Characterization of E. coli-derived recombinant human interferon-beta as compared with fibroblast human interferon-beta

Homogeneous E. coli-derived recombinant human interferon-beta (E. coli-rHuIFN-beta) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-beta (fibroblast HuIFN-beta). Purified E. coli-rHuIFN-beta and fibroblast HuIFN-beta exhibited a single band of Mr 19,000 and 23,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The primary structure of E. coli-rHuIFN-beta was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the 1H nuclear magnetic resonance (NMR) spectra of E. coli-rHuIFN-beta and fibroblast HuIFN-beta at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C18 column, the retention time of E. coli-rHuIFN-beta was longer than that of fibroblast HuIFN-beta. Moreover, although the isoelectric point of E. coli-rHuIFN-beta was pH 8.9, purified fibroblast HuIFN-beta exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the E. coli-rHuIFN-beta polypeptide folds similarly to fibroblast HuIFN-beta, and the carbohydrate moiety of natural HuIFN-beta has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.     

Conformations of fibroblast and E. coli-derived recombinant human interferon-beta s as studied by nuclear magnetic resonance and circular dichroism

The conformations of fibroblast and E. coli-derived recombinant human interferon-beta s were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.6. Both interferons have very similar conformations with high alpha-helix contents (approximately 70%). These results suggest that glycosylation does not appreciably change the conformation of human interferon-beta. Moreover, a slow conformational change is observed below pH 2.0, which induces the disruption of beta-sheets.     

Enzymatic modifications of human fibroblast and leukocyte interferons

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.     

Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions

Disulfide bond interchange has been pointed out as a considerable problem in preparing recombinant proteins from Escherichia coli cells. This has been reported in the system of reducing denaturation followed by a refolding process, where incorrectly folded molecules are sometimes produced. As the possibility of disulfide bond interchange may also arise in the cytoplasm of E. coli cells, the state of sulfhydryl groups of recombinant proteins obtained from a nonreducing and nondenaturing process should be examined. The state of sulfhydryl groups of E. coli-derived recombinant human interferon-beta 1, which had been purified under nonreducing and nondenaturing conditions, was examined by using the N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) labeling technique. Among the three cysteine residues in E. coli-derived human interferon-beta 1, the 17th cysteine was identified as being unpaired, as in the natural molecule. However, it was found that three isomers of the recombinant protein could be formed when the protein was denatured with 6 M guanidine hydrochloride. These three isomers were identified as having unpaired cysteine residues at positions 17, 31, and 141, respectively. These results indicate that disulfide bond interchange occurs in E. coli-derived recombinant human interferon-beta 1 under denaturing conditions in spite of the absence of a reducing agent.     

Conformation and stability of two recombinant human interferon-alpha analogs

Structural features of a recombinant E. coli derived interferon-alpha analog, interferon consensus1, was studied by circular dichroism and fluorescence spectroscopy. Circular dichroic spectra of the purified protein showed that it has about 70% alpha-helix and a distinct tertiary structure. These structural features are similar to those for a natural interferon-alpha subtype, interferon-alpha 2, indicating that the amino acid substitutions in interferon consensus1 apparently did not alter the protein structure. Another analog, interferon consensus5, which has Ser instead of Cys at residues 1 and 99 but is otherwise identical to interferon consensus1, was prepared to study the role of the disulfide bond between Cys 1 and 99. Circular dichroic and fluorescence spectra indicated similarity in the structure of these two analogs. However, interferon consensus1 was significantly more stable than interferon consensus5 against denaturation. pH unfolding experiments indicated that the former protein is more stable in the transition region by about 1.6 kcal/mol, which was interpreted in terms of the increased free energy of the denatured state due to an extra disulfide bond in interferon consensus1.     

Stimulation of microtubule assembly by recombinant human interferon-alpha and interferon-gamma

Interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) have been demonstrated to stimulate microtubule assembly measured in the in vitro assembly system. The process is substoichiometric occurring when the interferon concentrations are below that of tubulin. IFN-gamma is a more potent effector than IFN-alpha. The critical tubulin concentration describing microtubule assembly decreases from 1.5 mg/ml measured in the absence of added effector to 1.05 mg/ml and 1.3 mg/ml when measured in the presence of 2.16.10(-6) M IFN-gamma and 3.06.10(-6) M IFN-alpha, respectively.     

Differential effects of hyperthermia on human leukocyte production of interferon-alpha and interferon-gamma

Hyperthermia is being used clinically in the treatment of neoplasms. However, there are insufficient data regarding effects of hyperthermia on leukocyte functions potentially important in antitumor immunity. In order to provide such data, human mononuclear leukocytes were exposed to moderate (40.7 degrees C) and marked (42.7 degrees C) hyperthermia for 2 hr. Leukocyte viability, measured by dye exclusion, was not altered by such exposures. Exposure of the cells to moderate hyperthermia did not alter leukocyte production of interferon-alpha in response to influenza virus or interferon-gamma in response to the mitogen phytohemagglutinin. Exposure of the cells to marked hyperthermia significantly depressed production of interferon-alpha. In contrast, production of interferon-gamma was not altered by exposure of the leukocytes to marked hyperthermia. Many studies support a role for interferons (alpha as well as gamma) in antitumor immunity. The current and other data suggest that marked hyperthermia in cancer therapy should be applied locally whenever possible, rather than to the whole body, in order to limit adverse effects on immunity. The data suggest further that interferon-gamma may be a heat shock (stress) protein for human leukocytes.     

