Heterologous expression of photoactivated adenylyl cyclase (PAC) genes from the flagellate Euglena gracilis in insect cells.
The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha (Mr 105 kDa) and two PACbeta (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACalpha and PACbeta were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACbeta and PACbeta, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACalpha and PACbeta antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.
Ntefidou
Maria
M
Friedrich-Alexander Universität, Institut für Biologie, Erlangen, Germany.
Lüdtke
Timo
T
Ahmad
Margaret
M
Häder
Donat-Peter
DP
D000262:Adenylyl Cyclases / Q000235:genetics* / Q000528:radiation effects*; D000818:Animals; D017931:DNA Primers; D004789:Enzyme Activation / Q000187:drug effects; D005056:Euglena gracilis / Q000201:enzymology*; D005784:Gene Amplification; D007313:Insecta; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases