DNase I-Sepharose Research Articles

Expression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris.

The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.

Expression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris

The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.

Monitoring actin folding. Purification protocols for labeled proteins and binding to DNase I-sepharose beads.

Monitoring actin folding. Purification protocols for labeled proteins and binding to DNase I-sepharose beads.

Cotransport of glyceraldehyde-3-phosphate dehydrogenase and actin in axons of chicken motoneurons.

1. To study proteins transported with actin in axons, we pulse-labeled motoneurons in the chicken sciatic nerve with [35S]methionine and, 1-20 days later, isolated actin and its binding proteins by affinity chromatography of Triton soluble nerve extracts on DNase I-Sepharose. The DNase I-purified proteins were electrophoresed on two-dimensional gels and the specific activity of the radioactively labeled protein spots was estimated by fluorography. 2. In addition to actin, which binds specifically to DNase I, a small number of other proteins were labeled, including established actin monomer binding proteins and a protein of 36 kDa and pI 8.5. On the basis of its molecular mass, pI, amino acid composition, and immunostaining, the unrecognized protein was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 3. The high-affinity binding of GAPDH to actin was confirmed by incubation of Triton-soluble nerve extracts with either mouse anti-GAPDH (or antiactin) and indirect immunomagnetic separation with Dynabeads covalently linked to sheep anti-mouse antibody. Analysis by one-dimensional gel electrophoresis and immunoblotting showed that actin and GAPDH were the main proteins isolated by these methods. 4. Analysis of labeled nerves at 12 and 20 days after pulse labeling showed that GAPDH and actin were transported at the same rate, i.e., 3-5 mm/day, which corresponds to slow component b of axonal transport. These proteins were not associated with rapidly transported proteins that accumulated proximal to a ligation 7 cm from the spinal cord 9 hr after injection of radioactivity. 5. Our results indicate that GAPDH and actin are transported as a complex in axons and raise the possibility that GAPDH could act as a chaperone for monomeric actin, translocating it to intraaxonal sites for exchange with or assembly into actin filaments. Alternatively, actin could be involved in translocating and anchoring GAPDH to specialized sites in axons and nerve terminals that require a source of ATP by glycolysis.

Slow axonal transport of soluble actin with actin depolymerizing factor, cofilin, and profilin suggests actin moves in an unassembled form.

We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [35S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.

Identification of Actin Isoforms After in Situ Hydroxylamine Cleavage on Sequencer Membranes: Serum Actin Is a Cytoplasmic Isoform

Actin was isolated from fetal bovine serum by means of affinity chromatography on DNase I-Sepharose, reversed phase chromatography and SDS-gel electrophoresis. It was identified as a cytoplasmic isoform by direct micro sequence analysis of hydroxylamine cleavage products which had been generated in situ after covalent linkage of the protein to sequencer membrane disks through arylamine or isothiocyanato functionalities.

Actin in Allomyces arbuscula

A 42 kDa protein was isolated by affinity chromatography on DNAse I-Sepharose from 24-h-old mycelia of Allomyces arbuscula. It was identified as actin by immunoblots with monoclonal antibody probe against a chicken gizzard actin. This fungal actin has a pI of 5·9 and separates into two spots on two-dimensional polyacrylamide gel electrophoresis, suggesting its dual nature. It can polymerize into 8–10 nm filaments visualized by electron microscopy.

Localization of actin and characterization of its isoforms in the hyphae of Neurospora crassa

The actin of Neurospora crassa wild type Strain St. Lawrence has been purified, characterized and localized. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-Sepharose. This protein was identified as actin on immunoblots when an anti-actin monoclonal antibody raised against chicken gizzards was used as a probe. After two-dimensional gel electrophoresis three actin isoforms were detected. The distribution of actin in hyphae was examined by FITC-phalloidin staining of formaldehyde fixed hyphae. F-actin was found to be mainly concentrated in the hyphal tips in which it formed a uniform cap. Apical actin could be involved in hyphal morphogenesis, organelle motility and maintenance of polarity.

