Wan E W, Baneyx F
Department of Chemical Engineering, University of Washington, Seattle, Washington, 98195, USA.
Protein Expr Purif. 1998 Oct;14(1):13-22. doi: 10.1006/prep.1998.0941.
Overproduction of the third topological domain of the transmembrane protein TolA (TolAIII) in the periplasm of Escherichia coli confers a "leaky" phenotype to host cells by disrupting the integrity of the outer membrane and causing periplasmic proteins to leach into the growth medium. To examine the physiological consequences of TolAIII overexpression in more detail and assess the usefulness of this strategy for the release of periplasmic recombinant proteins into the extracellular fluid, we constructed a ColE1-compatible plasmid encoding a fusion between the ribose binding protein signal sequence and TolAIII under T7lac transcriptional control. About half of the total TolAIII synthesized in IPTG-induced cells aggregated in a precursor form in the cytoplasm. However, the majority of the mature protein was soluble and located in the extracellular fluid. TolAIII-overproducing cultures exhibited only slight growth defects upon entry into stationary phase but underwent extensive lysis when treated with 0.1% (w/v) SDS, and were unable to divide when supplemented with 0.02% SDS. The loss of outer membrane integrity resulted in long-term damage since cell viability was reduced by three orders of magnitude compared to control or uninduced cells. Overexpression of TolAIII did not significantly interfere with the translocation and processing of a plasmid-encoded fusion between the OmpA signal sequence and TEM-beta-lactamase but led to the release of most periplasmic proteins and 90% of the active enzyme into the extracellular fluid. Although the total levels of beta-lactamase accumulation in TolAIII-overproducing cultures was only 1.5- to 2-fold less than in control cells, the formation of periplasmic inclusions bodies was completely suppressed. A threshold concentration of TolAIII was necessary for efficient release of periplasmic proteins since the viability and detergent sensitivity of uninduced cells was comparable to that of control cultures and 80% of the beta-lactamase synthesized remained confined to the periplasm.
跨膜蛋白TolA的第三个拓扑结构域（TolAIII）在大肠杆菌周质中的过量表达，会破坏外膜的完整性，导致周质蛋白渗漏到生长培养基中，从而赋予宿主细胞一种“渗漏”表型。为了更详细地研究TolAIII过表达的生理后果，并评估该策略在将周质重组蛋白释放到细胞外液中的实用性，我们构建了一个与ColE1兼容的质粒，该质粒在T7lac转录控制下编码核糖结合蛋白信号序列与TolAIII之间的融合蛋白。在IPTG诱导的细胞中合成的总TolAIII中，约一半以前体形式聚集在细胞质中。然而，大多数成熟蛋白是可溶的，并位于细胞外液中。TolAIII过量产生的培养物在进入稳定期时仅表现出轻微的生长缺陷，但在用0.1%（w/v）SDS处理时会发生广泛裂解，并且在添加0.02% SDS时无法分裂。外膜完整性的丧失导致长期损伤，因为与对照或未诱导的细胞相比，细胞活力降低了三个数量级。TolAIII的过表达并未显著干扰OmpA信号序列与TEM-β-内酰胺酶之间质粒编码融合蛋白的转运和加工，但导致大多数周质蛋白和90%的活性酶释放到细胞外液中。尽管在TolAIII过量产生的培养物中β-内酰胺酶积累的总水平仅比对照细胞少1.5至2倍，但周质包涵体的形成被完全抑制。周质蛋白的有效释放需要TolAIII的阈值浓度，因为未诱导细胞的活力和对去污剂的敏感性与对照培养物相当，并且合成的80%的β-内酰胺酶仍局限于周质中。
