Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.     

Use of a protein-cellulose complex for inducing intensive antibody formation in mice

The protein-cellulose complex prepared by covalent immobilization of single protein molecules on insoluble cellulose particles was used for priming C57BL/6 and BALB/c mice. The serum antibody content and the number of spleen AFC were assayed after animals' boosting with the soluble protein. Such a complex was shown to have marked advantages over the same protein injected both in complete Freund's adjuvant and in a soluble form, in particular. The immune response of BALB/c mice was more than 10-fold higher than that of C57BL/6 animals.     

III. The RNA component of aminoacyl-tRNA synthetase complexes isolated from mouse liver. Absence of amino acid accepting activity.

A method is described which permits the simultaneous isolation and separation of insoluble aminoacyl-tRNA synthetase protein - RNA complexes containing high specific synthetase activity, and soluble tRNA which retains 25% to 50% of its specific amino acid accepting activity. A possible amino acid accepting activity of the RNA part of the insoluble aminoacyl-tRNA synthetase protein - RNA complex was investigated by assaying the unchanged complex and the RNA obtained after dissociation from the protein part of the synthetase complex. No amino acid accepting activity was found.     

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.     

Small heat-shock proteins function in the insoluble protein complex

Small heat-shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones. The current model proposes that sHSPs function to prevent irreversible aggregation of non-native proteins by forming soluble complex. The chaperone activity of sHSPs is usually determined by the capacity to suppress thermally or chemically induced protein aggregation. However, sHSPs were frequently found in the insoluble complex particularly in vivo. In this report, it is clearly revealed that the insoluble sHSP/substrate complex is formed when sHSP is overloaded with non-native substrates, which is the very case under in vivo conditions. The proposal that sHSPs function to prevent the protein aggregation seems misleading. sHSPs appear to promote the elimination of protein aggregates by incorporating into the insoluble protein complex.     

BOVINE ADRENAL MEDULLARY DOPAMINE-β-HYDROXYLASE: STUDIES ON INTERACTION WITH CONCANAVALIN A

Bovine adrenal medullary dopamine-β-hydroxylase binds with concanavalin A and forms an enzymically active precipitate. The formation of the insoluble complex is pH-dependent and can be inhibited by α-methyl-D-mannoside, D-mannose and D-glucose. The insoluble complex can be dissociated into two species with α-methyl-D-mannoside. From the results, it appears that the interaction between dopamine-β-hydroxylase and concanavalin A is due to the carbohydrate moiety of dopamine-β-hydroxylase. This property was used to purify the enzyme from a soluble lysate of chromaffin granules. Of all the proteins contained in the soluble lysate, dopamine-β-hydroxylase was the only one to be retained on a column of concanavalin A covalently bound to Sepharose 4B. The preparation of pure dopamine-β-hydroxylase exhibits a very high specific activity of 320 μmol of octopamine formed per 30 min per mg of protein.     

Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli

Sin Nombre virus is a member of the Hantavirus genus, family Bunyaviridae, and is an etiologic agent of hantavirus pulmonary syndrome. The hantavirus nucleocapsid (N) protein plays an important role in the encapsidation and assembly of the viral negative-sense genomic RNA. The Sin Nombre N protein was expressed as a C-terminal hexahistidine fusion in Escherichia coli and initially purified by nickel-affinity chromatography. We developed methods to extract the soluble fraction and to solubilize the remainder of the N protein using denaturants. Maximal expression of protein from native purification was observed after a 1.5-h induction with IPTG (2.4 mg/L). The zwitterionic detergent Chaps did not enhance the yield of native purifications, but increased the yield of protein obtained from insoluble purifications. Both soluble and insoluble materials, purified by nickel-affinity chromatography, were also subjected to Hi Trap SP Sepharose fast-flow (FF) chromatography. Both soluble and insoluble proteins had a similar A(280) profile on the Sepharose FF column, and both suggested the presence of a nucleic acid contaminant. The apparent dissociation constant of the N protein, purified by nickel-affinity and SP Sepharose FF chromatography, and the 5' end of the viral S-segment genome were measured using a filter binding assay. The N protein-vRNA complex had an apparent dissociation constant of 140 nM.     

