Voloshin Alexei M, Swartz James R
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biotechnol Bioeng. 2005 Aug 20;91(4):516-21. doi: 10.1002/bit.20528.
A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.
一种用于批量模式下通用无细胞系统放大的新方法被应用于大肠杆菌氯霉素乙酰转移酶（CAT）的表达以及一种正在开发作为B细胞淋巴瘤疫苗候选物（GLH）的GMCSF-scFv融合蛋白的表达。使用这种新的放大方法评估了两种不同的基于大肠杆菌的无细胞系统的性能。对两种产物和两种反应系统都测试了15至500微升的反应体积。在每种情况下，新的放大方法在每个反应体积下都保持了GLH和CAT的总、可溶性和活性体积产率。在500微升反应体积下，使用新的薄膜方法，PANOx SP系统产生了560±36微克/毫升的活性CAT和99±10微克/毫升的活性GLH蛋白，而500微升试管反应分别产生了250±42微克/毫升和72±7微克/毫升的活性CAT和GLH。同样，与Cytomim系统进行的500微升无细胞合成反应在薄膜中分别产生了481±38微克/毫升的活性CAT和109±15微克/毫升的活性GLH，相比之下，500微升试管反应中活性CAT为29±7微克/毫升，活性GLH蛋白为5±2微克/毫升。新的薄膜方法改善了Cytomim系统的氧气供应，并增加了疏水表面的可用性。分析表明，这些表面对蛋白质表达和折叠有显著益处。我们相信这种方法提供了一种通用的反应放大技术，适用于任何蛋白质靶点、无细胞系统和反应体积。
