Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei Gö1 in Escherichia coli.
Single subunits of the A1 ATPase from the archaeon Methanosarcina mazei Gö1 were produced in E. coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins. A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A1 domain and part of the stalk of the A1AO ATPase of M. mazei Gö1, was constructed, cloned into an expression vector and transformed into different strains of Escherichia coli. In any case, a functional, ATP-hydrolysing A1 ATPase was produced. Western blots demonstrated the production of subunits A, B, C, and F in E. coli, and minicell analyses suggested that subunits D, E, and G were produced as well. This is the first demonstration of a heterologous production of a functional ATPase from an archaeon. The A1 ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice. Inhibitor studies revealed that the heterologously produced A1 ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A1, but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol. The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes.
Lemker
T
T
Lehrstuhl für Mikrobiologie der Ludwig-Maximilians-Universität München, Maria-Ward-Str. 1a, D-80638 München, Germany.
Ruppert
C
C
Stöger
H
H
Wimmers
S
S
Müller
V
V
D019843:Archaeal Proteins; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019766:Genes, Archaeal; D007700:Kinetics; D017020:Methanosarcina / Q000201:enzymology* / Q000235:genetics*; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C113203:A1A0 ATPase, Methanosarcina mazei; D006180:Proton-Translocating ATPases