Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

Robert A. Setterquist; G. Kenneth Smith; Todd H. Oakley; Youn Hyung Lee; George E. Fox

doi:10.1016/S0378-1119(96)00402-7

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

Robert A. Setterquist
, G. Kenneth Smith
, Todd H. Oakley
, Youn Hyung Lee
, George E. Fox

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

Abstract

A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Original language	English
Pages (from-to)	237-242
Number of pages	6
Journal	Gene
Volume	183
Issue number	1-2
DOIs	https://doi.org/10.1016/S0378-1119(96)00402-7
Publication status	Published - 1996

Bibliographical note

Funding Information:
This work was supported by NASA grant NAGW-2108, EPA grant R-821205-010 and an Environmental Institute of Houston award to G.E.F. Participation by T.H.O. was facilitated by a grant from the NASA Planetary Biology Internship Program. G.K.S. was supported by a NASA Graduate Student Researcher's Fellowship, NGT-51085.

Keywords

5S rRNA
Heterologous ribosomes
Histidine-tag
IMAC
PCR cloning

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10.1016/S0378-1119(96)00402-7

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title = "Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies",

abstract = "A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.",

keywords = "5S rRNA, Heterologous ribosomes, Histidine-tag, IMAC, PCR cloning",

author = "Setterquist, \{Robert A.\} and Smith, \{G. Kenneth\} and Oakley, \{Todd H.\} and Lee, \{Youn Hyung\} and Fox, \{George E.\}",

note = "Funding Information: This work was supported by NASA grant NAGW-2108, EPA grant R-821205-010 and an Environmental Institute of Houston award to G.E.F. Participation by T.H.O. was facilitated by a grant from the NASA Planetary Biology Internship Program. G.K.S. was supported by a NASA Graduate Student Researcher's Fellowship, NGT-51085.",

year = "1996",

doi = "10.1016/S0378-1119(96)00402-7",

language = "English",

volume = "183",

pages = "237--242",

journal = "Gene",

issn = "0378-1119",

publisher = "Elsevier B.V.",

number = "1-2",

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TY - JOUR

T1 - Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

AU - Setterquist, Robert A.

AU - Smith, G. Kenneth

AU - Oakley, Todd H.

AU - Lee, Youn Hyung

AU - Fox, George E.

N1 - Funding Information: This work was supported by NASA grant NAGW-2108, EPA grant R-821205-010 and an Environmental Institute of Houston award to G.E.F. Participation by T.H.O. was facilitated by a grant from the NASA Planetary Biology Internship Program. G.K.S. was supported by a NASA Graduate Student Researcher's Fellowship, NGT-51085.

PY - 1996

Y1 - 1996

N2 - A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

AB - A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

KW - 5S rRNA

KW - Heterologous ribosomes

KW - Histidine-tag

KW - IMAC

KW - PCR cloning

UR - https://www.scopus.com/pages/publications/0030444362

U2 - 10.1016/S0378-1119(96)00402-7

DO - 10.1016/S0378-1119(96)00402-7

M3 - Article

C2 - 8996113

AN - SCOPUS:0030444362

SN - 0378-1119

VL - 183

SP - 237

EP - 242

JO - Gene

JF - Gene

IS - 1-2

ER -

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

Abstract

Bibliographical note

Keywords

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