Displaying human interleukin-2 on the surface of bacteriophage.
Previous attempts to produce active human Interleukin-2 (hIL-2) in E. coli have failed, due to its aggregation in the form of cytoplasmic inclusion bodies, and the inability of the protein to enter the periplasmic export pathway, when fused to bacterial signal sequences. We have reasoned that these limitations could be overcome by introducing changes in the signal sequence and/or in some hIL-2 residues, not critical for its biological activity; and proceeded to test this hypothesis using a phagemid vector carrying the pelB secretion signal sequence, and the filamentous phage display system. Deletion of the Pro +2 in hIL-2 led to the export of a correct size (processed) molecule to the bacterial periplasm of Su- cells by the phagemid vector. However, this was achieved under growth conditions that would not favor phage assembly in Su+ strains. Changing the hydrophobic core of the leader peptide reversed this situation and allowed phage assembly and display of a pIII/hIL-2 hybrid protein in TG1 cells. The phage-displayed hIL-2 is correctly folded, as judged by its ability to interact with a conformation-specific anti-hIL-2 monoclonal antibody, and maintains its biological activity when tested in a CTLL-2 cell proliferation assay. The changes introduced in hIL-2 and the signal sequence will make possible to use the powerful phage display technology for the selection of high-affinity variants from libraries of hIL-2 mutants.
Vispo
N S
NS
Pharmaceutical Division, Center for Genetic Engineering and Biotechnology, La Habana, Cuba. farma@ingen.cigb.edu.cu
Callejo
M
M
Ojalvo
A G
AG
Santos
A
A
Chinea
G
G
Gavilondo
J V
JV
Araña
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects
D007376:Interleukin-2; D021382:Protein Sorting Signals; D011133:Polysaccharide-Lyases; C074124:pectin lyase B