Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence

1995 ◽  
Vol 108 (3) ◽  
pp. 1083-1092 ◽  
Author(s):  
A. Vassilev ◽  
M. Kimble ◽  
C.D. Silflow ◽  
M. LaVoie ◽  
R. Kuriyama
Keyword(s):  
Molecular Mass ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Sedimentation Coefficient ◽  
Apparent Molecular Mass ◽  
Size Exclusion ◽  
Sf9 Cells ◽  
Dependent Manner ◽  
Monomeric Form ◽  
Gamma Tubulin

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

scholarly journals The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization

2001 ◽  
Vol 27 (1) ◽  
pp. 77-83 ◽  
Author(s):  
JL Thomas ◽  
JI Mason ◽  
G Blanco ◽  
ML Veisaga
Keyword(s):  
Molecular Mass ◽  
Hydroxysteroid Dehydrogenase ◽  
Size Exclusion ◽  
Sf9 Cells ◽  
Type I ◽  
Wild Type ◽  
Enzyme Protein ◽  
Wild Type Enzyme ◽  
Human Type ◽  
Type K

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.

scholarly journals Comparison of the activities of a multiple inositol polyphosphate phosphatase obtained from several sources: a search for heterogeneity in this enzyme

1995 ◽  
Vol 305 (2) ◽  
pp. 491-498 ◽  
Author(s):  
A Craxton ◽  
N Ali ◽  
S B Shears
Keyword(s):  
Molecular Mass ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyclonal Antiserum ◽  
Apparent Molecular Mass ◽  
Size Exclusion ◽  
Rat Tissues ◽  
Inositol Polyphosphate ◽  
Phosphatase Activities ◽  
Specific Activities

A multiple inositol polyphosphate phosphatase (formerly known as inositol 1,3,4,5-tetrakisphosphate 3-phosphatase) was purified approx. 22,000-fold from rat liver. The final preparation migrated on SDS/PAGE as a doublet with a mean apparent molecular mass of 47 kDa. Upon size-exclusion chromatography, the enzyme was eluted with an apparent molecular mass of 36 kDa. This enzyme was approximately evenly distributed between the ‘rough’ and ‘smooth’ subfractions of endoplasmic reticulum. There was a 20-fold range of specific activities of this phosphatase in CHAPS-solubilized particulate fractions prepared from the following rat tissues: liver, heart, kidney, testis and brain. However, each of these extracts contained different amounts of endogenous inhibitors of enzyme activity. After removal of these inhibitors by MonoQ anion-exchange chromatography, there was only a 2.5-fold range of specific activities; kidney contained the most and brain contained the least. We prepared and characterized polyclonal antiserum to the hepatic phosphatase, which immunoprecipitated 85-100% of both particulate and soluble phosphatase activities. The antiserum also immunoprecipitated, with equivalent efficacy, CHAPS-solubilized phosphatase activities from heart, kidney, testis, brain and erythrocytes (all prepared from rat). Our data strengthen the case that the function of the mammalian phosphatase is unrelated to the metabolism of Ca(2+)-mobilizing cellular signals. The CHAPS-solubilized phosphatase from turkey erythrocytes was not immunoprecipitated by the polyclonal antiserum, and is therefore an isoform that is structurally distinct, and possibly functionally unique.

scholarly journals Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

2021 ◽  
Author(s):  
Jelle De Vos ◽  
Patricia Pereira Aguilar ◽  
Christoph Köppl ◽  
Andreas Fischer ◽  
Clemens Grünwald-Gruber ◽  
...  
Keyword(s):  
Light Scattering ◽  
Process Control ◽  
Molecular Mass ◽  
Hydrophobic Interaction ◽  
Nucleocapsid Protein ◽  
Expression System ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Light Scattering Detection

<p>The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. <a>This protein has a strong tendency to dimerization and interaction with nucleic acids. A native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein were expressed in <i>E. coli</i> with a CASPON tag, using a growth-decoupled protein expression system<i>. </i>Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps</a>. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77%. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The HIC-MALS method enables to monitor the purity of the product and simultaneously access its molecular mass.</p>

scholarly journals Native Hydrophobic Interaction Chromatography Hyphenated to Multi-Angle Light Scattering Detection for In-Process Control of SARS-CoV-2 Nucleocapsid Protein Produced in Escherichia Coli

2021 ◽  
Author(s):  
Jelle De Vos ◽  
Patricia Pereira Aguilar ◽  
Christoph Köppl ◽  
Andreas Fischer ◽  
Clemens Grünwald-Gruber ◽  
...  
Keyword(s):  
Light Scattering ◽  
Process Control ◽  
Molecular Mass ◽  
Hydrophobic Interaction ◽  
Nucleocapsid Protein ◽  
Expression System ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Light Scattering Detection

<p>The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. <a>This protein has a strong tendency to dimerization and interaction with nucleic acids. A native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) method was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. High titers of the nucleocapsid protein were expressed in <i>E. coli</i> with a CASPON tag, using a growth-decoupled protein expression system<i>. </i>Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of chromatographic steps</a>. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77%. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The HIC-MALS method enables to monitor the purity of the product and simultaneously access its molecular mass.</p>

scholarly journals Changes in Apparent Molecular Mass of Pectin and Hemicellulose Extracts during Peach Softening

1998 ◽  
Vol 123 (3) ◽  
pp. 445-456 ◽  
Author(s):  
Supreetha Hegde ◽  
Niels O. Maness
Keyword(s):  
Molecular Mass ◽  
Sodium Carbonate ◽  
Prunus Persica ◽  
Apparent Molecular Mass ◽  
Size Exclusion ◽  
Wall Structure ◽  
Mass Peak ◽  
Exclusion Chromatography ◽  
Physical Entrapment ◽  
Second Peak

