Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling.

Janni Kristensen; Hans Uffe Sperling-Petersen; Kim Kusk Mortensen; Hans Peter Sørensen

doi:10.1016/j.ijbiomac.2005.10.011

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling.

Janni Kristensen, Hans Uffe Sperling-Petersen, Kim Kusk Mortensen, Hans Peter Sørensen

Nutritional Immunology

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

3 Citationer (Scopus)

Abstract

Udgivelsesdato: 2005-Dec-15

Originalsprog	Engelsk
Tidsskrift	International Journal of Biological Macromolecules
Vol/bind	37
Udgave nummer	4
Sider (fra-til)	212-7
Antal sider	5
ISSN	0141-8130
DOI	https://doi.org/10.1016/j.ijbiomac.2005.10.011
Status	Udgivet - 2005

Bibliografisk note

Keywords: Animals; Antibodies, Monoclonal; Escherichia coli; Escherichia coli Proteins; Green Fluorescent Proteins; Hybridomas; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Recombinant Proteins; Ribosomal Proteins

Adgang til dokumentet

10.1016/j.ijbiomac.2005.10.011

Citationsformater

@article{85ac2040ac0f11ddb5e9000ea68e967b,

title = "Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling.",

abstract = "We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.",

author = "Janni Kristensen and Sperling-Petersen, {Hans Uffe} and Mortensen, {Kim Kusk} and S{\o}rensen, {Hans Peter}",

note = "Keywords: Animals; Antibodies, Monoclonal; Escherichia coli; Escherichia coli Proteins; Green Fluorescent Proteins; Hybridomas; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Recombinant Proteins; Ribosomal Proteins",

year = "2005",

doi = "10.1016/j.ijbiomac.2005.10.011",

language = "English",

volume = "37",

pages = "212--7",

journal = "International Journal of Biological Macromolecules",

issn = "0141-8130",

publisher = "Elsevier",

number = "4",

}

TY - JOUR

T1 - Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling.

AU - Kristensen, Janni

AU - Sperling-Petersen, Hans Uffe

AU - Mortensen, Kim Kusk

AU - Sørensen, Hans Peter

N1 - Keywords: Animals; Antibodies, Monoclonal; Escherichia coli; Escherichia coli Proteins; Green Fluorescent Proteins; Hybridomas; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Recombinant Proteins; Ribosomal Proteins

PY - 2005

Y1 - 2005

N2 - We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.

AB - We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.

U2 - 10.1016/j.ijbiomac.2005.10.011

DO - 10.1016/j.ijbiomac.2005.10.011

M3 - Journal article

C2 - 16330094

SN - 0141-8130

VL - 37

SP - 212

EP - 217

JO - International Journal of Biological Macromolecules

JF - International Journal of Biological Macromolecules

IS - 4

ER -