Expression of biologically active rat apolipoprotein AIV in Escherichia coli

Min Liu; Nick Maiorano; Ling Shen; Kevin Pearson; Daisuke Tajima; Dian Ming Zhang; Stephen C. Woods; Randy J. Seeley; W. Sean Davidson; Patrick Tso

doi:10.1016/S0031-9384(02)00959-9

Expression of biologically active rat apolipoprotein AIV in Escherichia coli

Min Liu, Nick Maiorano, Ling Shen, Kevin Pearson, Daisuke Tajima, Dian Ming Zhang, Stephen C. Woods, Randy J. Seeley, W. Sean Davidson, Patrick Tso

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

27 Scopus citations

Abstract

Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.

Original language	English
Pages (from-to)	149-155
Number of pages	7
Journal	Physiology and Behavior
Volume	78
Issue number	1
DOIs	https://doi.org/10.1016/S0031-9384(02)00959-9
State	Published - Jan 2003

Bibliographical note

Funding Information:
The authors gratefully acknowledge the help of Dr. David Hui in providing the rat apo AIV cDNA. We also wish to thank Dr. Feng Wang for performing electrospray mass spectrometry and N-terminal sequence of the recombinant protein. This work was supported by research grants from National Institutes of Health DK54890, DK17844, DK56863, HL62542-01, DK54504, DK56910 and DK53444, as well as supported by Proctor and Gamble.

Funding

The authors gratefully acknowledge the help of Dr. David Hui in providing the rat apo AIV cDNA. We also wish to thank Dr. Feng Wang for performing electrospray mass spectrometry and N-terminal sequence of the recombinant protein. This work was supported by research grants from National Institutes of Health DK54890, DK17844, DK56863, HL62542-01, DK54504, DK56910 and DK53444, as well as supported by Proctor and Gamble.

Funders	Funder number
Proctor and Gamble
National Institutes of Health (NIH)	DK17844, DK54504, DK56910, DK53444, DK54890, HL62542-01
National Institute of Diabetes and Digestive and Kidney Diseases	P01DK056863

Keywords

Apolipoprotein A-IV
Escherichia coli
Food intake
Recombinant expression

ASJC Scopus subject areas

Experimental and Cognitive Psychology
Behavioral Neuroscience

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10.1016/S0031-9384(02)00959-9

Cite this

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title = "Expression of biologically active rat apolipoprotein AIV in Escherichia coli",

abstract = "Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.",

keywords = "Apolipoprotein A-IV, Escherichia coli, Food intake, Recombinant expression",

author = "Min Liu and Nick Maiorano and Ling Shen and Kevin Pearson and Daisuke Tajima and Zhang, {Dian Ming} and Woods, {Stephen C.} and Seeley, {Randy J.} and Davidson, {W. Sean} and Patrick Tso",

note = "Funding Information: The authors gratefully acknowledge the help of Dr. David Hui in providing the rat apo AIV cDNA. We also wish to thank Dr. Feng Wang for performing electrospray mass spectrometry and N-terminal sequence of the recombinant protein. This work was supported by research grants from National Institutes of Health DK54890, DK17844, DK56863, HL62542-01, DK54504, DK56910 and DK53444, as well as supported by Proctor and Gamble.",

year = "2003",

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doi = "10.1016/S0031-9384(02)00959-9",

language = "English",

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pages = "149--155",

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AU - Liu, Min

AU - Maiorano, Nick

AU - Shen, Ling

AU - Pearson, Kevin

AU - Tajima, Daisuke

AU - Zhang, Dian Ming

AU - Woods, Stephen C.

AU - Seeley, Randy J.

AU - Davidson, W. Sean

AU - Tso, Patrick

N1 - Funding Information: The authors gratefully acknowledge the help of Dr. David Hui in providing the rat apo AIV cDNA. We also wish to thank Dr. Feng Wang for performing electrospray mass spectrometry and N-terminal sequence of the recombinant protein. This work was supported by research grants from National Institutes of Health DK54890, DK17844, DK56863, HL62542-01, DK54504, DK56910 and DK53444, as well as supported by Proctor and Gamble.

PY - 2003/1

Y1 - 2003/1

N2 - Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.

AB - Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.

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KW - Escherichia coli

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KW - Recombinant expression

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Expression of biologically active rat apolipoprotein AIV in Escherichia coli

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

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