Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer-interface mutant.
In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure. A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer.
Borchert
T V
TV
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Pratt
K
K
Zeelen
J P
JP
Callens
M
M
Noble
M E
ME
Opperdoes
F R
FR
Michels
P A
PA
Wierenga
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase