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Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli

Svensson Bonde, Johan LU ; Andersson, Christer ; Reseland, JE ; Lyngstadaas, P and Bülow, Leif LU (2006) In Protein Expression and Purification 48(1). p.134-141
Abstract
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... (More)
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. (Less)
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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
chromatography, immobilised metal ion affinity, murine amelogenin, histidine, human osteoblasts, osteocalcin
in
Protein Expression and Purification
volume
48
issue
1
pages
134 - 141
publisher
Academic Press
external identifiers
  • wos:000238459900019
  • scopus:33646882130
  • pmid:16495078
ISSN
1046-5928
DOI
10.1016/j.pep.2006.01.005
language
English
LU publication?
yes
id
211df94f-9e12-47e5-afb4-e0d31fbb44fb (old id 405838)
date added to LUP
2016-04-01 11:53:46
date last changed
2020-06-10 02:24:06
@article{211df94f-9e12-47e5-afb4-e0d31fbb44fb,
  abstract     = {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.},
  author       = {Svensson Bonde, Johan and Andersson, Christer and Reseland, JE and Lyngstadaas, P and Bülow, Leif},
  issn         = {1046-5928},
  language     = {eng},
  number       = {1},
  pages        = {134--141},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli},
  url          = {http://dx.doi.org/10.1016/j.pep.2006.01.005},
  doi          = {10.1016/j.pep.2006.01.005},
  volume       = {48},
  year         = {2006},
}