Farkas Daniel, Franzén Lars-Gunnar, Hansson Örjan
Department of Chemistry, University of Gothenburg, P.O. Box 462, SE-40330 Gothenburg, Sweden.
Protein Expr Purif. 2011 Aug;78(2):156-66. doi: 10.1016/j.pep.2011.02.007. Epub 2011 Feb 25.
The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c₆ in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His₆ tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and ¹⁵N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.
光系统I亚基PsaF参与真核光合生物中电子供体蛋白质体蓝素和细胞色素c₆的对接。在此，我们报告了菠菜PsaF腔结构域（包含1 - 79位氨基酸残基）的表达、纯化及基本特性。重组蛋白利用pET32 Xa/LIC硫氧还蛋白融合系统在大肠杆菌BL21(DE3)中表达。硫氧还蛋白融合蛋白含有His₆标签，通过因子Xa进行蛋白酶消化，随后经固定化金属亲和层析将其去除并与PsaF分离。通过尺寸排阻色谱进一步纯化，从一升生长培养基中最终获得约6 mg的PsaF。经傅里叶变换离子回旋共振质谱验证，因子Xa处理后的PsaF具有正确的身份，该质谱还表明纯化后的蛋白在半胱氨酸残基6和38之间含有一个完整的二硫键。利用远紫外圆二色光谱进一步探究二级结构和折叠情况，结果表明其α - 螺旋含量与光系统I 3.3 Å分辨率的晶体结构一致，螺旋 - 卷曲转变温度为29℃。热荧光研究表明，二硫键对于维持蛋白的整体折叠是必需的，并且疏水区域在50 - 65℃会根据离子强度而暴露。所描述的表达和纯化程序可用于蛋白的同位素标记，¹⁵N - HSQC核磁共振研究表明，制备的蛋白不同构象之间存在缓慢或中等程度的交换，且它属于熔球态结构家族。最后，通过使用一种羧基和胺反应性零长度交联剂，我们证明重组蛋白通过一种特异性的、类似天然的静电相互作用与质体蓝素结合，从而证实了其功能。
