Glucokinase preparing process

Abstract

The invention discloses a method for expressing and preparing staphylokinase (Staphylokinase, SAK), comprising: 1. Construction of staphylokinase fusion protein expression engineering bacteria: constructing a recombinant plasmid containing the gene sequence of SEQ NO.1, and introducing the recombinant plasmid into Escherichia coli to obtain fermentation strains. 2. The preparation method includes: a. fermenting and expressing a soluble fusion protein having an amino acid sequence such as SEQ NO.2 by engineering bacteria; b. absorbing the fusion protein in step a with a nickel ion affinity gel column to extract the fusion protein; c. Dilute the fusion protein in step b with enterokinase, dilute appropriately, pass through the gel column in step b, and collect the permeate; d, concentrate the permeate through ultrafiltration in step c, and pass through the Q gel column to obtain the staphylokinase stock solution . The method of the invention has simple and reliable steps, and is especially suitable for large-scale production. The staphylokinase obtained by the method of the invention has high purity, high specific activity and good thrombolytic activity; it provides a new way for producing medicinal staphylokinase.

Description

technical field

[0001] The invention relates to a method for preparing staphylokinase (Staphylokinase, SAK), in particular to a method for expressing and purifying staphylokinase, which belongs to the field of bioengineering and pharmacy. Background technique

[0002] Staphylokinase is an extracellular protein synthesized by Staphylococcus aureus lysogenic phage (de Waart.J., K.C.Winkler and C.Grootsen Nature 1962, 195:407~408 Winkler K.C., deWaart.J., and C.Grootsen J. Gen. Microbiol. 1965, 39: 321-333 Kondo. I., Kiyotaka F., Infection and Immunity 1977, 18: 266-272). Since 1983 Sako (Sako.T., ​​Tsuchida.N Nucleic Acids Res.1983, 11:7679~7693), 1987 BehnkeD (Behnke D., Gerlach D.Mol.Gen.Genet.1987, 210:528~534 ), in 1992 CollenD (Collen D., Z.A.Zhao, P.Holvoet et al. Fibnolysis 1992, 6: 226-231) etc. successfully expressed the staphylokinase gene in Escherichia coli or Bacillus subtilis. The thrombus mechanism has been thoroughly studied. With the increase of animal expe...

Examples

Embodiment 1

[0049] [Example 1] Construction of engineering bacteria and expression of fusion protein

[0050] With the patent application publication number being CN1807603A engineering bacterium complete DNA as template, with primer 1, 2 as doing PCR amplification (primer 1:

[0051] 5'-AAAGGTACCGACGACGACGACAAGTCAAGTTCATTCGACAAAGGA-3'

[0052] Primer 2: 5'-AAAGAGCTCTCATTATTTTCTTTCTATAACAACCTT-3', primer 1 contains the KpnI site, primer 2 contains the SacI site) to obtain a fusion protein gene containing staphylokinase such as SEQ NO.1, after the obtained gene is sequenced and analyzed, cloned On the pET32(a) vector, the insertion site is KpnI / SacI, and the plasmid pET32(a) / Sak is constructed. The specific construction is shown in Figure 3.

[0053] The sequencing results are as follows: GGT ACC GAC GAC GAC GAC AAG TCA AGT TCA TTCGAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGT TAT TTT GAACCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GAT GGTAAA GGA AAT GAA TTG CTA TCC CCT CAT TAT ...

Embodiment 2

[0056] [Example 2] Preparation of Staphylokinase

[0057] 1. Materials and equipment

[0058] Bacteria-breaking tools: homogenizer;

[0059] SDS-PAGE electrophoresis scanner: DS-700Bio-Rad;

[0060] Chromatography column: 50×300mm, 100×300mm Shanghai Yarong Biochemical Instrument Factory;

[0061] Chromatography medium: Chelating-Sepharose Fast Flow and Q-Sepharose Fast Flow are both products of Pharmacia Biotech, and the particle diameter is 90 μm.

