Expression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris.
The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.
Verkhusha
Vladislav V
VV
Center for Molecular Medicine, Building B, Moscow State University, 119899, Moscow, Russia.
Shavlovsky
Mikhail M
MM
Nevzglyadova
Olga V
OV
Gaivoronsky
Andrey A
AA
Artemov
Aleksey V
AV
Stepanenko
Olga V
OV
Kuznetsova
Irina M
IM
Turoverov
Konstantin K
KK
D000199:Actins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins