Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants.

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.

Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system.

Almost all secretory proteins have a sequence consisting of 15-30 amino acids at the N-terminus (the so-called N-terminal signal peptide). Signal peptides direct the propeptide to the endoplasmic reticulum and through the secretory pathway. Although the sequences of signal peptides vary greatly, all contain a basic amino acid in the N-terminal region, followed by a hydrophobic core region. With the aim of improving the level of secretion of recombinant proteins in the baculovirus expression vector system (BEVS), we designed several signal peptides based on the signal peptide of silkworm SP1 by introducing the basic amino acid arginine into the N-terminal region and/or the polar amino acid asparagine into the C-terminal region of the silkworm SP1 signal peptide. Human interleukin (IL)-4, IL-13, and the extracellular domain of human IL-11 receptor alpha1 (IL-11Ralpha1) were fused to wild-type and modified SP1 signal peptides, and the effects that each signal peptide had on secretion were measured by enzyme-linked immunosorbent assay. Introduction of the basic amino acid arginine into the N-terminal region did not result in an increase in secretion of the recombinant proteins. On the other hand, introduction of the polar amino acid asparagine into the C-terminal region enhanced secretion of the recombinant proteins. Therefore, it is suggested that polar amino acids in the C-terminal region of signal peptides are important in the secretion of recombinant proteins in BEVS.

MEP HyperCel chromatography II: binding, washing and elution.

Binding, washing and elution conditions for MEP HyperCel chromatography were examined using two conditioned media (CM) containing monoclonal antibodies (humanized IgG1) and bovine serum albumin (BSA). Monoclonal antibodies derived from mammalian expression system bound to the column without pretreatment, although a majority of contaminating proteins present in the CM also showed binding. Inorganic salts, ethanol and glycerol were ineffective in eluting proteins under the conditions examined, suggesting that electrostatic or hydrophobic interactions are not a major factor for antibody binding to the MEP resin. Ethylene glycol, 2-propanol, urea and arginine were effective, to varying degrees, in elution of the bound proteins. The bound contaminating proteins and BSA were effectively eluted with ethylene glycol and the bound antibodies were finally eluted with aqueous arginine solutions at neutral pH. MEP showed selectivity toward BSA and hence utility for removing BSA from the samples. Interestingly, Fc-fusion proteins derived from silkworm larvae showed no detectable binding. Serum proteins present in silkworm larvae strongly competed with the Fc-fusion proteins and monoclonal antibody for binding to MEP resin, while the same Fc-fusion proteins can be readily purified in one-step by Protein-A resin, again confirming weak selectivity of the MEP resin.

Utilization of Arg-elution method for FLAG-tag based chromatography.

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein-protein or protein-ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5-1M arginine at pH 3.5-4.4, as reported in this paper.