Masuda K, Kamimura T, Kanesaki M, Ishii K, Imaizumi A, Sugiyama T, Suzuki Y, Ohtsuka E
Teijin Institute for Biomedical Research, Tokyo, Japan.
Protein Eng. 1996 Jan;9(1):101-6. doi: 10.1093/protein/9.1.101.
We have developed a high-level production system for the C-terminal domain of secretory leukoprotease inhibitor (SLPI) to investigate its pharmacological activities. A gene for the C-terminal domain of SLPI, (Asn55-Ala 107)SLPI, was constructed from chemically synthesized deoxyoligonucleotides. It was fused to a gene for the N-terminal portion of human growth hormone via a DNA sequence encoding Leu-Val-Pro-Arg, which can be cleaved by thrombin. The fused gene was expressed in Escherichia coli under the control of a trp promoter, and the fusion protein was obtained as an inclusion body. After sulfonation of the cysteine residues, the sulfonated fusion protein was cleaved at the desired site by thrombin. Sulfonated (Asn55-Ala107) SLPI was refolded in Tris buffer containing reduced and oxidized glutathione. The resulting (Asn55-Ala107) SLPI was purified by cation-exchange chromatography and reverse-phase high performance liquid chromatography. The final yield was 50 mg/I culture. (Asn55-Ala107) SLPI was as active against elastase as, but had less trypsin inhibitory activity than, native SLPI. This system is suitable for the large-scale production of the C-terminal domain of SLPI, which is an elastase-specific inhibitor.
我们开发了一种用于分泌型白细胞蛋白酶抑制剂（SLPI）C 末端结构域的高级生产系统，以研究其药理活性。从化学合成的脱氧寡核苷酸构建了 SLPI C 末端结构域（Asn55-Ala 107）SLPI 的基因。它通过编码可被凝血酶切割的 Leu-Val-Pro-Arg 的 DNA 序列与人生长激素 N 末端部分的基因融合。融合基因在 trp 启动子的控制下在大肠杆菌中表达，融合蛋白以包涵体形式获得。半胱氨酸残基磺化后，磺化的融合蛋白在所需位点被凝血酶切割。磺化的（Asn55-Ala107）SLPI 在含有还原型和氧化型谷胱甘肽的 Tris 缓冲液中重折叠。所得的（Asn55-Ala107）SLPI 通过阳离子交换色谱和反相高效液相色谱纯化。最终产量为 50 mg/I 培养物。（Asn55-Ala107）SLPI 对弹性蛋白酶的活性与天然 SLPI 相同，但对胰蛋白酶的抑制活性低于天然 SLPI。该系统适用于大规模生产 SLPI 的 C 末端结构域，它是一种弹性蛋白酶特异性抑制剂。
