Korea Science Academy, #899, Tanggam 3-Dong, Busanjin-Gu, Busan, 614-822, Korea.
Microb Cell Fact. 2009 Jan 29;8:11. doi: 10.1186/1475-2859-8-11.
ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.
The LARDs included four glycine-rich repeats comprising a beta-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD) to three types of proteins; green fluorescent protein (GFP), epidermal growth factor (EGF) and cytoplasmic transduction peptide (CTP). These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant.
The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.
三磷酸腺苷结合盒（ABC）转运蛋白在革兰氏阴性菌中同时通过内外膜将蛋白质分泌出去。荧光假单胞菌 SIK W1 的耐热脂肪酶（TliA）通过 ABC 转运蛋白分泌。TliA 的 C 末端有四个甘氨酸丰富重复（GGXGXD），这种结构出现在许多 ABC 转运蛋白分泌的蛋白质中。根据铜绿假单胞菌碱性蛋白酶（AprA）的结构，我们从 TliA 的同源模型中设计了脂肪酶 ABC 转运蛋白结构域（LARD），用于融合蛋白的分泌。
LARD 包括由β-折叠结构组成的四个甘氨酸丰富重复，添加到测试蛋白的 C 末端。融合蛋白和 LARD 之间要么添加 Pro-Gly 接头，要么添加因子 Xa 位点。我们将不同长度的 LARD（如 LARD0、LARD1 或整个 TliA（最长的 LARD））添加到三种类型的蛋白质上；绿色荧光蛋白（GFP）、表皮生长因子（EGF）和细胞质转导肽（CTP）。这些融合蛋白与荧光假单胞菌或菊欧文氏菌的 ABC 转运蛋白一起在大肠杆菌中表达。根据脂肪酶酶活性，融合蛋白通过 ABC 转运蛋白的整个 TliA 出口是明显的。在向大肠杆菌补充 ABC 转运蛋白后，GFP-LARD 和 EGF-LARD 被分泌到培养上清液中。
LARD 或整个 TliA 被添加到模型蛋白的 C 末端，使 GFP 和 EGF 等模型蛋白能够在大肠杆菌中表达，该大肠杆菌补充了 ABC 转运蛋白。这些结果为使用 LARD 或 TliA 作为 C 末端信号序列在假单胞菌中体外生产重组蛋白提供了可能性。
