Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization

TY - JOUR

T1 - Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization

AU - Pajęcka, Kamilla

AU - Nielsen, Camilla Wendel

AU - Hauge, Anne

AU - Zaganas, Ioannis

AU - Bak, Lasse Kristoffer

AU - Schousboe, Arne

AU - Plaitakis, Andreas

AU - Waagepetersen, Helle S

PY - 2014/3

Y1 - 2014/3

N2 - Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.

AB - Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.

KW - Adenosine Diphosphate

KW - Animals

KW - Cell Line

KW - Cytosol

KW - Glutamate Dehydrogenase

KW - Guanosine Triphosphate

KW - Histidine

KW - Humans

KW - Kinetics

KW - Mice

KW - Mitochondria

KW - Oligopeptides

KW - Protein Isoforms

KW - Recombinant Proteins

U2 - 10.1007/s11064-013-1042-z

DO - 10.1007/s11064-013-1042-z

M3 - Journal article

C2 - 23619558

SN - 0364-3190

VL - 39

SP - 487

EP - 499

JO - Neurochemical Research

JF - Neurochemical Research

IS - 3

ER -