Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin.
Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin.
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生产正确折叠的富含 [13C, 15N] 的重组豚鼠 [Val90]-α-乳清蛋白。
DOI：
10.1093/protein/10.4.455
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Anderson,S
中科院分区：
文献类型：
--
作者：
Kim,S;Baum,J;Anderson,S
将豚鼠α-乳蛋白(GpLA)的M90V突变体与葡萄球菌A蛋白的Ig G结合区(‘Z’区)进行融合，在大肠杆菌中进行细胞内表达，融合蛋白以包涵体形式表达，体外纯化和复性，经CNBR裂解得到天然α-乳清蛋白。重组M90V gpLA与天然gpLA在刺激半乳糖转移酶合成乳糖的能力上基本一致，重组分子和天然分子也具有相似的圆二色谱、热熔解图谱和核磁共振谱。然而，由于Met-->；Val突变，观察到C-螺旋残基中的酰胺质子的化学位移发生了轻微的扰动。在规定的培养基中，该表达系统能够生产高度浓缩的15N和13C，15N标记的gpLA。这种材料的使用将使gpLA的天然和熔融球状状态的溶液构象可以通过高分辨率多维核磁共振来表征。
The M90V mutant of guinea pig alpha-lactalbumin (gpLA) was expressed intracellularly in Escherichia coli using a gpLA gene fusion to the IgG-binding ('Z') domain coding sequences of staphylococcal protein A. The fusion protein was expressed as an inclusion body, then purified and refolded in vitro; CNBr cleavage of the fusion polypeptide yielded native alpha-lactalbumin. The recombinant M90V gpLA was virtually identical with natural gpLA with respect to its ability to stimulate lactose synthesis by galactosyl transferase and the recombinant and natural molecules also exhibited similar circular dichroism spectra, thermal melting profiles and NMR spectra. However, modest perturbations in the chemical shifts of amide protons in the C-helix residues, attributable to the Met-->Val mutation, were observed. In defined media, this expression system enabled the production of highly-enriched 15N- and 13C, 15N-labeled gpLA. Use of this material will allow the solution conformations of the native and molten globule states of gpLA to be characterized by high-resolution multidimensional NMR.