Pectin and hemicellulose were solubilized from cell walls of peach [Prunus persica (L.) Batsch] fruit differing in firmness by extraction with imidazole and sodium carbonate (pectin extracts), followed by a graded series of potassium hydroxide (hemicellulose extracts). The extracts were subjected to size exclusion chromatography. In imidazole extracts, as fruit softened, there was an increase in proportion of a large apparent molecular mass peak, with a galacturonosyl to rhamnosyl residue ratio resembling a rhamnogalacturonan-like polymer. A smaller apparent molecular mass peak was enriched in galacturonic acid and probably represented a broad polydisperse peak derived from more homogalacturonan-like polymers. In sodium carbonate extracts, a homogalacturonan-like polymer appeared to elute primarily as a higher apparent molecular mass constituent, which increased in quantity relative to other constituents as fruit softened. In cold 1 mol·L-1 KOH extracts three peaks predominated. A xyloglucan-like polymer appeared to elute predominantly in the second peak and fucose was strongly associated with it. In 4 mol·L-1 KOH extracts (tightly bound hemicellulose) the higher apparent molecular mass peak was predominantly acidic and presumably of pectic origin. The smaller apparent molecular mass peaks were composed primarily of neutral sugars, the second peak became smaller and the third peak larger as fruit softened. The ability to separate pectin and xyloglucan-like polymer as two separate fractions based on charge suggests that the nature of any pectin-hemicellulose interaction in this fraction is probably one of physical entrapment of pectin fractions by hemicellulose and not principally by covalent crosslinking between the two polysaccharide classes in peach. Flesh firmness serves as an important determinant of quality in peaches. Our results indicate that apparent molecular mass of both pectins and hemicelluloses changed as peaches softened, resulting in alteration of cell wall structure and associated with decreased tissue cohesion.

Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3485-3499 ◽  
Author(s):  
R. Kuriyama ◽  
S. Dragas-Granoic ◽  
T. Maekawa ◽  
A. Vassilev ◽  
A. Khodjakov ◽  
...  
Keyword(s):  
Cho Cells ◽  
Sequence Similarity ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sf9 Cells ◽  
Coding Region ◽  
Expression Library ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Calculated Molecular Mass

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Development of a Viral-Like Particle Candidate Vaccine Against Novel Variant Infectious Bursal Disease Virus

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 142
Author(s):  
Yulong Wang ◽  
Nan Jiang ◽  
Linjin Fan ◽  
Li Gao ◽  
Kai Li ◽  
...  
Keyword(s):  
Disease Virus ◽  
Infectious Bursal Disease Virus ◽  
Protective Efficacy ◽  
Expression System ◽  
Size Exclusion ◽  
Candidate Vaccine ◽  
Infectious Bursal Disease ◽  
Immune Protection ◽  
Bursal Disease ◽  
Novel Variant

Infectious bursal disease (IBD), an immunosuppressive disease of young chickens, is caused by infectious bursal disease virus (IBDV). Novel variant IBDV (nVarIBDV), a virus that can evade immune protection against very virulent IBDV (vvIBDV), is becoming a threat to the poultry industry. Therefore, nVarIBDV-specific vaccine is much needed for nVarIBDV control. In this study, the VP2 protein of SHG19 (a representative strain of nVarIBDV) was successfully expressed using an Escherichia coli expression system and further purified via ammonium sulfate precipitation and size-exclusion chromatography. The purified protein SHG19-VP2-466 could self-assemble into 25-nm virus-like particle (VLP). Subsequently, the immunogenicity and protective effect of the SHG19-VLP vaccine were evaluated using animal experiments, which indicated that the SHG19-VLP vaccine elicited neutralization antibodies and provided 100% protection against the nVarIBDV. Furthermore, the protective efficacy of the SHG19-VLP vaccine against the vvIBDV was evaluated. Although the SHG19-VLP vaccine induced a comparatively lower vvIBDV-specific neutralization antibody titer, it provided good protection against the lethal vvIBDV. In summary, the SHG19-VLP candidate vaccine could provide complete immune protection against the homologous nVarIBDV as well as the heterologous vvIBDV. This study is of significance to the comprehensive prevention and control of the recent atypical IBD epidemic.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Structural models for the self-assembly and microtubule interactions of gamma-, delta- and epsilon-tubulin

2001 ◽  
Vol 114 (2) ◽  
pp. 413-422 ◽  
Author(s):  
Y.F. Inclan ◽  
E. Nogales
Keyword(s):  
Self Assembly ◽  
Structural Models ◽  
Dependent Manner ◽  
Gamma Delta ◽  
Sequence Comparisons ◽  
Alpha Tubulin ◽  
A Cell ◽  
Gamma Tubulin ◽  
Cycle Dependent ◽  
Key Residues

alphabeta-tubulin heterodimers self-assemble to form microtubules nucleated by gamma-tubulin in the cell. Gamma-tubulin is believed to recruit the alphabeta-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of delta-tubulin and epsilon-tubulin. delta-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and epsilon-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of alphabeta tubulin as a model, we have analyzed the sequences of gamma-, delta- and epsilon-tubulin in regions corresponding to different polymerization interfaces in the tubulin alphabeta dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. delta-tubulin most likely interacts longitudinally with alpha-tubulin at the minus ends of microtubules, while epsilon-tubulin most likely binds to the plus end of beta-tubulin. Conservation of key residues in gamma-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.

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