Rice allergenic proteins, 14-16 kDa albumin and alpha-globulin, remain insoluble in rice grains recovered from rice miso (rice-containing fermented soybean paste)

The rice grains (RG) and rice seed proteins remaining in rice miso were investigated with a view point to the potential allergenicity of rice miso. RG ranging from 36 to 180 mg dry weight per g dry miso were separated from several samples of commercially available rice miso. Scanning electron microscopy of the recovered RG indicated that starch granules disappeared almost completely while protein bodies remained intact in RG. Most of the major seed proteins were extracted from RG by heating with 1% SDS/2% 2-mercaptoethanol and detected by SDS-polyacrylamide gel electrophoresis. Major rice allergenic proteins, 14-16 kDa albumin (Alb14-16) and alpha-globulin (alpha-Glb) were also detected by immunoblotting using the specific antisera, and their contents were estimated to be 1.7 to 9.0 and 1 to 7 mg protein per g dry RG respectively. However, the major rice proteins, including glutelin and prolamin, in RG were insoluble in salt, alcohol, and urea solutions, but soluble in 6 M guanidine hydrochloride (Gu-HCl). By immunoblotting and ELISA, no Alb14-16 and only a slight amount of alpha-Glb were detected even in the 6 M Gu-HCl fraction, indicating that these major allergenic proteins are denatured and are present in an insoluble form in rice miso.     

pUBEX/pUBSEX: a versatile expression vector system for production of fusion and nonfusion proteins in Escherichia coli.

Despite the large number of expression vectors now available, none provide the facility of allowing fusion and nonfusion protein production from the same vector system. In some situations it is preferable to obtain an insoluble fusion protein, in others a soluble nonfusion protein may be required. We have designed, constructed and tested a modification of the pEX vectors, in which it is possible to express the product of a suitably inserted cDNA either as part of a Cro-beta-galactosidase (Cro-beta Gal) fusion or as a delta Cro fusion which contains only nine noninsert-encoded amino acids at its N terminus. The conversion from Cro-beta Gal to delta Cro fusion protein production is achieved by a simple intramolecular deletion of lacZ sequence from the pUBEX vector, to create the pUBSEX variant. Plasmid pUBEX can be induced to produce large amounts of insoluble Cro-beta Gal fusion proteins, whereas pUBSEX will produce predominantly soluble delta Cro fusion proteins.     

Isolation of substrates of epidermal transglutaminase from bovine epidermis.

Substrates of the transglutaminase specific to epidermis were identified by fluorescent labeling of bovine epidermal homogenates with dansyl cadaverine. This lysine analog was preferentially incorporated into a soluble protein of 150,000 MW. A highly insoluble protein was also labeled; this protein was solubilized and extracted following chemical cleavage with cyanogen bromide. The soluble and insoluble substrates of epidermal transglutaminase were immunochemically related, as shown by precipitation in agar or by chromatography on antibody affinity columns. They were distinguished from the fibrous, α-helical and sulfur-rich matrix proteins of skin as well as from fibrinogen and cold insoluble globulin of plasma.     

Characterization of an insoluble adenosine deaminase complexing protein from human kidney

Evidence for the presence of an insoluble form of adenosine deaminase complexing protein in human kidney has been obtained. An initial study demonstrated that binding of monomeric adenosine deaminase to particulate material from kidney was saturable and could be blocked by preincubating the enzyme with soluble complexing protein. Treatment of particulate material with deoxycholate, followed by immunoassay of the detergent extract, confirmed the presence of an insoluble form of complexing protein in the kidney. Several other human organs examined by this technique contained smaller amounts of insoluble complexing protein. Complexing protein isolated from the soluble and particulate fractions of kidney homogenates were found to be structurally similar. The proteins had the same subunit M_r and showed complete crossreactivity with antiserum to soluble complexing protein. Indirect immunoperoxidase staining of renal cortical tissue revealed that complexing protein was concentrated in the brush border of the proximal tubules. These results indicate that (a) the soluble and insoluble forms of complexing protein from human kidney may be products of the same gene(s) and (b) a portion of the complexing protein in human kidney is bound to the brush border membranes of cells lining the proximal tubules.     

