George Stearns

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit.

Stem cell factor (SCF) is an early‐acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane‐bound forms. It transduces signals by ligand‐ mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet‐derived growth factor (PDGF), macrophage colony‐stimulating factor, Flt‐3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand‐binding portions composed of immunoglobulin‐like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 Å resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric ‘head‐to‐head’ association. Using various prior observations, we have located potential Kit‐binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt‐1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt‐1, and a similar model can be made for the complex of PDGF with its receptor.

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.

Identification of interleukin 6 as a synergistic factor for the differentiation-inducing effect of TNF on leukemic ML-1 cells

Recombinant TNF was capable of inducing differentiation of human leukemic ML-1 cells in the monocytic pathway. Recombinant interleukin 6 did not have the activity but it could significantly increase the activity of recombinant TNF. Both of these molecules were found to play a similar role in PWM-induced conditioned medium from human lymphocytes (LCM). The differentiation inducing effect of LCM could be partially neutralized by antibody to interleukin 6. Fractionation of LCM also identified interleukin 6 as the factor that synergizes with TNF.

Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Comparative analysis of the effects of recombinant cytokines on the growth and differentiation of ML-1, a human myelogenous leukemic cell line.

We have investigated the effect of a number of cytokines on the human acute myelomonocytic leukemic cell line, ML-1. The differentiation inducing effects of interleukins (IL-1 beta, IL-3 and IL-6), colony stimulating factors (GCSF and GMCSF), TNF, LIF and IFNg, were studied either individually or in combination. Criteria for monocytic differentiation were as follows: an increase in the percentage of cells reducing nitroblue tetrazolium (NBT) salt, an increase in the alkaline phosphatase activity as well as the appearance of macrophagic phenotype. Among all the cytokines tested, only TNF was found to induce differentiation and to inhibit growth of ML-1 cells. IL-3, IL-6, interferon gamma, GCSF and to a smaller extent IL-1 beta and GMCSF synergized the differentiation inducing activity of TNF.

Use of LC/MS peptide mapping for characterization of isoforms in 15N-Labeled recombinant human leptin

Publisher Summary Recombinant human leptin has been recently cloned and expressed in E. coli, and it effectively demonstrates adiposity in mice through modulation of appetite and metabolism. The molecule contains four methionine residues at positions 1, 54, 68, and 136. This chapter discusses the separation and characterization of three norleucine-incorporated recombinant human leptins that are uniformly labeled with 15N isotope or double labeled with 15N and 13C isotopes. The extent of incorporation at each methionine residue can be determined by reverse-phase high-performance liquid chromatography (HPLC) and amino acid analysis methods. The norleucine incorporation was observed preferentially occurring at the internal Met residues. The misincorporation of norleucine for methionine is known to occur in bacteria when high level synthesis of recombinant proteins is induced in minimal medium fermentation. This misincorporation is detected in the production of 15N-labeled recombinant human leptin produced using minimal medium conditions, however, is not present in the clinical samples produced using other fermentation conditions. The mechanism for the misincorporation is believed to involve the de novo synthesized norleucine that bypasses the leucine biosynthetic pathway and enters directly into the incorporation pathway by associating with tRNAMet in the acylation reaction. The level of incorporation as well as the distribution of norleucine for the methionine residues, however, has varied for different recombinant proteins.