Purification and characterization of progenipoietins produced in Escherichia. coli.
The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. These unique proteins have previously been shown to induce the proliferation of multiple cell lineages. The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described. These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity. Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains. ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different. Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable. Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation. This study describes the purification and physico-chemical properties of this class of proteins generated using an E. coli expression system.
Violand
Bernard N
BN
Pharmacia Corporation, 700 Chesterfield Village Parkway, Mail Zone AA2I, St. Louis, MO 63198, USA. bernard.n.violand@pharmacia.com
Minnerly
John C
JC
Feng
Yiqing G
YG
Polazzi
Joseph O
JO
Mathis
Karl J
KJ
Thomas
Kristin E
KE
Zeng
David L
DL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003115:Colony-Stimulating Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; C434603:progenipoietin-1