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

Producción científica: Article › revisión exhaustiva

27 Citas (Scopus)

Resumen

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.

Idioma original	English
Páginas (desde-hasta)	149-155
Número de páginas	7
Publicación	Physiology and Behavior
Volumen	78
N.º	1
DOI	https://doi.org/10.1016/S0031-9384(02)00959-9
Estado	Published - ene 2003

Nota bibliográfica

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.

Financiación

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.

Financiadores	Número del financiador
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

ASJC Scopus subject areas

Experimental and Cognitive Psychology
Behavioral Neuroscience

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10.1016/S0031-9384(02)00959-9

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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.",

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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.

KW - Apolipoprotein A-IV

KW - Escherichia coli

KW - Food intake

KW - Recombinant expression

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Expression of biologically active rat apolipoprotein AIV in Escherichia coli

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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