Rippmann J F, Klein M, Hoischen C, Brocks B, Rettig W J, Gumpert J, Pfizenmaier K, Mattes R, Moosmayer D
Institut für Industrielle Genetik, Universität Stuttgart, 70569 Stuttgart, Germany.
Appl Environ Microbiol. 1998 Dec;64(12):4862-9. doi: 10.1128/AEM.64.12.4862-4869.1998.
Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.
最近有研究表明，奇异变形杆菌的L型细胞（L VI）缺乏周质区室，可有效地用于异源蛋白的生产和分泌。为了寻找重组抗体的新型表达系统，我们比较了大肠杆菌JM109和奇异变形杆菌L VI中单链可变片段（scFv）的生产水平，这两种菌株表达了四种具有潜在临床意义的不同scFv，它们在表达水平以及周质表达时形成聚集体的倾向方面存在差异。大肠杆菌中所有分析的scFv的生产都受到异源产物严重毒性作用的限制，表现为培养生长受到抑制以及周质空间中形成不溶性聚集体，从而限制了活性产物的产量。相比之下，L型细胞在所有测试的scFv的生产条件下均表现出几乎不受限制的生长。此外，奇异变形杆菌L VI的表达实验导致每升培养基中scFv浓度在40至200毫克范围内（相应的体积产量比大肠杆菌JM109高33至160倍），具体取决于所表达的抗体。在转运抑制实验中，scFv构建体的分泌被证明是一种与信号切割偶联的主动运输。我们推测，新合成产物直接释放到大量生长培养基中有利于折叠成天然活性结构。在奇异变形杆菌L VI上清液中观察到的scFv有限聚集（以一级动力学方式发生）被发现是由于scFv的内在特性，而与宿主细胞的表达过程无关。发现奇异变形杆菌L VI上清液有利于scFv的纯化。两步色谱法产生了具有高抗原结合活性的均一scFv，这从与真核细胞的结合实验中可以看出。
