Department of Chemistry, Duncan Hall 612, One Washington Square, San José State University, San José, CA, 95192, USA.
BMC Biochem. 2018 Dec 18;19(1):12. doi: 10.1186/s12858-018-0101-0.
Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.
Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.
The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host.
研究埃及伊蚊中参与重要生物过程的蛋白质和酶受到可直接从蚊子中分离出的蛋白质数量的限制。此外，参与消化血餐蛋白的消化酶（中肠蛋白酶）需要更氧化的环境才能使二硫键正确折叠。因此，在大肠杆菌中表达外源蛋白的重组技术被证明可以有效地生产毫克数量的表达产物。然而，由于最常用的菌株细胞质具有还原性，因此重组蛋白酶的可溶性表达受到阻碍。幸运的是，现在有新的大肠杆菌菌株具有更氧化的细胞质，以确保二硫键的正确折叠。
利用具有更氧化细胞质的大肠杆菌菌株（New England Biolabs 的 Shuffle® T7）和细菌生长温度的变化，已经实现了四种最丰富表达的埃及伊蚊中肠蛋白酶（AaET、AaSPVI、AaSPVII 和 AaLT）的可溶性表达。之前尝试用全长酶原形式和具有异源肠激酶切割位点的修饰假原肽表达这些蛋白酶，导致重组蛋白表达不溶性。与更氧化的细胞质和生长温度的变化相结合，有助于提高大肠杆菌中酶原（无信号肽）天然前肽蛋白酶的可溶性。此外，该方法导致蛋白酶在细菌表达过程中自动催化激活，并可观察到 BApNA 活性。测试了细菌生长诱导后不同的时间点，以确定观察到无活性（酶原）物种向活性形式转变的时间。这有助于使用镍亲和层析仅纯化和分离无活性的酶原形式。
在大肠杆菌中可溶性表达重组蛋白酶的困难是由天然的还原细胞质引起的。然而，使用具有更氧化细胞质的细菌菌株，可以实现重组可溶性表达，但仅与细菌生长温度的变化一致。本文所述的方法应为利用大肠杆菌作为宿主重组表达埃及伊蚊蛋白酶或依赖二硫键的蛋白质提供一个简单的起点。
