TY - JOUR
T1 - Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli
AU - Svensson Bonde, Johan
AU - Andersson, Christer
AU - Reseland, JE
AU - Lyngstadaas, P
AU - Bülow, Leif
PY - 2006
Y1 - 2006
N2 - Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M 180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E coli cells expressing the histidine tagged amelogenin rp(H)M 180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M 180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin. (c) 2006 Elsevier Inc. All rights reserved.
AB - Amelogenin is a dental enamel matrix protein involved in formation of dental enamel. In this study, we have expressed two different recombinant murine amelogenins in Escherichia coli: the untagged rM179, and the histidine tagged rp(H)M180, identical to rM179 except that it carries the additional N-terminal sequence MRGSHHHHHHGS. The effects of the histidine tag on expression levels, and on growth properties of the amelogenin expressing cells were studied. Purification of a crude protein extract containing rp(H)M 180 was also carried out using IMAC and reverse-phase HPLC. The results of this study showed clearly that both growth properties and amelogenin expression levels were improved for E coli cells expressing the histidine tagged amelogenin rp(H)M 180, compared to cells expressing the untagged amelogenin rM179. The positive effect of the histidine tag on amelogenin expression is proposed to be due to the hydrophilic nature of the histidine tag, generating a more hydrophilic amelogenin, which is more compatible with the host cell. Human osteoblasts treated with the purified rp(H)M 180 showed increased levels of secreted osteocalcin, compared to untreated cells. This response was similar to cells treated with enamel matrix derivate, mainly composed by amelogenin, suggesting that the recombinant protein is biologically active. Thus, the histidine tag favors expression and purification of biologically active recombinant amelogenin. (c) 2006 Elsevier Inc. All rights reserved.
KW - chromatography
KW - immobilised metal ion affinity
KW - murine amelogenin
KW - histidine
KW - human osteoblasts
KW - osteocalcin
U2 - 10.1016/j.pep.2006.01.005
DO - 10.1016/j.pep.2006.01.005
M3 - Article
C2 - 16495078
SN - 1046-5928
VL - 48
SP - 134
EP - 141
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -