A simple method for non-denaturing purification of biotin-tagged proteins through competitive elution with free biotin

被引：5

作者：

Lin, Kui ^{[1
]}

Yan, Qin ^{[2
]}

Mitchell, Audrey ^{[2
]}

Funk, Natasha ^{[2
]}

Lu, Catherlin ^{[2
]}

Xiao, Hao ^{[2
,3
]}

机构：

[1] Guangxi Zhuang Autonomous Reg Ctr Anal & Test Res, Guangxi 530022, Peoples R China

[2] ImmuneChem Pharmaceut Inc, Burnaby, BC V5J 3J1, Canada

[3] Gaungxi Univ, Fac Anim Sci & Technol, Nanning 530015, Guangxi, Peoples R China

来源：

BIOTECHNIQUES | 2020年 / 68卷 / 01期

关键词：

affinity chromatography; anti-biotin antibody agarose; biotinylated protein; competitive elution; immunoprecipitation; purification; BIOTINYLATION;

D O I：

10.2144/btn-2019-0088

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The use of avidin or streptavidin in the purification of biotinylated proteins has been highly restricted due to the harsh and denaturing elution conditions. Here, we use biotinylated bovine serum albumin as a working model to demonstrate a simple and rapid method for biotin-tagged protein purification under non-denaturing conditions. The biotinylated bovine serum albumin is specifically bound to the anti-biotin antibody agarose beads and competitively eluted with free biotin under near-neutral conditions. The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer and allowing the buffer to incubate with agarose beads for 30 min prior to elution. The elution recovery rate is over 85% without apparent protein denaturation. The method is applicable for both immunoprecipitation and column chromatography. METHOD SUMMARY We describe a method for the purification of biotin-tagged proteins under non-denaturing conditions with no change in protein natural structure or function. Anti-biotin antibody agarose is employed to specifically bind the biotinylated proteins, followed by competitive elution with free biotin at near-neutral conditions. This approach is applicable for both immunoprecipitation and column chromatography.

引用

页码：41 / 44

页数：4