TY - JOUR
T1 - A statistical approach for optimization of RANKL overexpression in Escherichia coli
T2 - Purification and characterization of the protein
AU - Papaneophytou, Christos P.
AU - Rinotas, Vagelis
AU - Douni, Eleni
AU - Kontopidis, George
PY - 2013
Y1 - 2013
N2 - Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3 mM, a post-induction temperature of 25 C and a post-induction time of 6.5 h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1 ± 0.5 μM.
AB - Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3 mM, a post-induction temperature of 25 C and a post-induction time of 6.5 h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1 ± 0.5 μM.
KW - Induction conditions
KW - RANKL
KW - Receptor activator of nuclear factor-κB (RANK)
KW - Response surface methodology
UR - http://www.scopus.com/inward/record.url?scp=84877847675&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2013.04.005
DO - 10.1016/j.pep.2013.04.005
M3 - Article
C2 - 23623854
AN - SCOPUS:84877847675
SN - 1046-5928
VL - 90
SP - 9
EP - 19
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -