High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.
LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.
Samal
B B
BB
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320.
Arakawa
T
T
Boone
T C
TC
Jones
T
T
Prestrelski
S J
SJ
Narhi
L O
LO
Wen
J
J
Stearns
G W
GW
Crandall
C A
CA
Pope
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D006131:Growth Inhibitors / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008027:Light; D008222:Lymphokines / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D012542:Scattering, Radiation; D012689:Sequence Homology, Nucleic Acid; D017550:Spectroscopy, Fourier Transform Infrared; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D006131:Growth Inhibitors; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines