A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins.
Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.
Velappan
Nileena
N
B Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Fisher
Hugh E
HE
Pesavento
Emanuele
E
Chasteen
Leslie
L
D'Angelo
Sara
S
Kiss
Csaba
C
Longmire
Michelle
M
Pavlik
Peter
P
Bradbury
Andrew R M
AR
D003593:Cytoplasm / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D017912:Inovirus / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins