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

• Published in: Biotechnology and bioengineering 1999-11, Vol.65 (4), p.382-388
• Main Authors: Davis, Gregory D., Elisee, Claude, Newham, Denton M., Harrison, Roger G.
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Animals; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; bacterioferritin; Biological and medical sciences; Biotechnology; Cattle; Cytochrome b Group - chemistry; Cytochrome b Group - genetics; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins; Ferritins - chemistry; Ferritins - genetics; Fundamental and applied biological sciences. Psychology; fusion protein; Genetic engineering; Genetic technics; Growth Hormone - chemistry; Growth Hormone - genetics; GrpE protein; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Hormones; Humans; Interferon-gamma - chemistry; Interferon-gamma - genetics; Interleukin-3 - chemistry; Interleukin-3 - genetics; Interleukin-3 - isolation & purification; Mathematical models; Methods. Procedures. Technologies; Modification of gene expression level; NusA protein; Peptide Elongation Factors; Probability; Protein Engineering - methods; Purification; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Solubility; soluble protein expression; Statistics; Thioredoxins - chemistry; Thioredoxins - genetics; Transcription Factors - chemistry; Transcription Factors - genetics; Transcriptional Elongation Factors
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19991120)65:4<382::AID-BIT2>3.0.CO;2-I

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.