Receptor-mediated endocytosis as a selection force to enrich bacteria expressing rhodostomin on their surface.
Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999]. To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E. coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive bacteria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates. We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles.
Chang
H H
HH
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan, ROC.
Shih
K N
KN
Lo
S J
SJ
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008565:Membrane Proteins / Q000096:biosynthesis; D008647:Mesocricetus; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D010641:Phenotype; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D019151:Peptide Library; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C072026:rhodostomin