Hayashi Masahiro, Iwamoto Shigehisa, Sato Shinya, Sudo Shigeo, Takagi Mari, Sakai Hiroshi, Hayakawa Tohru
Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Protein Expr Purif. 2013 Apr;88(2):230-4. doi: 10.1016/j.pep.2013.01.011. Epub 2013 Feb 8.
Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.
胱抑素C是一种由多种人体组织产生的半胱氨酸蛋白酶抑制剂。胱抑素C的血液浓度取决于肾小球滤过率，是肾功能障碍的内源性标志物。因此，具有高免疫原性的重组胱抑素C蛋白在诊断市场上有需求。在本研究中，为建立一个高效的生产系统，根据大肠杆菌基因的密码子偏好设计并合成了一个合成胱抑素C基因。重组胱抑素C作为与肽标签4AaCter的融合蛋白表达，该标签有助于在大肠杆菌细胞中形成蛋白质包涵体。与4AaCter标签融合显著提高了胱抑素C的生产水平，无需复杂的纯化步骤即可获得高度纯化的蛋白。最终产物的纯度和产量分别估计为87±5%和7.1±1.1mg/升培养物。我们的方法制备的重组胱抑素C与天然人胱抑素C一样对抗胱抑素C抗体有反应。我们的结果表明，使用4AaCter标签的蛋白质生产系统可能是制备大量抗原蛋白的有力手段。
