Anthony W. Yem

On-resin biotinylation of chemically synthesized proteins for one-step purification☆

A general, convenient, one-step purification procedure for chemically synthesized proteins present in low yields using on-resin biotinylation is reported. The protein, terminally deprotected and neutralized on-resin, is stirred in dimethylformamide and then biotinylated with N-hydroxysuccinimidobiotin (2 mg/mg protein on-resin) for 24 h at 45 degrees C. Following low/high hydrogen fluoride cleavage (J. P. Tam, W. F. Heath, and R. B. Merrifield (1983) J. Amer. Chem. Soc. 105, 6442-6455) the crude cleavage product was applied to an avidin agarose column. The column was washed with phosphate-buffered saline until all unbound materials had been eluted off. Then the biotinylated protein was eluted with 0.1 M glycine HCl, pH 2.0. A pilot experiment with two unrelated peptides on-resin established the experimental conditions for biotinylation. We then demonstrated that the chemically synthesized 153 residue [Asp205]-interleukin-1 beta (117-269), present in less than 1% yield in the crude HF cleavage mixture, could be purified to homogeneity in one step. In addition 70 and 114 residue synthetic fragments, (200-269) and (156-269), were also purified in this manner. Biotinylation on-resin appears to be an attractive method of purifying low yield chemically synthesized proteins and for preparing proteins with biotinyl moieties at specific locations such as the amino terminus.

Solution structure of human interleukin-1 receptor antagonist protein.

Interleukin‐1 receptor antagonist protein (IRAP) is a naturally occurring inhibitor of the interleukin‐1 receptor. In contrast to IL‐1β IRAP binds to the IL‐1 receptor but does not elicit a physiological response. We have determined the solution structure of IRAP using NMR spectroscopy. While the overall topology of the two 153‐residue proteins is quite similar, functionally critical differences exist concerning the residues of the linear amino acid sequence that constitute structurally homologous regions in the two proteins. Structurally homologous residues important for IL‐1 receptor binding are conserved between IRAP and IL‐1β. By contrast, structurally homologous residues critical for receptor activation are not conserved between the two proteins.

Proton, carbon, and nitrogen chemical shifts accurately delineate differences and similarities in secondary structure between the homologous proteins IRAP and IL-1β

Summary1Hα,13Cα, and15Nα secondary chemical shifts, defined as the difference between the observed value and the random coil value, have been calculated for interleukin-1 receptor antagonist protein and interleukin-1β. Averaging of the secondary chemical shifts with those of adjacent residues was used to smooth out local effects and to obtain a correlation dependent on secondary structure. Differences and similarities in the placement of secondary structure elements in the primary segdences of these structurally homologous proteins are manifested in the smoothed secondary chemical shifts of all three types of nuclei. The close correlation observed between the secondary chemical shifts and the previously defined locations of secondary structure, as defined by traditional methods, exemplifies the advantage of chemical shifts to delineate regions of secondary structure.

FKBP-12 is not an Inhibitor of Protein Kinase C

It was recently noted that the amino acid sequence of FK506 binding protein (FKBP-12) is nearly identical to that of an endogenous inhibitor of protein kinase C, PKCI-2. To follow up on this observation, we have tested the hypothesis that FKBP-12 is an inhibitor of PKC. The kinase activity of rat brain protein kinase C (PKC) was not inhibited by the presence of up to 700 micrograms recombinant human FKBP-12 per ml, in either the presence or absence of FK506. FKBP-12 also did not affect PMA-induced phosphorylation of an endogenous PKC substrate, an 80 kDa protein, in permeabilized cells. To test whether FKBP-12 could account for endogenous PKC inhibitory activity in cells, Jurkat cell lysate was chromatographed on an anion exchange column. A peak of PKC inhibitory activity was eluted at approximately 200 mM NaCl. As shown by both Western blots and FK506 binding activity, FKBP-12 was eluted only in the flow-through and wash fractions. These results demonstrate that FKBP-12 is clearly distinct from endogenous PKC inhibitory activity.

Characterization of the tryptophan environments of interleukins 1α and 1β by fluorescence quenching and lifetime measurements

Abstract The tryptophan environments of interleukins 1α and 1β, immunomodulatory proteins with similar biological activities but only 25% sequence homology, were characterized by steady-state and dynamic fluorescence measurements. Both proteins exhibited similar emission maxima, but the emission intensity of IL-1β was greatly enhanced by increasing the ionic strength of the medium, whereas that of IL-1α was unaffected. The two cytokines were also similarly quenched by the polar quencher acrylamide, but differences were observed for the ionic quenchers iodide and cesium. The fluorescence intensity decays of both cytokines were characterized by two (long and short) component lifetimes. However, the average lifetime of IL-1β (4.4 ns) was much longer than that of IL-1α (1.93 ns). Taken together with the results of steady-state measurements, we suggest that the single tryptophan of IL-1β is statically quenched by neighboring charged residues, whereas the tryptophan fluorescence of IL-1α is unaffected by ionic strength, and that the tryptophans of the two proteins have different accessibilities to ionic quenchers. The results are discussed in terms of similarities and differences in the tryptophan environments of the two proteins.

A Two Step Purification of Recombinant Human Interleukin-1β Expressed in E. Coli

Recombinant human interleukin-1β has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1β is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1β (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 × 107 units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1β sera.

Chemical modification of Interleukin-1β: Biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein

We have modified recombinant human Interleukin-1β using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide atpH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI=6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1β. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1β and may be important for the binding of IL-1 to its receptor.

Isolation and bioactivities of three IL-1β N-terminal variants

Three distinct N-terminal variants of rhIL-1β can be generated by expression of the IL-1β gene in E. coli; the naturally occurring Ala1 species, Met0-Ala1 and des-Ala1 proteins. Since most studies with rhIL-1β have used a mixture of two or more variants, we have evaluated their individual bioactivities. The variants were resolved by cation exchange HPLC. Bioactivity measurement on murine thymocytes gave a potency order of Ala1 > des-Ala1 > Met0-IL-1β. Analysis using human T-cells co-stimulated with PMA showed a potency order of Ala1 > des-Ala1 > Met0-IL-1β. Thus changes in the N-terminal amino acid of IL-1β changes the activity of the protein. Since murine and human T-cells respond similarly, the interactions between the N-terminus of rhIL-1β and their receptors probably occur through comparable mechanisms.

A Soluble Binding Assay for Measuring 3H-FK506 Binding to the HSP56 Immunophilin

Heat shock protein 56 (hsp56) was previously identified as an immunophilin based on its ability to specifically bind to FK506-Affi-Gel 10. In this report, we have quantitated human Jurkat T cell hsp56 binding to 3H-FK506, as well as to the immunosuppressant rapamycin. Binding was measured utilizing immunoadsorbed hsp56, and, in addition, we demonstrate that 3H-FK506 binds to hsp56 in solution. Hsp56 bound to an antibody-Sepharose column binds 3H-FK506 with an affinity of 19.4 +/- 4.6 nM, as compared to 23.2 +/- 6.8 nM for soluble hsp56. In competition experiments, the apparent affinity constant for rapamycin was 11.6 +/- 2.8 nM, using immobilized hsp56, and 17.3 +/- 7.7 nM, using the soluble hsp56 preparation. These results demonstrate that hsp56 binds FK506 and rapamycin with similar affinities, and suggest that hsp56 may play a role in mediating the cellular function of both of these drugs.