin (N-Lg-Flo1p) as Ligand
[0035] Get the N-Lg-Flo1p refolding liquid 8mL that prepares in Example 1 (contain N-Lg-Flo1p protein 16.5mg), purify through Ni-NTA Sepharose 6FF affinity column, use the equilibrium of 5 times column volume Wash the column with buffer solution, collect the washing solution until no protein flows out, measure the content of miscellaneous protein and unbound N-Lg-Flo1p with Q5000 ultra-micro nucleic acid protein analyzer, and calculate the concentration of N-Lg-Flo1p in Ni-NTA agarose. The adsorption amount on gel 6FF was 12.1 mg / mL. Thus, an affinity adsorbent with N-Lg-Flo1p as a ligand is obtained.
Embodiment 3
[0036] Example 3 Adsorption of mannan oligosaccharides with N-Lg-Flo1p-Ni-NTA Sepharose 6FF affinity adsorbent
[0037] Take 1 mL of N-Lg-Flo1p-Ni-NTA Sepharose 6FF affinity adsorbent, add to 10 mL of adsorption buffer (20 mmol / L Tris-HCl buffer, pH7.5, containing 0.2mol / L NaCl, 2mmol / L CaCl 2 ), shake for 1 h on a shaker with a rotational speed of 100 r / min. The affinity adsorbent was loaded into a column, and 5 column volumes were eluted with 20 mM Tris-HCl buffer solution containing 0.2M NaCl, pH 4.5, to collect mannooligosaccharides. The content of mannooligosaccharides in the eluent was determined by anthrone colorimetry, and the calculated adsorption capacity was 10.14 mg / (mL adsorbent).
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