A functional chimaeric S-layer-enhanced green fluorescent protein to follow the uptake of S-layer-coated liposomes into eukaryotic cells.
The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.
Ilk
Nicola
N
Center for Ultrastructural Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Natural Resources and Applied Life Sciences, Gregor Mendelstr. 33, A-1180 Vienna, Austria. ilk@edv1.boku.ac.at
Küpcü
Seta
S
Moncayo
Gerald
G
Klimt
Sigrid
S
Ecker
Rupert C
RC
Hofer-Warbinek
Renate
R
Egelseer
Eva M
EM
Sleytr
Uwe B
UB
Sára
Margit
M
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D008081:Liposomes; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D049452:Green Fluorescent Proteins