Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification.
Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.
Zanette
D
D
Lepetit Research Center, Gerenzano, VA, Italy.
Dundon
W
W
Soffientini
A
A
Sottani
C
C
Marinelli
F
F
Akeson
A
A
Sarubbi
E
E
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D008252:Macaca fascicularis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D011994:Recombinant Proteins; D012795:Sialoglycoproteins