Myscofski D M, Dutton E K, Bolken T C, Franke C A, Hruby D E
Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-3804, USA.
Protein Expr Purif. 2000 Oct;20(1):112-23. doi: 10.1006/prep.2000.1275.
Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacterial expression vector for secreted or surface-anchored recombinant proteins. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.
戈登氏链球菌（S. gordonii）已被用作分泌型或表面锚定重组蛋白的革兰氏阳性细菌表达载体。将革兰氏阳性细菌的N端信号序列与目标蛋白融合是有效输出所必需的全部条件。该系统被称为表面蛋白表达系统（SPEX），已用于表达各种用途的蛋白质。在本研究中，通过构建表达多组氨酸标签融合蛋白的载体，对SPEX系统进行了进一步开发。构建了带有N端或C端组氨酸标签的SPEX载体。对化脓性链球菌M6蛋白的C重复区域（CRR）和金黄色葡萄球菌核酸酶A（NucA）酶进行了表达测试。使用金属亲和色谱法（MAC）纯化融合蛋白。结果表明，融合蛋白在N端或C端位置带有His标签的情况下从戈登氏链球菌中表达并分泌出来，并且可以使用MAC进行纯化。通过免疫印迹和ELISA分析确定，M6融合蛋白在表达和纯化后保留了免疫反应性。此外，NucA融合蛋白在MAC纯化后保留了功能活性。使用MAC分别纯化M6-His和NucA-His融合蛋白约15倍和10倍，蛋白质回收率约为30%。本研究表明，N端或C端位置的多组氨酸标签是从重组戈登氏链球菌培养物上清液中纯化分泌型异源蛋白的可行方法。本研究进一步说明了SPEX系统在蛋白质分泌表达和纯化方面的价值。
