Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli

Claudia T. Evans; Daniel D. Owens; Clive A. Slaughter; Paul A. Srere

doi:10.1016/S0006-291X(88)81006-4

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli

Claudia T. Evans, Daniel D. Owens, Clive A. Slaughter, Paul A. Srere

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

14 Scopus citations

Abstract

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an M_r of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The V_max and K_m of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.

Original language	English (US)
Pages (from-to)	1231-1238
Number of pages	8
Journal	Biochemical and Biophysical Research Communications
Volume	157
Issue number	3
DOIs	https://doi.org/10.1016/S0006-291X(88)81006-4
State	Published - Dec 30 1988
Externally published	Yes

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

Access to Document

10.1016/S0006-291X(88)81006-4

Cite this

@article{296fa0bdfc6544689dc9df8962b43710,

title = "Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli",

abstract = "A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.",

author = "Evans, {Claudia T.} and Owens, {Daniel D.} and Slaughter, {Clive A.} and Srere, {Paul A.}",

note = "Funding Information: Acknowled~ents. We are indebted to Penny Kerby for secretarial assistance, Michael Vasko (VA Medical Center, Dallas, TX) for computer analysis of kinetic data and to Carolyn R. MoOmaw and Bikash C. Pramanik for the chromatographic resolution of PTC-TMK from pig heart citrate synthase and confirmation of its identity by thermospray LC/MS. This research was supported by grants from the Veterans Administration and U.S. Public Health Service.",

year = "1988",

month = dec,

day = "30",

doi = "10.1016/S0006-291X(88)81006-4",

language = "English (US)",

volume = "157",

pages = "1231--1238",

journal = "Biochemical and Biophysical Research Communications",

issn = "0006-291X",

publisher = "Academic Press Inc.",

number = "3",

}

TY - JOUR

T1 - Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli

AU - Evans, Claudia T.

AU - Owens, Daniel D.

AU - Slaughter, Clive A.

AU - Srere, Paul A.

N1 - Funding Information: Acknowled~ents. We are indebted to Penny Kerby for secretarial assistance, Michael Vasko (VA Medical Center, Dallas, TX) for computer analysis of kinetic data and to Carolyn R. MoOmaw and Bikash C. Pramanik for the chromatographic resolution of PTC-TMK from pig heart citrate synthase and confirmation of its identity by thermospray LC/MS. This research was supported by grants from the Veterans Administration and U.S. Public Health Service.

PY - 1988/12/30

Y1 - 1988/12/30

N2 - A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.

AB - A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.

UR - http://www.scopus.com/inward/record.url?scp=0024205292&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024205292&partnerID=8YFLogxK

U2 - 10.1016/S0006-291X(88)81006-4

DO - 10.1016/S0006-291X(88)81006-4

M3 - Article

C2 - 3144969

AN - SCOPUS:0024205292

SN - 0006-291X

VL - 157

SP - 1231

EP - 1238

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 3

ER -

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli

Abstract

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this