阅读说明：本技术 可降解微球及其应用 (Degradable microspheres and application thereof ) 是由 王建斌 王宏伟 尹俊龙 田梦 于 2019-05-31 设计创作，主要内容包括：一种可降解微球,其包括通过交联剂交联的聚合物分子。聚合物分子和/或交联剂含有敏感化学键,敏感化学键能够经化学和/或光处理而断裂从而导致可降解微球的降解。一种从样品中分离目标蛋白的方法,该方法利用可降解微球的降解替代蛋白纯化中的洗脱步骤,从而能够选用亲和力更强的目标蛋白和亲和配体组合以提高蛋白纯化效率。该方法适合于多个蛋白样品的高通量制备,例如为电镜观察或质谱测量提供蛋白样品。(A degradable microsphere comprising polymer molecules crosslinked by a crosslinking agent. The polymer molecules and/or the cross-linking agent contain sensitive chemical bonds that can be broken by chemical and/or light treatment resulting in degradation of the degradable microspheres. A method for separating target protein from a sample utilizes degradation of degradable microspheres to replace an elution step in protein purification, so that a combination of the target protein with stronger affinity and an affinity ligand can be selected to improve the protein purification efficiency. The method is suitable for high-throughput preparation of a plurality of protein samples, for example, protein samples are provided for electron microscope observation or mass spectrometry.)

A degradable microsphere comprising polymer molecules cross-linked by a cross-linking agent, wherein the polymer molecules and/or the cross-linking agent contain sensitive chemical bonds that can be broken by chemical and/or light treatment resulting in degradation of the degradable microsphere.

The degradable microspheres of claim 1, wherein the sensitive chemical bonds are provided by a compound selected from the group consisting of cystamine based compounds, o-nitroanisole based compounds or o-nitrobenzyl alcohol based compounds, glycolide or lactide, and polypeptides with internal proteolytic enzyme cleavage sites.

The degradable microsphere of claim 1, wherein the polymer molecule is selected from the group consisting of polyacrylic acids, polyacrylates, polyacrylamides, polyvinyl alcohols, and polyethylene glycol compounds; the cross-linking agent is selected from cystamine compounds, o-nitroanisole compounds or o-nitrobenzyl alcohol compounds, glycolide or lactide and polypeptides with proteolytic enzyme sites.

The degradable microsphere of claim 1, wherein the cross-linking agent is cystamine bisacrylamide.

The degradable microsphere of claim 4, wherein the polymer molecule is a copolymer of acrylamide and cystamine acrylamide.

The degradable microspheres of claim 5, which are prepared by reacting in water-in-oil reaction droplets, wherein the aqueous phase comprises acrylamide, cystamine bisacrylamide, cystamine acrylamide, and ammonium persulfate.

The degradable microspheres of claim 6, wherein the aqueous phase comprises 6% acrylamide, 0.2% cystamine bisacrylamide, 0.5% ammonium persulfate, and 75mM cystamine acrylamide by weight.

The degradable microsphere of claim 1, having a diameter of 1 μm to 100 μm.

A method of isolating a protein of interest from a sample comprising the protein of interest, comprising the steps of:

1) attaching affinity ligands for the target protein to degradable microspheres;

2) contacting the sample with the degradable microspheres so that the target protein is attached to the degradable microspheres by the affinity ligand;

3) separating the degradable microspheres from the sample; and

4) degrading the degradable microspheres by chemical and/or light treatment to obtain the target protein.

The method of claim 9, wherein the degradable microspheres comprise polymer molecules crosslinked by a crosslinking agent, wherein the polymer molecules and/or the crosslinking agent contain sensitive chemical bonds that can be broken by chemical and/or light treatment resulting in degradation of the degradable microspheres.

The method of claim 10, wherein the sensitive chemical bond is provided by a compound selected from the group consisting of cystamine-based compounds, o-nitroanisole-based compounds or o-nitrobenzyl alcohol-based compounds, glycolide or lactide, and polypeptides with an internal proteolytic enzyme cleavage site.

The method of claim 10, wherein the polymer molecules are selected from the group consisting of polyacrylics, polyacrylates, polyacrylamides, polyvinyl alcohols, and polyethylene glycols; the cross-linking agent is selected from cystamine compounds, o-nitroanisole compounds or o-nitrobenzyl alcohol compounds, glycolide or lactide and polypeptides with proteolytic enzyme sites.

The method of claim 10, wherein the cross-linking agent is cystamine bisacrylamide.

The method of claim 13, wherein the polymer molecule is a copolymer of acrylamide and cystamine acrylamide.

The method of claim 9, wherein the degradable microspheres are prepared by reacting in water-in-oil reaction droplets, wherein the aqueous phase comprises acrylamide, cystamine bisacrylamide, cystamine acrylamide, and ammonium persulfate.

The method of claim 15, wherein the aqueous phase comprises 6% acrylamide, 0.2% cystamine bisacrylamide, 0.5% ammonium persulfate, and 75mM cystamine acrylamide by weight.

The method of claim 14, 15 or 16, wherein step 1) is accomplished by linking the affinity ligand to the amino group on the cystamine acrylamide in the degradable microspheres by the coupling agent glutaraldehyde.

The method of claim 9, wherein the degradable microspheres have a diameter of 1 μ ι η to 100 μ ι η.

The method of claim 9, wherein the target protein is tagged with a Flag tag and the affinity ligand is an anti-Flag antibody; or the target protein is provided with a Strep tag, and the affinity ligand is a Streptactin protein.

The method of claim 9, wherein step 3) is accomplished by chromatography or centrifugation.

A method for observing a target protein by an electron microscopy technique, comprising isolating the target protein by the method of any one of claims 9 to 20 and observing or structurally analyzing the target protein by electron microscopy.

A method of analyzing a target protein by mass spectrometry comprising isolating the target protein by the method of any one of claims 9 to 20 and analyzing the target protein by a mass spectrometer.

The method of claim 22, wherein the analysis comprises detecting the molecular weight, mutation, post-translational modification, or polymerization state of the protein of interest.

The method of claim 23, wherein the post-translational modification is an acylation modification, an alkylation modification, a biotinylation modification, a proximity modification, a glycosylation modification, a phosphorylation modification, an esterification modification, a nitrosylation modification, a ubiquitination modification, a small ubiquitin-related modification (SUMO) modification, an amination modification, a hydroxylation modification, or a carboxylation modification.

A method for the qualitative or quantitative detection of a protein of interest in a sample, comprising the step of isolating said protein of interest from said sample by the method of any one of claims 9 to 20.