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Applied Biochemistry and Biotechnology

, Volume 67, Issue 3, pp 199–214 | Cite as

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

  • Sean Merlin
  • Edwin Rowold
  • Ann Abegg
  • Cathleen Berglund
  • Jon Klover
  • Nick Staten
  • John P. McKearn
  • Stephen C. Lee
Original Articles

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.

Index Entries

Phage display cytokine receptor growth factor proliferation biopanning 

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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Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Sean Merlin
    • 1
  • Edwin Rowold
    • 1
  • Ann Abegg
    • 1
  • Cathleen Berglund
    • 1
  • Jon Klover
    • 1
  • Nick Staten
    • 1
  • John P. McKearn
    • 1
  • Stephen C. Lee
    • 1
  1. 1.Searle Research and DevelopmentMonsanto CompanySt. LouisUSA

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