The Rubisco complex protein: A protein induced by fruit removal that forms a complex with ribulose-1,5-bisphosphate carboxylase/oxygenase

Continuous removal of fruits from soybean plants (Glycine max [L.] Merr.) causes a redistribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) from the soluble to the insoluble phase of leaf extracts. The extent of this redistribution is genotype-dependent. We previously reported that insoluble Rubisco occurs in a high-molecular-mass complex together with a protein composed of 30-kDa subunits (S.J. Crafts-Brander et al., Planta, 183, 300–306). In the present study, the Rubisco Complex Protein (RCP), was isolated from the Rubisco-RCP complex by gel-filtration chromatography in 4 M urea. Under these conditions, RCP migrated with an apparent molecular mass of 120 kDa, indicating that the protein maintains a tetrameric structure even in 4 M urea. Once freed of urea, purified RCP was soluble, but formed insoluble complexes with Rubisco from soybean, tobacco and spinach when RCP and Rubisco were incubated in a ratio of 11 by weight. Purified Rubisco and RCP also associated into a high-molecular-mass complex when either component was in several-fold excess, but in this case the complex was soluble. Similarly, the amount of Rubisco sequestered as an insoluble Rubisco:RCP complex in leaf extracts of different soybean genotypes was related to the relative amounts of Rubisco and RCP present in the extracts. Thus, with both purified components and in leaf extracts, formation of an insoluble complex between Rubisco and RCP required a precise stoichiometry. Antibodies directed against purified RCP detected an accumulation of RCP in soybean leaves around the time of flowering. The RCP was also detected in petioles, stems, and pod walls of soybean, but not seeds. Fruit removal caused a marked increase in the amount of RCP in the leaves to levels as high as 15% of the total soluble protein. The accumulation of RCP in response to source:sink manipulations was similar to soybean vegetative storage proteins (VSPs). However, immunogold-localization showed that RCP was located in the cytosol of leaves, compartmentalized separate from both Rubisco and the VSPs. Thus, the physiological relevance of the specific association between RCP and Rubisco is obscure.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RCP rubisco complex protein - VSPs vegetative storage proteins Kentucky Agricultural Experiment Station Journal Article No. 93-3-162We wish to acknowledge L.F. Staples and J.C. Anderson for their expert technical assistance. Electron microscopy was performed by the Nano-Probe Laboratory, Lucille Parker Markey Cancer Center, University of Kentucky. We thank Dr. K.C. Vaughn, USDA-ARS, for providing guidance pertaining to immunogold-localization procedures.     

Effect of protein malnutrition on the metabolism of dermal collagen in the rat

The effect of protein malnutrition on the metabolism of collagen was studied in young female albino rats after a single injection of 3H-proline by determining the specific as well as total activities of 3H-hydroxyproline in the skin collagen fractions and in the urine. a) Compared to controls, the total activity of 3H-hydroxyproline in the soluble collagen and in the urine was significantly lower in the deficient group at 12 hrs. after the administration of 3H-proline. b) The urinary excretion of hydroxyproline and the total activity of urinary 3H-hydroxyproline measured after four weeks of labelled proline injection were also considerably decreased in the protein-deficient animals. c) When the total radioactivities of both soluble and insoluble collagen are expressed as a percentage of the sum of both, the recorded activity was more in soluble and less in insoluble collagen at 12 and 120 hrs. after the administration of 3H-proline, due to the influence of protein malnutrition. The results of the present investigation therefore clearly indicate that the synthesis of collagen is decreased and accompanied by a retardation in the maturation of soluble to insoluble collagen in the protein-deficient animals compared to controls. In addition, protein deficiency is accompanied by decreased rates of catabolism of both soluble and insoluble collagen.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Effect of phospholipase A treatment of low density lipoproteins on the dextran sulfate--lipoprotein interaction

