摘要
采用金属螯合亲和层析技术纯化了N-末端组氨酸(His)标记的重组蛋白Ax Ce SD。以2000 ku的水溶性葡聚糖Dextran T2000为基质,亚氨基二乙酸(IDA)作为螯合剂,Cu2+做亲和配基制备了能特异性吸附重组蛋白His标记的水溶性葡聚糖亲和-超滤载体,并探讨了p H、离子浓度、吸附时间以及温度等因素对水溶性葡聚糖亲和-超滤载体吸附重组蛋白Ax Ce SD的影响。结果表明,在一定范围内,随着p H和温度的升高亲和-超滤载体对Ax Ce SD吸附量增加,而随着离子浓度的增加其对Ax Ce SD吸附量减少;亲和-超滤载体对Ax Ce SD的吸附在30 min内能够达到平衡。应用Langmuir方程拟合了等温条件下亲和-超滤载体对Ax Ce SD的吸附曲线,得到最大理论吸附量为125.15 mg/g,解离常数为3.259×10-6 mol/L,说明亲和-超滤载体对His标记的重组Ax Ce SD的吸附为以螯合作用为主的特异性吸附。
N-terminal histidine(His)6-tagged recombinant protein Ax Ce SD was purified by affinity chromatography. The affinityultrafiltration escort, Cu2+-IDA-Dextran T2000 specifically able to adsorb the His-tag of recombinant protein, was synthesized using water-soluble Dextran T2000 as the matrix(molecular weight 2000 ku), iminodiacetic acid as the chelating agent, and Cu2+ as the affinity ligand. The effects of p H, ionic strength, adsorption time, and reaction temperature on the adsorption characteristics of the escort with N-terminal His-tagged recombinant Ax Ce SD were investigated. The results showed that the adsorption capacity of the affinity-ultrafiltration escort for Ax Ce SD increased with the increases in p H and temperature within a certain range, but decreased as the ionic strength increased. Moreover, the adsorption equilibrium could be achieved within 30 min. The isotherm adsorption curve was fitted to the Langmuir model. The maximum adsorption yield was 125.15 mg/g and the dissociation constant was 3.259×10-6 mol/L, indicating that the specific adsorption of the affinity-ultrafiltration escort to recombinant Ax Ce SD was mainly from chelation.
出处
《现代食品科技》
EI
CAS
北大核心
2015年第5期65-70,共6页
Modern Food Science and Technology
基金
高等学校博士学科点专项科研基金资助课题(博导类)(20130172110018)
