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1.
The human, bovine, murine and rat interferon (IFN)-alpha families contain 4 conserved cysteines located at positions 1, 29, 99 and 139 that are involved in disulfide bridges. Rat and murine IFN-alpha subspecies carry a fifth Cys (Cys-86) which is not conserved in bovine and human IFN-alpha subspecies except for human IFN-alpha 1. Changing Cys-86 in rat IFN-alpha 1 into Ser or Tyr virtually abolished antiviral activity. As shown by others, the substitution of Cys-86 to Ser in human IFN-alpha 1 had no pronounced effect on activity. This suggests that in contrast to human and bovine IFN-alpha, Cys-86 in rodent IFN-alpha plays a crucial role in receptor binding. Changing Cys-1 to Gly in rat IFN-alpha 1 also destroyed activity, in agreement with results obtained in the human IFN-alpha 1 system. 相似文献
2.
Croguennec T Bouhallab S Mollé D O'Kennedy BT Mehra R 《Biochemical and biophysical research communications》2003,301(2):465-471
The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented. 相似文献
3.
Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon 总被引:1,自引:0,他引:1
S Pestka B Kelder J A Langer T Staehelin 《Archives of biochemistry and biophysics》1983,224(1):111-116
Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification. 相似文献
4.
Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form. The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain. By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure. This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid. Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure. A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C. 相似文献
5.
A F Sheval'e V V Samukov V I Ofitserov V V Kalashnikov G A Mizenko A A Kolokol'tsov 《Bioorganicheskaia khimiia》1990,16(7):916-925
Three peptides corresponding to the sequences 124-144, 124-138, 129-144 of the human leukocyte interferon alpha 2 (IFN-alpha 2) were synthesized. The synthesis was performed by DCC-HOBT coupling of protected peptide segments in solution. The segments were obtained by the active ester coupling methodology using base-labile 2-[4-(phenylazobenzyl)sulfonyl]ethyl (Pse) group as carboxyterminal protection. After complete deprotection with 1 M methanesulphonic acid in trifluoroacetic acid--thioanisol--m-cresol mixture the peptides were purified by reversed-phase chromatography. The studies of interaction of the peptides with rabbit antiserum against IFN-alpha 2 revealed at least one minor antigenic determinant within the 124-144 region of IFN-alpha 2 amino acid sequence. Rabbit antisera developed against peptides 124-138 and 129-144 showed ability of binding recombinant IFN-alpha 2 and neutralizing its antiviral activity. Free peptides or their conjugates with bovine serum albumine did not display antiviral activity, neither could they inhibit the activity of IFN-alpha 2. 相似文献
6.
C A Lunn J Fossetta N Murgolo P J Zavodny D Lundell S K Narula 《Protein engineering》1992,5(3):249-252
We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity. 相似文献
7.
The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68. 相似文献
8.
We have previously reported the antiproliferative activity of synthetic sequences 29-35 and 122-139 of the interferon-alpha2b (IFN-alpha2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-alpha2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on (125)I-IFN-alpha2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-alpha2b peptides behaved as partial agonists of the IFN-alpha2b molecule and may be of interest for drug design purposes. 相似文献
9.
Watson WH Pohl J Montfort WR Stuchlik O Reed MS Powis G Jones DP 《The Journal of biological chemistry》2003,278(35):33408-33415
Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals. 相似文献
10.
Gene expression and antiviral activity of alpha/beta interferons and interleukin-29 in virus-infected human myeloid dendritic cells 总被引:5,自引:0,他引:5
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Osterlund P Veckman V Sirén J Klucher KM Hiscott J Matikainen S Julkunen I 《Journal of virology》2005,79(15):9608-9617
Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response. 相似文献
11.
Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired. 相似文献
12.
Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues 总被引:21,自引:0,他引:21
A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists. 相似文献
13.
14.
Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond 总被引:12,自引:0,他引:12
M W Pantoliano R C Ladner P N Bryan M L Rollence J F Wood T L Poulos 《Biochemistry》1987,26(8):2077-2082
Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers. 相似文献
15.
Determination of disulfide array and subunit structure of taste-modifying protein, miraculin 总被引:2,自引:0,他引:2
The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity. 相似文献
16.
