Large-Scale Generation of Recombinant Granulin Peptides in E. coli
- 538 Downloads
Generating milligram quantities of correctly folded granulin molecules with properly formed disulfide bonds and biologically relevant activities may represent a considerable challenge. Here I describe a protocol for obtaining well-folded human granulins A, C, and F by expressing them as thioredoxin fusion proteins in Origami (DE3) Escherichia coli cells promoting disulfide bond formation in the cytoplasm environment. The thioredoxin tag is removed by proteolytic cleavage with enterokinase and granulins which are purified by reversed-phase HPLC. Well-folded disulfide species display lower retention time than misfolded species and therefore can be readily purified.
Key wordsGranulin Progranulin Large-scale bacterial expression Disulfide bond Purification HPLC
- 13.Stark GR, Stein WH, Moore S (1960) Reactions of the cyanate present in aqueous urea with amino acids and proteins. J Biol Chem 235:3177–3181Google Scholar