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New fusion protein systems designed to give soluble expression inEscherichia coli

1999; Wiley; Volume: 65; Issue: 4 Linguagem: Inglês

10.1002/(sici)1097-0290(19991120)65

1097-0290

Gregory D. Davis, Claude Elisee, Denton M. Newham, Roger G. Harrison,

Protein purification and stability

Three native E. coli proteins—NusA, GrpE, and bacterioferritin (BFR)—were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37°C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37°C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-γ were also expressed in E. coli at 37°C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 382–388, 1999.