Localization of actin and characterization of its isoforms in the hyphae ofNeurospora crassa

The actin of Neurospora crassa wild type Strain St. Lawrence has been purified, characterized and localized. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-Sepharose. This protein was identified as actin on immunoblots when an anti-actin monoclonal antibody raised against chicken gizzards was used as a probe. After two-dimensional gel electrophoresis three actin isoforms were detected. The distribution of actin in hyphae was examined by FITC-phalloidin staining of formaldehyde fixed hyphae. F-actin was found to be mainly concentrated in the hyphal tips in which it formed a uniform cap. Apical actin could be involved in hyphal morphogenesis, organelle motility and maintenance of polarity.

Actin in Mucor rouxii.

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.

Actin inMucor rouxii

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.

Characterization of acidic actin in mouse sarcoma 180 cells.

Mouse sarcoma 180 cells have a polypeptide that has the same molecular weight as actin but it is more acidic than alpha-actin. Its tryptic peptide pattern on reversed-phase HPLC was very similar to that of beta + gamma-actin, an actin sample prepared by affinity chromatography on DNase I-Sepharose contained the acidic polypeptide, and monoclonal anti-actin antibody reacted with it; therefore, the polypeptide is considered an actin isoform. The mRNA for this variant actin was identified by analyzing the polypeptides translated in vitro, which indicated that the variant actin is not a post-translationally modified form of any known actin. The variant actin was not stained by polyclonal anti-gizzard actin antibody which reacts with gamma-cytoplasmic, alpha-smooth and gamma-smooth muscle actins, nor by polyclonal anti-skeletal muscle actin antibody which reacts with skeletal, cardiac and alpha-smooth muscle actins. These results suggest that this variant actin is related to beta-cytoplasmic actin or, is a novel species whose N-terminal amino acid sequence is not Glu-Glu-Glu.

Preferential association of acidic actin with nuclei and Nuclear matrix from mouse leukemia L5178Y cells

Nuclear matrix prepared from mouse leukemia L5178Y cells contained not only the two common actin isomers, beta and gamma actins, but also two additional acidic species of actin (pI 5.1 and 5.3). An anti-actin antibody recognized these acidic species as well as beta and gamma actins on a nitrocellulose filter following western blotting of two-dimensional electrophoresis. These acidic species were co-purified with beta and gamma actins using DNase I-Sepharose affinity chromatography on the nuclear matrix. Limited digestion of the acidic actin with protease V8 or trypsin gave very similar peptide fragments as did digestion of beta and gamma actins. These acidic actins were found to be distributed in the nuclear fraction, but were scarcely detectable in the cytoplasmic fraction. One of the acidic actins (pI 5.3) was found in all subnuclear fractions (DNase extract, high-salt extract and nuclear matrix), while the other species, the most acidic actin (pI 5.1), was localized predominantly in the nuclear matrix.

Gelsolin, a Ca2+-dependent actin-binding protein in a hamster insulin-secreting cell line.

Using a gel overlay technique we have previously described a 90,000-mol wt actin-binding protein in a number of hormone-secreting tissues and tentatively identified this protein as gelsolin. Gelsolin is a protein that cuts or solates cross-linked actin filaments and can also serve as a nucleating site for actin polymerization. The objective of this study was to isolate this protein from a hamster insulin-secreting (HIT) cell line and compare the immunologic properties and peptide maps of purified rabbit macrophage gelsolin, human platelet gelsolin, and the HIT cell 90,000-mol wt protein. DNase I-Sepharose retained the HIT cell actin-binding proteins in 1 mM CaCl2; some of the 90,000-mol wt protein could then be eluted with 1 mM EGTA. The remaining actin-binding proteins were eluted using a buffer containing SDS. The EGTA peak fractions contained two major protein bands of Mr = 90,000 and 42,000, which suggested that a 90,000-mol wt-actin complex was eluted from the DNase I-Sepharose column. Specific antibodies to the human platelet and rabbit macrophage gelsolins bound to the 90,000-mol wt bands in the eluates, but did not crossreact with other actin-binding proteins. Indirect immunofluorescence using an anti-human platelet gelsolin antibody localized the 90,000-mol wt protein to stress fibers that were also stained with phalloidin, which suggested that gelsolin is associated with actin in vivo. Tryptic peptide maps of all three radioiodinated gelsolins were virtually indistinguishable. Thus, gelsolin is a highly conserved gene product found in at least three diverse cell types, an insulin-secreting beta cell line, macrophages, and platelets, and may link a transient increase in Ca2+ cellular levels with changes in actin polymerization and/or the gel-sol state of these cells.