The effect of phospholipase A on the interaction of low density lipoproteins of the S(f) 0-10 class with dextran sulfate was studied in phosphate buffer of pH 7.4, ionic strength 0.1, by chemical, spectrophotometric, and centrifugal methods. When low density lipoproteins that had been treated with phospholipase A were substituted for untreated lipoproteins, the amount of insoluble dextran sulfate-lipoprotein complex formed was greatly reduced. Hydrolysis of over 20% of the lecithin and phosphatidyl ethanolamine constituents of the lipoproteins prevented the formation of insoluble complex. However, even the lipoproteins in which almost all the phosphoglycerides were hydrolyzed produced soluble complex, which was converted to insoluble complex upon addition of magnesium sulfate. It is apparent that the lipoproteins altered extensively by treatment with phospholipase A retain many characteristic properties of native low density lipoproteins. Fatty acids, but not lysolecithin, released by the action of phospholipase A interfered with the formation of insoluble complex; this interference was due to association of the fatty acids with the lipoproteins. With increases in the concentration of the associated fatty acids, the amounts of magnesium ion required for the conversion of soluble complex to insoluble complex increased progressively. Charge interaction is evidently of paramount importance in the formation of sulfated polysaccharide-lipoprotein complexes.     

New fusion protein systems designed to give soluble expression in Escherichia coli.

Three native E. coli proteins-NusA, GrpE, and bacterioferritin (BFR)-were studied in fusion proteins expressed in E. coli for their ability to confer solubility on a target insoluble protein at the C-terminus of the fusion protein. These three proteins were chosen based on their favorable cytoplasmic solubility characteristics as predicted by a statistical solubility model for recombinant proteins in E. coli. Modeling predicted the probability of soluble fusion protein expression for the target insoluble protein human interleukin-3 (hIL-3) in the following order: NusA (most soluble), GrpE, BFR, and thioredoxin (least soluble). Expression experiments at 37 degrees C showed that the NusA/hIL-3 fusion protein was expressed almost completely in the soluble fraction, while GrpE/hIL-3 and BFR/hIL-3 exhibited partial solubility at 37 degrees C. Thioredoxin/hIL-3 was expressed almost completely in the insoluble fraction. Fusion proteins consisting of NusA and either bovine growth hormone or human interferon-gamma were also expressed in E. coli at 37 degrees C and again showed that the fusion protein was almost completely soluble. Starting with the NusA/hIL-3 fusion protein with an N-terminal histidine tag, purified hIL-3 with full biological activity was obtained using immobilized metal affinity chromatography, factor Xa protease cleavage, and anion exchange chromatography.     

Patterns of storage protein and triacylglycerol accumulation during loblolly pine somatic embryo maturation

Conifer somatic embryo germination and early seedling growth are fundamentally different than in their zygotic counterparts in that the living maternal megagametophyte tissue surrounding the embryo is absent. The megagametophyte contains the majority of the seed storage reserves in loblolly pine and the lack of the megagametophyte tissue poses a significant challenge to somatic embryo germination and growth. We investigated the differences in seed storage reserves between loblolly pine mature zygotic embryos and somatic embryos that were capable of germination and early seedling growth. Somatic embryos utilized in this study contained significantly lower levels of triacylglycerol and higher levels of storage proteins relative to zygotic embryos. A shift in the ratio of soluble to insoluble protein present was also observed. Mature zygotic embryos had roughly a 3:2 ratio of soluble to insoluble protein whereas the somatic embryos contained over 5-fold more soluble protein compared to insoluble protein. This indicates that the somatic embryos are not only producing more protein overall, but that this protein is biased more heavily towards soluble protein, indicating possible differences in metabolic activity at the time of desiccation.     

A simple in vivo assay for increased protein solubility.

Low solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.     

Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.

Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.