Zhang Chenming, Lillie Raymond, Cotter Jackie, Vaughan David
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210 Seitz Hall, Blacksburg, VA 24061, USA.
J Chromatogr A. 2005 Mar 25;1069(1):107-12. doi: 10.1016/j.chroma.2004.10.018.
Tobacco is widely used as a model plant for feasibility studies of recombinant protein production from transgenic plants. However, dealing with large quantities of biomass to recover recombinant proteins is a challenge for down-stream processing. In this study, the effect of isoelectric precipitation on native tobacco protein was first studied. Among the three acids studied, hydrochloric acid is shown to be more effective than acetic or citric acid, and at pH 4, 60% of native tobacco protein was precipitated by HCl. Egg white lysozyme was used as the model protein to test the feasibility of polyelectrolyte precipitation in protein recovery from tobacco extract. Precipitation of lysozyme at pH 7 was shown ineffective probably because of the interference of polyphenolic acids. However, after isoelectric precipitation at pH 5 poly(acrylic) acid (PAA) was shown to precipitate 85% of the soluble lysozyme when the polymer dosage was increased to 1.5 mg polymer/mg lysozyme, while negligible amounts of native tobacco protein was co-precipitated. Lysozyme precipitation by PAA in tobacco extract obtained at pH 5 was also studied, and lysozyme yield was significant improved.
烟草被广泛用作转基因植物生产重组蛋白可行性研究的模式植物。然而，处理大量生物质以回收重组蛋白是下游加工面临的一项挑战。在本研究中，首先研究了等电沉淀对天然烟草蛋白的影响。在所研究的三种酸中，盐酸比乙酸或柠檬酸更有效，在pH 4时，60%的天然烟草蛋白被盐酸沉淀。蛋清溶菌酶用作模型蛋白，以测试聚电解质沉淀从烟草提取物中回收蛋白的可行性。在pH 7时溶菌酶沉淀显示无效，可能是由于多酚酸的干扰。然而，在pH 5进行等电沉淀后，当聚合物用量增加到1.5 mg聚合物/mg溶菌酶时，聚丙烯酸（PAA）可沉淀85%的可溶性溶菌酶，而天然烟草蛋白的共沉淀量可忽略不计。还研究了在pH 5获得的烟草提取物中PAA对溶菌酶的沉淀作用，溶菌酶产量显著提高。
