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Gene and method thereof for expressing protein

A gene and amino acid technology, applied in the field of cell-free synthesis, can solve the problems of difficult protein expression, strong hydrophobicity, non-expression, etc., and achieve the effect of overcoming low expression and good immune activity

Inactive Publication Date: 2017-05-31
WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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Problems solved by technology

[0003] Skeletal muscle receptor tyrosine kinase (MuSK) is an antigenic protein present in mammals. MuSK is a transmembrane protein associated with acetylcholine receptors. In the development of skeletal muscle, MuSK is the establishment of postsynaptic Myasthenia gravis (MG) is an autoimmune disease caused by autoantibodies that affects the neuromuscular junction (NMJ), muscle-specific kinase antibody (MuSKAb ) plays an important role in the onset and progression of myasthenia gravis. At present, the role of MuSK antibody in myasthenia gravis is still one of the hotspots in international research, and this research has a certain demand for MuSK protein Quantity. However, the protein is difficult to express in common expression systems, and it is almost not expressed due to its strong hydrophobicity

Method used

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  • Gene and method thereof for expressing protein
  • Gene and method thereof for expressing protein
  • Gene and method thereof for expressing protein

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Embodiment 2

[0051] Steps 1-4 are the same as Steps 1-4 in Embodiment 1,

[0052] 5. Precipitation treatment

[0053] The pellet was resuspended with PBS, centrifuged, and the supernatant was discarded, and this step was repeated twice. Resuspend the pellet with 1 mL of 1.5% SKL solution, and add DTT to a final concentration of 5 mM. 30°C, 220rpm, shake for 1h until the precipitate is completely dissolved. Centrifuge and take the supernatant. Add 20% PEG4000 to a final concentration of 2%, 50 mM GSSG to a final concentration of 5 mM, and 100 mM GSSH to a final concentration of 8 mM. Place the centrifuge tube at 4°C for 12 hours. The protein after standing was taken out and dialyzed against TE buffer for 3 days. TE buffer was changed twice during dialysis. Centrifuge to obtain the supernatant. This is the final MuSK protein solution.

[0054] In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examp...

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Abstract

The invention discloses a gene of skeletal muscle receptor tyrosine kinase and a preparation method of protein expressed by the gene. The method comprises the following steps: (1) synthesizing and cloning of the skeletal muscle receptor tyrosine kinase gene; (2) constructing recombinant plasmid; (3) preparing escherichia coli S30 extract; (4) semicontinious acellular protein synthesis reaction; (5) sedimentation treatment. An improved acellular synthesis method is adopted in the method so as to synthesize skeletal muscle receptor tyrosine kinase in vitro, and the expression efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of cell-free synthesis, and in particular relates to a skeletal muscle receptor tyrosine kinase gene and a method for preparing the protein expressed by the gene through an improved cell-free synthesis system. Background technique [0002] Natural inner membrane proteins exist in the lipid environment of biological membranes, but the current technology for studying inner membrane proteins is to disperse them in an aqueous solution environment first, and operate with reference to the method of water-soluble proteins. In order to effectively study inner membrane proteins, it is necessary to dissolve them in an aqueous environment. This is currently usually accomplished by adding detergents to allow the formation of soluble complexes of the inner membrane proteins and lipids in water. Solubilization of inner membrane proteins is a crude process during which protein denaturation and / or aggregation often occurs....

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/70
CPCC12N9/1205C12N15/70C12N2800/101C12N2800/22C12Y207/01112
Inventor 华权高沈鹤霄马峰罗绍祥肖颖舒芹徐春雷王静邓陈玲易汪雪李昆鹏
Owner WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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