Abstract
A deletion derivative of the cytokine human interleukin-3 (hIL-315–125, comprising amino acids 15–125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble cytokine, phage-presented hIL-315–125 exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-315–125 species (designated hIL-315–125 ε) had 20 amino acids appended to its C-terminus; hIL-315–125 ε did not exhibit reduced bioactivity. hIL-315–125-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.
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Abbreviations
- aa:
-
amino acid
- Ab:
-
antibody
- AML:
-
acute myelogenous leukemia cell
- Ap:
-
ampicillin
- BBS:
-
borate buffered saline
- BHK:
-
baby ham-ster kidney cell
- bp:
-
base pair(s)
- BSA:
-
bovine serum albumin
- bST:
-
bovine somatotropin (growth hormone)
- CAPS:
-
3-(cyclohexylamino)-1-propane-sulphionic acid
- Cb:
-
carbenicillin
- cpm:
-
counts per minute
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DPBS:
-
Dulbecco’s phosphate buffered saline
- ELISA:
-
enzyme-linked immunosorbent assay
- FBS:
-
fetal bovine serum
- hCNTF:
-
human ciliary neurotrophic factor
- hG-CSF:
-
human granulocyte colony-stimulating factor
- hGH:
-
human growth hormone
- hGM-CSF:
-
human granulocyte-macrophage colony-stimulating factor
- hIL-3:
-
human interleukin-3
- hIL-3R:
-
receptor for human interleukin-3
- hIL-5:
-
human interleukin-5
- Km:
-
kanamycin
- pIII:
-
tail protein of filamentous bacteriophage
- PCR:
-
DNA polymerase chain reaction
- PEG:
-
polyethylene glycol
- SDS:
-
sodium dodecyl sulfate
- TU:
-
titerable unit of phage
- V:
-
volts.
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Merlin, S., Rowold, E., Abegg, A. et al. Phage presentation and affinity selection of a deletion mutant of human lnterleukin-3. Appl Biochem Biotechnol 67, 199–214 (1997). https://doi.org/10.1007/BF02788798
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DOI: https://doi.org/10.1007/BF02788798