[0062] Chromatographic detection system: UV detector (HD-93-1 type) Shanghai Jinda Biochemical Instrument Factory.

[0063] 2. Preparation process

[0064] 1) Broken engineering bacteria

[0065] Get the engineering bacteria with a wet weight of 500g from the fermentation, suspend the bacteria with 2500ml pH8.0 10mM phosphate buffer, break the bacteria, centrifuge to obtain 2300ml of supernatant, filter through 0.45 μm, put on a metal chelation column to extract the fusion protein, and The sequence is shown in SEQ NO.2.

...

technical field

[0001] The invention relates to a method for preparing staphylokinase (Staphylokinase, SAK), in particular to a method for expressing and purifying staphylokinase, which belongs to the field of bioengineering and pharmacy. Background technique

[0002] Staphylokinase is an extracellular protein synthesized by Staphylococcus aureus lysogenic phage (de Waart.J., K.C.Winkler and C.Grootsen Nature 1962, 195:407~408 Winkler K.C., deWaart.J., and C.Grootsen J. Gen. Microbiol. 1965, 39: 321-333 Kondo. I., Kiyotaka F., Infection and Immunity 1977, 18: 266-272). Since 1983 Sako (Sako.T., ​​Tsuchida.N Nucleic Acids Res.1983, 11:7679~7693), 1987 BehnkeD (Behnke D., Gerlach D.Mol.Gen.Genet.1987, 210:528~534 ), in 1992 CollenD (Collen D., Z.A.Zhao, P.Holvoet et al. Fibnolysis 1992, 6: 226-231) etc. successfully expressed the staphylokinase gene in Escherichia coli or Bacillus subtilis. The thrombus mechanism has been thoroughly studied. With the increase of animal expe...

Examples

Embodiment 1

[0049] [Example 1] Construction of engineering bacteria and expression of fusion protein

[0050] With the patent application publication number being CN1807603A engineering bacterium complete DNA as template, with primer 1, 2 as doing PCR amplification (primer 1:

[0051] 5'-AAAGGTACCGACGACGACGACAAGTCAAGTTCATTCGACAAAGGA-3'

[0052] Primer 2: 5'-AAAGAGCTCTCATTATTTTCTTTCTATAACAACCTT-3', primer 1 contains the KpnI site, primer 2 contains the SacI site) to obtain a fusion protein gene containing staphylokinase such as SEQ NO.1, after the obtained gene is sequenced and analyzed, cloned On the pET32(a) vector, the insertion site is KpnI / SacI, and the plasmid pET32(a) / Sak is constructed. The specific construction is shown in Figure 3.

[0053] The sequencing results are as follows: GGT ACC GAC GAC GAC GAC AAG TCA AGT TCA TTCGAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGT TAT TTT GAACCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GAT GGTAAA GGA AAT GAA TTG CTA TCC CCT CAT TAT ...

Embodiment 2

[0056] [Example 2] Preparation of Staphylokinase

[0057] 1. Materials and equipment

[0058] Bacteria-breaking tools: homogenizer;

[0059] SDS-PAGE electrophoresis scanner: DS-700Bio-Rad;

[0060] Chromatography column: 50×300mm, 100×300mm Shanghai Yarong Biochemical Instrument Factory;

[0061] Chromatography medium: Chelating-Sepharose Fast Flow and Q-Sepharose Fast Flow are both products of Pharmacia Biotech, and the particle diameter is 90 μm.

[0062] Chromatographic detection system: UV detector (HD-93-1 type) Shanghai Jinda Biochemical Instrument Factory.

[0063] 2. Preparation process

[0064] 1) Broken engineering bacteria

[0065] Get the engineering bacteria with a wet weight of 500g from the fermentation, suspend the bacteria with 2500ml pH8.0 10mM phosphate buffer, break the bacteria, centrifuge to obtain 2300ml of supernatant, filter through 0.45 μm, put on a metal chelation column to extract the fusion protein, and The sequence is shown in SEQ NO.2.

...

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