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Phage presentation and affinity selection of a deletion mutant of human lnterleukin-3

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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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Authors and Affiliations

  1. Searle Research and Development, Monsanto Company, 700 Chesterfield Parkway, 63198, St. Louis, MO, USA

    Sean Merlin, Edwin Rowold, Ann Abegg, Cathleen Berglund, Jon Klover, Nick Staten, John P. McKearn & Stephen C. Lee

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  1. Sean Merlin
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  2. Edwin Rowold
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  3. Ann Abegg
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  4. Cathleen Berglund
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  5. Jon Klover
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  6. Nick Staten
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  7. John P. McKearn
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  8. Stephen C. Lee
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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

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