Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain.
We have recently shown that rabbit actin can be introduced by electroporation into the Chlamydomonas ida5 mutant lacking conventional actin and rescue its mutant phenotype [Hayashi et al., 2001: Cell Motil. Cytoskeleton 49:146-153]. In this study, we explored the possibility of using electroporation for functional assay of a recombinant protein. The p28 light chain of inner-arm dyneins was expressed in Escherichia coli, purified to homogeneity, and introduced by electroporation into a non-motile mutant ida4oda6 that lacks it. Because this protein was insoluble in the low ionic strength solution used in the previous study, electroporation was performed at physiological ionic strength in the presence of Ca(2+). Most cells shed their flagella after electroporation. Reflagellation took place within 3 h and up to 30% of the cells became motile, indicating that the introduced p28 retained its functional activity. Fluorescently-labeled p28 was equally effective; in this case fluorescence was observed along the flagella. The presence of Ca(2+) and deflagellation appeared to be important for efficient protein delivery, because a triple mutant with the fa1 mutation deficient in the flagellar shedding mechanism recovered motility only very poorly. Similar results were obtained with other combinations of recombinant proteins and mutants. This study thus demonstrates the feasibility of using electroporation for activity assays of recombinant proteins.
Hayashi
Masahito
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Japan.
Yanagisawa
Haru-Aki
HA
Hirono
Masafumi
M
Kamiya
Ritsu
R
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D001681:Biological Assay / Q000379:methods; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002696:Chlamydomonas / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000172:deficiency* / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D005407:Flagella / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics; D020127:Recovery of Function / Q000187:drug effects / Q000235:genetics
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C096191:light chain protein p28, Chlamydomonas; D004398:Dyneins; D002118:Calcium