Systematic optimization of active protein expression using GFP as a folding reporter.
Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.
Omoya
Kentaro
K
Department of Pediatrics, Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu, Gifu 501-1194, Japan.
Kato
Zenichiro
Z
Matsukuma
Eiji
E
Li
Ailian
A
Hashimoto
Kazuyuki
K
Yamamoto
Yutaka
Y
Ohnishi
Hidenori
H
Kondo
Naomi
N
D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D020382:Interleukin-18 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D020382:Interleukin-18; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase