New fusion protein systems designed to give soluble expression in Escherichia coli.
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 degrees 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 degrees 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-gamma were also expressed in E. coli at 37 degrees 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.
Davis
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GD
School of Chemical Engineering and Materials Science, University of Oklahoma, 100 East Boyd Street, Norman, Oklahoma 73019-1004, USA.
Elisee
C
C
Newham
D M
DM
Harrison
R G
RG
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002417:Cattle; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005293:Ferritins / Q000737:chemistry / Q000235:genetics; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010445:Peptide Elongation Factors; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D006360:Heat-Shock Proteins; D007377:Interleukin-3; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D013879:Thioredoxins; D007371:Interferon-gamma; D013006:Growth Hormone; D005293:Ferritins; C018032:bacterioferritin