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Method for purifying sea-mussel mucin by carboxymethyl ion exchange chromatography

An ion exchange chromatography and mussel mucin technology, which is applied in the field of protein purification, can solve problems such as low yield of mussel mucin, and achieve the effects of high selectivity and high yield

Active Publication Date: 2009-01-21
JIANGYIN USUN BIOCHEMICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to overcome the problem of low yield of mussel mucin purified by traditional ion exchange and gel filtration methods, using carboxymethyl ion exchange gel CM Sepharose FF or CM Sepharose HP with agarose matrix in combination with gel filtration media Separation and purification of mussel mucin with Superdex 200 or Sephacryl S-200 can improve the selectivity of separation and purification and increase the yield of active mussel mucin

Method used

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  • Method for purifying sea-mussel mucin by carboxymethyl ion exchange chromatography
  • Method for purifying sea-mussel mucin by carboxymethyl ion exchange chromatography

Examples

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Effect test

Embodiment 1

[0027] Example 1. Using CM Sepharose FF medium in combination with Superdex 200 medium to separate and purify mussel mucin

[0028] 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;

[0029] 2) centrifuge at 12000r / min for 35min, and keep the supernatant;

[0030] 3) Sephadex G-25 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;

[0031] 4) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration membrane;

[0032] 5) CM Sepharose FF ion exchange medium is used, the mobile phase is pH4, and the adsorption mobile phase is 0.1M sodium chloride, and the elution is performed by a linear gradient of 0.5-1.2M sod...

Embodiment 2

[0040] Example 2. Using CM Sepharose HP in combination with Sephacryl S-200 medium to separate and purify mussel mucin

[0041] 1) Using 1% perchloric acid as the extraction solvent, add 100 g of mussel foot silk to 300 ml of extraction solvent, extract at 15°C for 15 minutes, use a stirrer to crush the frozen mussel foot silk at high speed and suspend it in the extraction solvent;

[0042] 2) Remove the residue by filtering with a 45 μm filter membrane, and keep the supernatant;

[0043] 3) Sephadex G-50 was used to remove small molecular compounds, the mobile phase was 30mM sodium acetate buffer solution with pH 3.0, 0.2M sodium chloride was added, and the breakthrough peak was collected;

[0044] 4) Concentrate fractions with Millipore CENTRIPLUS ultrafiltration membrane;

[0045] 5) Using CM Sepharose HP ion exchange medium, mobile phase pH 5, adsorption mobile phase is 0.3M sodium chloride, elution is 0.5-1.2M sodium chloride linear gradient elution within 10-20 column v...

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Abstract

The invention relates to a method for using a carboxymethyl ion exchange chromatography to purify mussel mucin. Mussel mucin contains a group of L-3,4- -Dihydroxyphenylalanine group (L-DOPA), the lysine thereof has strong positive charges and is capable of forming static bonds. The adoption of CM series which is an ion exchange medium connected with a carboxymethyl group (O-CH2COO<->) for separation and purification of mussel mucin has higher selectivity and higher yielding rate, as compared with other ion exchange media. Extraction with a strong acid is adopted to eliminate small-molecular compounds from a desalting column, a carboxylmethyl ion exchange medium with agar as the matrix is used to combine gel filtering medium to separate and purify mussel mucin, and an acetic acid-urea- polyacrylamide gel electrophoresis is used to differentiate mussel mucin through a specific chromogenesis with nitro blue tetrazolium, to improve the yielding rate of active mussel mucin.

Description

technical field [0001] The invention relates to a protein purification method, in particular to a method for purifying mussel mucin using carboxymethyl ion exchange chromatography. Background technique [0002] Mussel Adhesive Protein (MAP), also known as Mytilus edulisfoot protein (Mefp), comes from the marine shellfish Mytilus edulis, which has the ability to withstand the impact of waves in offshore waters. A protein glue is produced and stored in special glands, which is released by 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 each other due to oxidat...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K4/12
Inventor 孟桂凤邢思亮
Owner JIANGYIN USUN BIOCHEMICAL TECH CO LTD
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