Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs

Srikanta Ghosh, Rajarshi Ghosh, Pradip Das, Dhrubajyoti Chattopadhyay

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.

Original languageEnglish (US)
Pages (from-to)40-48
Number of pages9
JournalProtein Expression and Purification
Volume21
Issue number1
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Small Nuclear RNA
Giardia
Purification
Small Nucleolar RNA
RNA
Giardia lamblia
Proteins
Eukaryota
Recombinant Proteins
Dilatation and Curettage
Recombinant proteins
Cloning
RNA Precursors
Electrophoretic Mobility Shift Assay
Processing
Chelation
Chromatography
Electrophoresis
Protein Binding
Escherichia coli

Keywords

  • Fibrillarin
  • Giardia lamblia
  • rRNA
  • snRNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs. / Ghosh, Srikanta; Ghosh, Rajarshi; Das, Pradip; Chattopadhyay, Dhrubajyoti.

In: Protein Expression and Purification, Vol. 21, No. 1, 2001, p. 40-48.

Research output: Contribution to journalArticle

Ghosh, Srikanta ; Ghosh, Rajarshi ; Das, Pradip ; Chattopadhyay, Dhrubajyoti. / Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs. In: Protein Expression and Purification. 2001 ; Vol. 21, No. 1. pp. 40-48.
@article{3745fe1226964567aa65be42ef98d69f,
title = "Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs",
abstract = "Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.",
keywords = "Fibrillarin, Giardia lamblia, rRNA, snRNA",
author = "Srikanta Ghosh and Rajarshi Ghosh and Pradip Das and Dhrubajyoti Chattopadhyay",
year = "2001",
doi = "10.1006/prep.2000.1319",
language = "English (US)",
volume = "21",
pages = "40--48",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs

AU - Ghosh, Srikanta

AU - Ghosh, Rajarshi

AU - Das, Pradip

AU - Chattopadhyay, Dhrubajyoti

PY - 2001

Y1 - 2001

N2 - Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.

AB - Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.

KW - Fibrillarin

KW - Giardia lamblia

KW - rRNA

KW - snRNA

UR - http://www.scopus.com/inward/record.url?scp=0034961958&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034961958&partnerID=8YFLogxK

U2 - 10.1006/prep.2000.1319

DO - 10.1006/prep.2000.1319

M3 - Article

VL - 21

SP - 40

EP - 48

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -