New fusion protein systems designed to give soluble expression in Escherichia coli

✍ Scribed by Gregory D. Davis; Claude Elisee; Denton M. Newham; Roger G. Harrison

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 214 KB
• Volume: 65
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19991120)65:4<382::aid-bit2>3.0.co;2-i

✦ Synopsis

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-␥ 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 Nterminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.