Kun Tian, Xiaoyun Feng, Qin Du, Chuhang Liao, Xiaohua Ren
Dept. of Stomatology, Hospital of University of Electronic Science and Technology of China UESTC & Sichuan Provincial People's Hosptial, Chengdu 610072, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2017 Feb 1;35(1):63-67. doi: 10.7518/hxkq.2017.01.009.
Recombinant human leucine-rich amelogenin peptide (LRAP) was studied by cryogenic transmission electron microscopy (TEM); evaluation focused on its self-assembly and crystal growth in vitro.
Human LRAP was recombined through prokaryotic expression vector pCold-SUMO and transformed into Escherichia coli BL21plys to acquire purified proteins. Cryogen TEM recorded assembly and self-assembling of LRAP from pH 3.5 to pH 8.0, and the hydroxyapatite crystal growth in the mixture of LRAP protein solution and artificial saliva was observed using TEM and selected area electron diffraction.
More than 90% purity LRAP was expressed, purified and identified as described in methods. LRAP linked into oligomers, nanospheres, nanochains, and microribbons, whereas pH value increased from 3.5 to 8.0. Mature hydroxyapatite crystal growth was guided in artificial saliva filled with calcium phosphate.
CONCLUSIONS: LRAP is simplified amelogenin functional domain and conserved the basic characters of amelogenin such as self-assembling and inducing crystallization along c axis. In the area of acellular synthesis of hydroxyapatite using extracellular enamel matrix protein, LRAP is one of candidate repair materials for irregular hard tissue defection. .
采用低温透射电子显微镜（TEM）研究重组人富含亮氨酸釉原蛋白肽（LRAP）；评估重点在于其体外自组装和晶体生长情况。
通过原核表达载体pCold-SUMO重组人LRAP，并将其转化至大肠杆菌BL21plys中以获得纯化蛋白。低温TEM记录了LRAP在pH 3.5至pH 8.0范围内的组装和自组装情况，并使用TEM和选区电子衍射观察了LRAP蛋白溶液与人工唾液混合体系中羟基磷灰石晶体的生长情况。
如方法中所述，表达、纯化并鉴定出纯度超过90%的LRAP。随着pH值从3.5升高到8.0，LRAP连接形成寡聚体、纳米球、纳米链和微带。在充满磷酸钙的人工唾液中引导了成熟羟基磷灰石晶体的生长。
LRAP是简化的釉原蛋白功能结构域，保留了釉原蛋白的基本特性，如自组装和沿c轴诱导结晶。在利用细胞外釉质基质蛋白进行羟基磷灰石无细胞合成领域，LRAP是不规则硬组织缺损的候选修复材料之一。