An actin-depolymerizing protein (depactin) from starfish oocytes: properties and interaction with actin.

Physico-chemical properties and interaction with actin of an actin-depolymerizing protein from mature starfish oocytes were studied. This protein, which is called depactin, exists in a monomeric form under physiological conditions. Its molecular weight is approximately 20,000 for the native protein and approximately 17,000 for denatured protein. The Glu + Asp/Lys + Arg molar ratio of this protein is 1.55. The apparent pl of the denatured depactin is approximately 6. The extent of actin polymerization is reduced by the presence of depactin; however, the rate of polymerization seems to be accelerated as measured spectrophotometrically at 238nm. This effect is interpreted to indicate that depactin cut the newly formed filaments into small fragments, thereby increasing the number of the filament ends to which monomers are added. The apparent critical concentration of actin for polymerization, as determined by viscometry or flow birefringence measurement, is increased by the presence of depactin in a concentration-dependent manner. Raising the pH of the solution does not reverse the action of depactin. The molar ratio of actin and depactin, which interact with each other, is estimated to be 1:1 by means of a cross-linking experiment using a water-soluble carbodiimide. Depactin binds to a DNase I-Sepharose column via actin and is selectively eluted with 0.6 M KCl or 0.6 M Kl. The association constant between actin and depactin is estimated, using the column, to be 2-3 X 10(6) M-1. The content of depactin in the high-speed supernatant of the oocyte extract is determined to be 1%; this can act upon approximately 63% of the actin in the supernatant.

Rat pancreas actin: Purification and characterization

Isolation of rat pancreas actin was performed with three different technics: polymerization-depolymerization method, affinity chromatography on DNase I-Sepharose 4B or ion exchange chromatography on DEAE-cellulose. Inhibition of DNase I activity, localization by SDS polyacrylamide slab gel electrophoresis and presence of microfilaments allowed its identification. Affinity process led us to obtain actin which kept inhibitory activity (30,000 U per mg) on DNase I when using vacuum dialysis. Actin eluted from DEAE-cellulose associated reversibly in 50–70 Å microfilaments in the presence of phalloidin, was pure at 95% and had a satisfactory inhibitor activity (77,000 U per mg).

Selective isoactin release from cultured embryonic skeletal muscle cells.

The culture medium of embryonic quail myoblasts, labeled for 24 h with [35S]L-methionine, was analyzed by two-dimensional gel autoradiography. The major polypeptide observed had a 43,000 molecular weight and an isoelectric point of 5.4. This polypeptide could be specifically adsorbed to DNAse-I Sepharose. A tryptic peptide map of the [35S]methionine-labeled peptides of intracellular actin and the extracellular major polypeptide were virtually identical. These findings identify the released polypeptide as actin. A comparison of two-dimensional gel patterns of intracellular and extracellular labeled polypeptides showed a large number of differences indicating the actin release did not result from general cellular breakdown. The released actin was not filamentous as judged by its behavior during Bio-Gel A-5m chromatography (Bio-Rad Laboratories, Richmond, Calif.) The released actin did not originate solely from contaminating fibroblasts in the culture because actin was also observed in the medium in clonal myoblast cultures and in purified myotube preparations. Finally, the nonmuscle isoactins, as opposed to muscle alpha-isoactin, were released preferentially. These results indicate that within the developing muscle cell where both muscle and nonmuscle specific isoactins are simultaneously present, the different isoactins may be physically or functionally compartmentalized with the nonmuscle isoactins existing primarily at or near the cell surface.

An actin-like protein is a component of axonemes from Chlamydomonas flagella

Several lines of evidence indicate that a component of axonemes from Chlamydomonas flagella is similar to actin from rabbit skeletal muscle. The polypeptide has an apparent molecular weight of 42,000 and in a pH gradient has the electrophoretic behavior of beta-actin. It was co-polymerized with rabbit actin and purified by affinity chromatography on DNase I-Sepharose. Incomplete proteolysis of mixed 35S-labeled axonemal protein and cold rabbit actin formed similar sets of peptides as analyzed by one-dimensional gel electrophoresis. The actin-like protein and the tubulin appear to be present in the axoneme in the molar ratio 1:60.