Marjorie E. Winkler

Human transforming growth factor-α: Precursor structure and expression in E. coli

Abstract Transforming growth factor-α (TGF-α) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-α from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-α mRNA. A cloned TGF-α cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-α produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.

Accurate measurements of long-range heteronuclear coupling constants from homonuclear 2D NMR spectra of isotope-enriched proteins

Long-range heteronuclear coupling constants are of high interest for studies of protein structure and dynamics. Today, typical sets of conformational constraints for protein structures consist of a large number of NOE constraints and a small number of dihedral angle constraints on C#J and x ’ obtained from homonuclear proton-proton coupling constants. The resultant structures show well-defined overall polypeptide folds. However, local backbone structures are not determined as precisely as may be possible with additional data. Furthermore, many side chains are less well defined than the backbone. While amino acid side chains may often adopt multiple conformations, there are no good approaches for characterizing their distributions in proteins. Measurements of heteronuclear coupling constants would provide a whole new class of constraints that could significantly improve protein structures at the local level, where improvement is needed most. The most promising approach is to measure “N-‘H vicinal coupling constants. This would lead to the following achievements. (i) Measurements of the coupling constants between the peptide nitrogen and the P-protons, combined with measurements of homonuclear coupling constants between CYand P-protons, can provide a characterization of the side-chain dihedral angle x1 and stereospecific assignments of @-methylene protons. For mobile side chains, these data can also be used to determine distributions of rotamer populations. This approach is an improvement over previous strategies which have used homonuclear coupling constants and NOES to achieve stereospecific assignments ( I). (ii) Stereospecific assignments of ,&methylene proton resonances, in turn, would provide a more precise interpretation of intraresidue HN-HB and sequential HO-HN NOES, which depend on the values of the intervening dihedral angles 4, x ’ and x ‘, 1+4, respectively. (iii) Measurements of the coupling constants between the a-proton of residue i and the peptide nitrogen of residue i + 1, combined with sequential NOES, should provide improved determinations of the backbone dihedral angle

Purification of plasmid DNA by tangential flow filtration

J~. (iv) Vicinal couplings between methylene protons and side-chain amide or guanido nitrogens could also provide Asn x *, Gln x 3, and Arg x 4 dihedral angle constraints.

Folding of Eukaryotic Proteins Produced in Escherichia Coli

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.

Contribution of the C-terminal dipeptide of transforming growth factor-α to its activity: biochemical and pharmacologic profiles

Although intracellular expression in E. coli may result in accumulation of the eukaryotic protein in inclusion bodies, the protein may often be recovered by first solubilizing with denaturant followed by refolding. Some general guidelines for developing a refolding procedure are apparent but the specific protocol must be empirically determined for each protein. Convenient and rapid assays for detecting native protein are critical for developing a refolding procedure. Maintaining solubility during refolding is a common feature of recovery processes. Proper folding should be assessed by a number of methods including activity, spectroscopic and stability measurements. For some proteins, properly folded protein may be obtained by secretion from E. coli; however, secretion does not ensure correct folding and protection from proteolytic degradation.

Transforming growth factor-alpha: a more potent angiogenic mediator than epidermal growth factor

We have used a matrix of biological (two distinct guinea-pig stomach contractile smooth muscle preparations) and biochemical (human placental membrane receptor binding and phosphorylation) assays to evaluate the activity profiles of epidermal growth factor-urogastrone (EGF-URO, mouse and human), transforming growth factor-alpha (TGF-alpha, human) and the TGF-alpha derivative lacking the C-terminal dipeptide, Leu49-Ala50, TGF-alpha-(1-48). In the longitudinal muscle (LM) bioassay, the relative potencies of the peptides were: EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), with relative activity ratios (EC50S) of approximately 1:3:16. In the LM assay system, TGF-alpha-(1-48) was a partial agonist. In the circular muscle (CM) bioassay, the relative order of potencies was: TGF-alpha- greater than EGF-URO greater than TGF-alpha-(1-48), with EC50S of about 1:2:7. In the CM assay, all three peptides were full agonists, even though EGF-URO caused an intense desensitization of the tissue whereas TGF-alpha and TGF-alpha-(1-48) did not. The relative affinities of the peptides in the placenta membrane binding assay, EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), were in good qualitative and quantitative agreement with the LM (but not the CM) bioassay, with relative KDS in the proportions of about 1:3:17. In the phosphorylation assay, using either the phosphorylated EGF-URO receptor or calpactin-II as an index of receptor kinase activation, the relative potencies of the peptides, EGF-URO greater than TGF-alpha greater than TGF-alpha-(1-48), were also qualitatively in accord with the relative potencies measured in the LM and ligand binding assays (but not in the CM preparation); however, quantitatively, the relative potency ratios (EC50S) observed in the phosphorylation assay (1:2:3) were somewhat out of keeping with the relative values observed in the LM and ligand binding assays. All three peptides were full agonists in the phosphorylation assay. Our data point to the importance of the C-terminal dipeptide, Leu49-Ala50 of TGF-alpha in terms of the binding affinity and intrinsic activity of this polypeptide; and our work provides further evidence for the distinct nature of the EGF-URO/TGF-alpha receptor system present in the CM bioassay preparation. The biological/biochemical activity profiles documented for the three polypeptides can serve as a basis for the further evaluation of other synthetic and naturally occurring members of the EGF-URO/TGF-alpha family of polypeptides.