Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins

Petr N. Datskevich, Evgeny V. Mymrikov, Nikolai N. Sluchanko, Anton Shemetov, Maria V. Sudnitsyna, Nikolai B. Gusev

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

Original languageEnglish (US)
Pages (from-to)45-54
Number of pages10
JournalProtein Expression and Purification
Volume82
Issue number1
DOIs
StatePublished - Mar 1 2012
Externally publishedYes

Fingerprint

Small Heat-Shock Proteins
Proteins
Housekeeping
Ion Exchange
Ammonium Sulfate
Cytoskeleton
Gel Chromatography
Apoptosis

Keywords

  • Chaperone-like activity
  • Enhanced fluorescent proteins
  • Human small heat shock proteins
  • Oligomeric state

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins. / Datskevich, Petr N.; Mymrikov, Evgeny V.; Sluchanko, Nikolai N.; Shemetov, Anton; Sudnitsyna, Maria V.; Gusev, Nikolai B.

In: Protein Expression and Purification, Vol. 82, No. 1, 01.03.2012, p. 45-54.

Research output: Contribution to journalArticle

Datskevich, Petr N. ; Mymrikov, Evgeny V. ; Sluchanko, Nikolai N. ; Shemetov, Anton ; Sudnitsyna, Maria V. ; Gusev, Nikolai B. / Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins. In: Protein Expression and Purification. 2012 ; Vol. 82, No. 1. pp. 45-54.
@article{520af5d844734ef0a5ddde5bd344fc92,
title = "Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins",
abstract = "Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.",
keywords = "Chaperone-like activity, Enhanced fluorescent proteins, Human small heat shock proteins, Oligomeric state",
author = "Datskevich, {Petr N.} and Mymrikov, {Evgeny V.} and Sluchanko, {Nikolai N.} and Anton Shemetov and Sudnitsyna, {Maria V.} and Gusev, {Nikolai B.}",
year = "2012",
month = "3",
day = "1",
doi = "10.1016/j.pep.2011.11.004",
language = "English (US)",
volume = "82",
pages = "45--54",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins

AU - Datskevich, Petr N.

AU - Mymrikov, Evgeny V.

AU - Sluchanko, Nikolai N.

AU - Shemetov, Anton

AU - Sudnitsyna, Maria V.

AU - Gusev, Nikolai B.

PY - 2012/3/1

Y1 - 2012/3/1

N2 - Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

AB - Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.

KW - Chaperone-like activity

KW - Enhanced fluorescent proteins

KW - Human small heat shock proteins

KW - Oligomeric state

UR - http://www.scopus.com/inward/record.url?scp=82655176602&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=82655176602&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2011.11.004

DO - 10.1016/j.pep.2011.11.004

M3 - Article

C2 - 22100527

AN - SCOPUS:82655176602

VL - 82

SP - 45

EP - 54

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -