Warren Travis K, Lund S Amanda, Jones Kevin F, Hruby Dennis E
Siga Technologies, Inc., 4575 SW Research Way, Suite 230 Corvallis, OR 97333, USA.
Protein Expr Purif. 2005 Apr;40(2):319-26. doi: 10.1016/j.pep.2004.10.019.
While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.
虽然大肠杆菌表达系统已被广泛用于生产异源蛋白，但这些系统在生产特定蛋白产物方面存在局限性，包括表达不佳、不溶性蛋白表达形成包涵体和/或截短产物的表达。使用表面蛋白表达（SPEX）系统，染色体整合的异源基因由天然感受态革兰氏阳性菌戈登链球菌表达并分泌到培养基中。在发现大肠杆菌是生产足够量完整产物的不合适表达系统后，我们成功利用SPEX生产了异源抗原BH4XCRR，该抗原是根据与化脓性链球菌M蛋白C重复区域同源的序列设计的。为了进一步提高戈登链球菌对该产物的产量，我们试图开发一种用于生产和分泌异源蛋白的新系统。我们观察到，在各种生长条件下，戈登链球菌分泌高水平的172 kDa蛋白，通过N端序列分析鉴定为葡糖基转移酶GTF。在此，我们报告了一种基于质粒的表达系统的开发，命名为PLEX，我们用它来提高戈登链球菌对BH4XCRR的产量。将戈登链球菌染色体上包含正调控基因rgg、推定的gtfG启动子和gtfG分泌信号序列的区域克隆到大肠杆菌/链球菌穿梭质粒pVA838中。此外，将bh4xcrr结构基因克隆到同一质粒中，位于rgg和gtfG的下游且读码框一致。将该质粒构建体转化到戈登链球菌中，在转化体的培养上清液中检测到的BH4XCRR浓度高于表达相同产物的SPEX菌株的上清液。使用涉及疏水相互作用和凝胶过滤色谱的可扩展两步纯化工艺，很容易从培养上清液中纯化出BH4XCRR。
