Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci.
Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.
Andréoni
C
C
Centre d'Immunologie Pierre Fabre, Saint Julien en Genevois, France.
Goetsch
L
L
Libon
C
C
Samuelson
P
P
Nguyen
T N
TN
Robert
A
A
Uhlén
M
M
Binz
H
H
Ståhl
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D008863:Microspheres; D017457:Receptors, Albumin / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis*; D012709:Serum Albumin / Q000378:metabolism; D013210:Staphylococcus / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D017457:Receptors, Albumin; D011994:Recombinant Proteins; D012709:Serum Albumin