Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin-5 from Escherichia coli.
Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.
Graber
P
P
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Bernard
A R
AR
Hassell
A M
AM
Milburn
M V
MV
Jordan
S R
SR
Proudfoot
A E
AE
Fattah
D
D
Wells
T N
TN
D000818:Animals; D001402:B-Lymphocytes; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015848:Interleukin-5 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012645:Selenomethionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D015848:Interleukin-5; D011994:Recombinant Proteins; C080478:selenomethionyl interleukin-5; D012645:Selenomethionine