title: Expression of soluble proteins in Escherichia coli by linkage with the acidic propiece of eosinophil major basic protein.
abstract: An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP. It was reasoned that constructs of this acidic domain with small heterologous cationic proteins expressed in E. coli could result in soluble expression while preventing trafficking and packaging into insoluble inclusion bodies. This has been demonstrated using four examples: complement C5a, CCL18, fibroblast growth factor-β, and leukemia inhibitory factor, whose isoelectric points range from 8.93 to 9.59. Further general applicability of this technique has been shown by using two different expression systems, one which encodes an amino-terminal oligo-histidine leash, and another that codes for an amino-terminal glutathione-S-transferase. Thus the utility of coupling MAP to cationic polypeptides for the purpose of soluble heterologous protein expression in E. coli has been demonstrated.
authors.affiliation: Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, United States. rdiscipio@tpims.org
authors.forename: Richard G
authors.initials: RG
authors.lastname: DiScipio
authors.forename: Sophia K
authors.initials: SK
authors.lastname: Khaldoyanidi
authors.forename: Ingrid U
authors.initials: IU
authors.lastname: Schraufstatter
mesh_terms: D019742:Chemokines, CC / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D015936:Complement C5a / Q000235:genetics / Q000302:isolation & purification; D047093:Eosinophil Major Basic Protein / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D019742:Chemokines, CC; D053605:Leukemia Inhibitory Factor; D011993:Recombinant Fusion Proteins; D005346:Fibroblast Growth Factors; D015936:Complement C5a; D047093:Eosinophil Major Basic Protein