Reagentless identification of human bifidobacteria by intrinsic fluorescence

A new identification method for bifidobacteria species from the human gastrointestinal tract was developed based on the measurement and statistical analysis of the intrinsic fluorescence of aromatic amino acids (AAA) and nucleic acids (NA), following their excitation at 250 nm. The model was constructed by recording the fluorescence spectra of 53 Bifidobacterium strains of 10 different species, including the corresponding type strains, and validated by analyzing the spectra data from nine further problem strains. Principal components analysis (PCA) and factorial discriminant analysis (FDA) of the results showed the technique to distinguish between the isolates at the species level; the Bifidobacterium pseudolongum subspecies (globosum and pseudolongum) could also be distinguished. The proposed method provides a powerful, inexpensive and convenient means of rapidly identifying intestinal bifidobacteria, which could be of help for large probiotic surveys.     

Expression and refolding of recombinant human fibroblast procathepsin D

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.     

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.     

Production of recombinant human interferon-alpha 1 by Escherichia coli using a computer-controlled cultivation process.

Genetically engineered E. coli K12 BMH-71-18 with plasmid PBV-867 was used for constitutive expression of human interferon-alpha 1 (IFN) with a defined medium. A manual, time-based, fed-batch cultivation process produced a cell density of 26.3 g l-1 (OD550 89), an IFN activity of 1.55 x 10(8) IU l-1 and a specific IFN productivity of 0.65 x 10(6) IU g-1. An analysis was conducted to characterize the problems involved in the high density microbial processes of recombinant protein production. The strategy suggested by the analysis is to establish a nutrient feeding profile that improves both the plasmid stability and the overall productivity of IFN. The nutrient feeding procedure developed here was based on the growth dynamics and a glucose consumption model. By using this procedure to continuously supply nutrients during cultivations, cell density reached 58 to 80 g l-1 and the specific IFN productivities of these runs were increased over that of the manual process. Nutrient feeding rates were found to affect the specific IFN productivity substantially. The optimized process achieved an IFN activity of 1.26 x 10(9) IU l-1, a cell density of 58 g l-1 and a specific IFN productivity of 2.2 x 10(7) IU g-1. More significantly, the overall productivity IU l-1 h-1 of the optimized, computer-controlled cultivation process was increased 12.9-fold over that of the manual cultivation process.     

Photodegradation mechanism and reaction kinetics of recombinant human interferon-alpha2a.

The photodegradation mechanism of recombinant human interferon-alpha2a (IFNalpha2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNalpha2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNalpha2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNalpha2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNalpha2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.     

Effect of human nasal secretions on the antiviral activity of human fibroblast and leukocyte interferon.

Many normal human nasal secretions contain an inhibitor of human fibroblast IF. This inhibitor had no effect on human leukocyte IF. The amount of inhibition of fibroblast IF increased with increasing quantities of nasal secretions. Also, the inhibition could be overcome with increasing concentrations of IF.     

Analysis of oligomeric forms of recombinant human leukocyte interferons

Using SDS-PAAG electrophoresis, gel-permeation HPLC and immunoblotting, it was demonstrated that homogeneous preparations of human leukocyte interferons (alpha-INF)-A, -N and -I1 obtained from the biomass of the corresponding producer strains (Pseudomonas sp.) contained several oligomeric forms produced by way of S-S intermolecular cross-linkage and making up to 10-15%, 4-7% and 2-5% of the total monomeric form content in the protein preparations. Immunologic testing with the use of MAB NK-2 and [125I]NK-2 showed that the oligomeric forms of alpha-INF-A, -N and -I1 were present in the protein preparations at all purification stages and seemed to be formed at early steps of interferon synthesis in the cell. The effects of limited proteolysis as well as of acid, alkaline and thermodenaturation on the aggregation and oligomerization of alpha-INF-A were studied. SDS-PAAG electrophoresis performed in the absence of the reducing agents showed that upon denaturation of 10% TCA, the amount of the oligomeric forms in the preparations of homogeneous and especially partly proteolytic INF was significantly increased. The causes and the putative mechanisms of aggregation and oligomerization of INF are discussed.     

Limited proteolysis of human leukocyte interferon-alpha2 and localization of the antigenic determinant binding the monoclonal antibodies

Large peptide fragments of human leucocyte interferon-alpha 2 (INF-alpha 2) were obtained by limited proteolysis with trypsin, pepsin, thermolysine and Bacillus amyloliquefaciens intracellular serine proteinase. The ability of the fragments to bind murine monoclonal antibodies NK2 raised against INF-alpha 2 was studied by the immunoblotting technique. The region of sequence 110-149 is the most sensitive to proteolytic attack, being probably exposed on the surface of the INF-alpha 2 molecule. INF-alpha 2 fragments 1-139, 1-147, 1-149 are capable of binding antibodies, whereas fragments 1-109 and 1-112 do not bind antibodies NK2. A comparison of the primary structure of human leucocyte and murine leucocyte INF families in the region of sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequences to bind antibodies NK2 demonstrated that the antigenic determinant for antibodies NK2 is the sequence Glu114-Asp115-Ser116-Ile117 of the INF-alpha 2 molecule.     

Antitumor, antiviral and natural killer-inducing activities of human recombinant interferon-alpha in the guinea pig

It was found that human recombinant interferon-alpha (Hu rIFN-alpha) was biologically active in the guinea pig. Namely, intratumor injection of either Hu rIFN-alpha A or A/D regressed tumors and prevented metastasis of guinea-pig line 10 tumor. These rIFNs augmented splenic natural killer activity of the guinea pig and reduced the cytopathic effect of virus on guinea-pig cells. Hu IFN-gamma showed no such activity.     

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.