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Purification of truncated and mutated chemotaxis inhibitory protein of Staphylococcus aureus—an anti-inflammatory protein

Gustafsson, Erika LU ; Forsberg, Cecilia; Haraldsson, Karin; Lindman, Stina LU ; Ljung, Lill and Furebring, Christina LU (2009) In Protein Expression and Purification 63(2). p.95-101
Abstract
The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration.... (More)
The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield™ flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2™.



The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS31–113 and wild-type CHIPS1–121. We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS31–113 and wild-type CHIPS1–121. The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Inflammation, Refolding, Purification, Inclusion bodies
in
Protein Expression and Purification
volume
63
issue
2
pages
95 - 101
publisher
Academic Press
external identifiers
  • wos:000261629800005
  • pmid:18950716
  • scopus:56349105099
ISSN
1046-5928
DOI
10.1016/j.pep.2008.09.017
language
English
LU publication?
yes
id
d7b30717-cb88-467c-aae0-806216d7824b (old id 1260548)
date added to LUP
2008-10-30 12:32:45
date last changed
2017-01-01 04:34:26
@article{d7b30717-cb88-467c-aae0-806216d7824b,
  abstract     = {The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield™ flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2™.<br/><br>
<br/><br>
The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS31–113 and wild-type CHIPS1–121. We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS31–113 and wild-type CHIPS1–121. The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.},
  author       = {Gustafsson, Erika and Forsberg, Cecilia and Haraldsson, Karin and Lindman, Stina and Ljung, Lill and Furebring, Christina},
  issn         = {1046-5928},
  keyword      = {Inflammation,Refolding,Purification,Inclusion bodies},
  language     = {eng},
  number       = {2},
  pages        = {95--101},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Purification of truncated and mutated chemotaxis inhibitory protein of Staphylococcus aureus—an anti-inflammatory protein},
  url          = {http://dx.doi.org/10.1016/j.pep.2008.09.017},
  volume       = {63},
  year         = {2009},
}