Department of Life Sciences, Imperial College London, London, UK.
Methods Mol Biol. 2022;2466:93-109. doi: 10.1007/978-1-0716-2176-9_7.
A completely automated purification of glutathione-S-transferase (GST) fusion proteins, either in soluble form or after renaturation of insoluble inclusion bodies, is described. Depending on the expression levels and the amount of glutathione affinity matrix employed, the protocol yields approximately 30-100 μg of purified GST-fusion protein from 2 mL microplate cultures. The high yield is facilitated by employing an efficient chemical/enzymatic lysis procedure for preparing bacterial cell lysates. Insoluble GST-fusion proteins are automatically refolded by a high-throughput robotic microdialysis procedure that also assesses the degree of successful refolding by integrated GST enzymatic assays and quantitation of soluble protein successfully recovered after affinity purification. For soluble GST-fusion proteins the purification procedure is normally completed within 60 min, whereas urea-based denaturation-renaturation strategies typically require an additional 18 h. The integration of quantitation of cell growth and affinity-purified GST-fusion protein yield allows direct comparisons of different expression constructs and the yield of soluble GST-fusion proteins to be optimized in a systematic manner.
本文描述了一种完全自动化的谷胱甘肽-S-转移酶（GST）融合蛋白的纯化方法，无论是可溶性形式还是不可溶性包涵体复性后的形式。根据表达水平和使用的谷胱甘肽亲和基质的量，该方案从 2 mL 微孔板培养物中可获得约 30-100 μg 的纯化 GST 融合蛋白。通过采用高效的化学/酶裂解程序制备细菌细胞裂解物，高产量得以实现。不溶性 GST 融合蛋白通过高通量机器人微透析程序自动复性，该程序还通过集成的 GST 酶测定和定量成功回收的可溶性蛋白来评估复性的成功程度。对于可溶性 GST 融合蛋白，纯化程序通常在 60 分钟内完成，而基于尿素的变性-复性策略通常需要额外的 18 小时。细胞生长和亲和纯化 GST 融合蛋白产量的定量的整合允许直接比较不同的表达构建体，并以系统的方式优化可溶性 GST 融合蛋白的产量。
