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Method for separating and purifying sea-mussel mucin by using mixing adsorption chromatography
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A technology for separation and purification of mussel mucin, which is applied in the field of protein separation and purification, can solve the problems of low yield of mussel mucin, and achieve the effects of high selectivity, high protein yield and simple process
Active Publication Date: 2011-10-12
JIANGYIN USUN BIOCHEMICAL TECH CO LTD
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[0010] The purpose of the present invention is to overcome the problem of low yield of mussel mucin purified by traditional methods, and to provide a high-selectivity, high-efficiency Yield mussel mucin separation and purification method, process simplification, cost reduction
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Embodiment 1
[0026] Embodiment 1, using Superose 12 medium mixed adsorption chromatography to separate mussel mucin
[0027] 1) Using 0.5% perchloric acid and 2% acetic acid as the extraction solvent, add 100g mussel foot silk to 400ml extraction solvent, extract at 18°C for 12min, use a stirrer to crush the frozen mussel foot silk at high speed and suspend it evenly in in the extraction solvent;
[0028] 2) centrifuge at 12000r / min for 35min, and keep the supernatant;
[0029] 3) Sephadex G-50 was used to remove small molecular compounds, the mobile phase was 20mM sodium acetate buffer solution with pH 2.5, 0.15M sodium chloride was added, and the breakthrough peak was collected;
[0030] 4) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration membrane;
[0031] 5) Superose 12, column volume 50ml, eluent including 0.15M sodium chloride, 0-300ml, trifluoroacetic acid 0.02-2% for gradient elution;
[0032] 6) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration me...
Embodiment 2
[0038] Example 2, using Superose 12 pg medium mixed adsorption chromatography to separate mussel mucin
[0039] 1) Using 1% perchloric acid as the extraction solvent, add 100g mussel foot silk to 300ml extraction solvent, extract at 15°C for 15 minutes, use a stirrer to crush the frozen mussel foot silk at high speed to evenly suspend it in the extraction solvent;
[0040] 2) Remove the residue by filtering with a 45 μm filter membrane, and keep the supernatant;
[0041] 3) Sephadex G-25 was used to remove small molecule compounds, the mobile phase was 30mM sodium acetate buffer at pH 3.0, 0.2M sodium chloride was added, and the breakthrough peak was collected;
[0042] 4) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration membrane;
[0043] 5) Use Superose 12 pg, column volume 50ml, eluent includes 0.2M sodium chloride, 0-300ml, acetic acid 0-20% gradient elution;
[0044] 6) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration membrane;
[0045] 7)...
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Abstract
The invention relates to a method for separating and purifying mussel mucin by using a mixed adsorption chromatography. Mussel mucin contains a group of L-3,4- -Dihydroxyphenylalanine (L-DOPA), a phenohydroxyl group thereof can act as the supplier for hydrogen bond, the benzene ring thereof can generate a hydrophobic effect, and the lysine thereof with strong positive charges is capable of forming a static bond. On the basis of the properties of mussel mucin, a mixed adsorption chromatography (i.e. the adsorption chromatography based on three principles of adsorption with hydrogen bond, adsorption with hydrophobic effect, and static adsorption) is adopted to overcome the problem of low yielding rate of mussel mucin in the prior art for separating and purifying mussel mucin. An strong acidextraction is adopted to eliminate small-molecular compounds from a desalting column, an argar medium with high concentration and high cross-linking degree to separate and purify mussel mucin, and anacetic acid-urea- polyacrylamide gel electrophoresis is used to differentiate mussel mucin through specific chromogenesis with nitro blue tetrazolium. Three principles adopted with one separation medium to separate mussel mucin achieve high selectivity, simplify the purification technology, and decrease production cost.
Description
technical field [0001] The invention relates to a method for separating and purifying proteins, in particular to a method for separating and purifying mussel mucin using mixed adsorption chromatography. Background technique [0002] Mussel adhesive protein (MAP), also called Mytilus edulis foot protein (Mefp), comes from the marine shellfish Mytilus edulis, which has the ability to withstand the impact of waves in the offshore, It produces and stores a protein glue in special glands, which is released by the filaments onto solid surfaces such as rocks to form a water-resistant bond and fix itself. Studies on glass have shown that protein glue forms plaques, extending from them, with strong tensile strength (10 6 -10 7 newton meter -2 ), while the intraplaque material contains mussel mucin MAP. [0003] Mussel mucin contains L-3,4-dihydroxyphenylalanine (L-DOPA), which is formed by the action of tyrosinase on tyrosine residues. The L-DOPA residues are cross-linked to eac...
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