N Dhanasekaran M Wessling-Resnick D J Kelleher G L Johnson A E Ruoho 《The Journal of biological chemistry》1988,263(34):17942-17950
A heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine (125-ACTP), has been synthesized. 125I-ACTP has been used to derivative reduced sulfhydryls of the retinal G protein, transducin (Gt), to form a mixed disulfide bond under mild, nondenaturing conditions (pH 7.4, 4 degrees C). The resulting disulfide was easily cleaved using reducing reagents. A 200-fold molar excess of 125I-ACTP relative to Gt resulted in the incorporation of 1-1.3 mol of the 125I-N-(3-iodo-4-azidophenylpropionamido)cysteine moiety of ACTP into Gt alpha. In contrast to 125I-ACTP, dithionitrobenzoate and dithiopyridone derivatized six sulfhydryls in native Gt. Incubation of a 10-fold molar excess of 125I-ACTP relative to Gt resulted in the derivatization of 0.75-0.9 and 0.1 mol of reduced sulfhydryls/mol Gt alpha and beta, respectively. Gt gamma was not derivatized by 125I-ACTP. Thus, Gt alpha was preferentially derivatized by 125I-ACTP. Tryptic digestion and amino acid sequencing of Gt alpha indicated that both Cys-347 near the carboxyl terminus and Cys-210 between the second and third consensus sequences forming the GTP-binding site were derivatized by 125I-ACTP in a ratio of approximately 70 and 30%, respectively. Thus, both Cys-210 and Cys-347 are labeled, even though derivatization by 125I-ACTP does not exceed 1 mol of SH/mol Gt alpha. It appears that derivatization of one sulfhydryl, either Cys-210 or Cys-347, excludes labeling of the second cysteine either by steric hindrance or induced conformational change making the second cysteine inaccessible to 125I-ACTP. Consistent with this finding was the observation that pertussis toxin-catalyzed ADP-ribosylation of Cys-347 inhibited 125I-ACTP derivatization of Cys-210. Derivatization of Gt alpha at either Cys-210 or Cys-347 by 125I-ACTP inhibited rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate binding to Gt, mimicking the effect of ADP-ribosylation of Cys-347 by pertussis toxin. ACTP contains a radioiodinated phenylazide moiety which, upon activation, can cross-link the derivatized cysteine to an adjacent polypeptide domain. Following reduction of the disulfide, the [125I] iodophenyl moiety will be transferred to the azide-inserted polypeptide. When photoactivation of the phenylazide moiety of 125I-ACTP after sulfhydryl derivatization was performed, insertion of the Cys-347 which contains Cys-210, was found.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
17.
Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation 总被引:1,自引:0,他引:1
Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation. 相似文献
18.
Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin 总被引:1,自引:0,他引:1
The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha. 相似文献
19.
Fluorescence measurements and H/2H exchange experiments monitored by mass spectrometry have been applied to investigate the influence of the conserved disulfide bridges on the folding behavior and in vitro aggregation properties of the scFv fragment of the antibody hu4D5-8. A set of four proteins, carrying none, one, or both of the disulfide bridges have been compared regarding their stabilities, folding kinetics and tendency to aggregate. The results show that refolding of all four scFvs is ultimately limited by a slow proline isomerization in the VLdomain, since the native cis -conformation of proline L95 seems to be a prerequisite for formation of the native interface. Starting from short-term denatured protein, with the proline residues in their native conformation, a kinetically trapped intermediate is populated depending on the conditions, whose rate of conversion is slower than that of the fast-folding molecules. According to deuteron protection patterns determined by mass spectrometry, those domains retaining the disulfide bridge are able to form stable native-like structure, independent of native interface formation. The disulfide-free domains, in contrast, require the native interface for sufficient stabilization. The resistance of the scFvs towards aggregation seems to be critically dependent on the presence of the disulfide bridge in the VHdomain, and thus on the ability of the VHdomain to form stable structure prior to interaction with the VLdomain. The presence of a stable VLdomain in combination with a disulfide-free VHdomain appears to further promote aggregation, indicating the involvement of structured domains in the aggregates. 相似文献
20.
J C Cross C E Farin S F Sharif R M Roberts 《Molecular reproduction and development》1990,26(2):122-128
Antiviral activity has been found in conceptus and placental tissues in numerous species, including mice, pigs, sheep, cattle and humans. In sheep and cattle, the antiviral activity is due to an interferon alpha (IFN-alpha), but in other species the nature of the protein(s) responsible for placental activity is unknown. The objectives of this study were to determine if the constitutive antiviral activity associated with the mouse conceptus is produced as early as the peri-implantation period, and to determine if the activity is due to an IFN-alpha or -beta. Conceptus and placental tissue explants released antiviral activity from Day 4 through at least Day 16 of gestation as measured in an agar overlay bioassay employing CHO cells challenged with vesicular stomatitis virus. This activity was neutralized by antiserum against MuIFN-alpha/beta. The same antiserum failed, however, to immunoprecipitate radiolabeled proteins from medium collected from Day 4 blastocysts cultured in the presence of L-[35S]-methionine. S1 nuclease analysis of placental RNA and screening of ectoplacental cone and extraembryonic ectoderm cDNA libraries with MuIFN-alpha and -beta probes failed to detect IFN related mRNAs, even under relatively non-stringent conditions of hybridization. Thus, while antiviral activity is produced by peri-implantation conceptuses in several diverse mammalian species, it does not appear to be due to a conserved type of IFN in all these species. 相似文献