Cell-free production of aggregation-prone proteins in soluble and active forms

We have developed an efficient cell-free protein synthesis system for the production of soluble and active eukaryotic proteins that are predominantly produced as inclusion bodies in bacteria. S30 extracts (indicating the supernatant of cell homogenate when centrifuged at 30,000g) for cell-free protein synthesis were prepared from Escherichia coli that was modified to overexpress a set of chaperones (GroEL/ES or DnaK/J-GrpE) and disulfide isomerase (leader sequence-free mature DsbC expressed in the cytoplasm). The solubility and biological activity concentration (biological activity per unit volume of cell-free protein synthesis reaction mixture) of the protein synthesized by the new cell-free protein synthesis system showed a dramatic improvement. Solubility enhancement was most dramatic with the existence of DnaK/J-GrpE. It shows that the co-translational interaction with DnaK/J-GrpE prior to folding trial is important in maintenance of the aggregation-prone protein in a folding-competent soluble state. For maximizing the biological activity concentration of the expressed protein, the additional presence of GroEL/ES and DsbC was required. When human erythropoietin was expressed in the developed cell-free protein synthesis system including endogenously overexpressed chaperones and/or DsbC, the biological activity concentration of erythropoietin was enhanced by 700%. It implies that the post-translational folding and disulfide bond reshuffling as well as co-translational folding are important in acquiring functionally active protein from cell-free expression system. This is the first report of using S30 extracts including endogenously overexpressed chaperones and/or disulfide isomerase for the efficient production of soluble and active proteins in cell-free protein synthesis. This new cell-free protein synthesis system was capable of introducing much larger amounts of chaperones and disulfide isomerase compared to a conventional method that supplements them separately. The developed cell-free protein synthesis system supported efficient expression of the eukaryotic proteins in soluble and active forms without the need of any exogenous addition or coexpression of folding effectors.     

Translation in vitro and regulation of mouse liver S-adenosylmethionine synthetase messenger RNA

Total RNA was isolated from adult mouse liver tissues. The alpha- and beta-form isozymes of S-adenosylmethionine synthetase existing in liver were synthesized in a reticulocyte lysate cell-free system under the direction of total RNA and were immunoprecipitated with antibody to the beta-form. The newly synthesized and the in vivo labeled S-adenosylmethionine synthetase subunits were compared by SDS-polyacrylamide gel electrophoresis. Both the alpha- and beta-forms consist of the same size Mr 48 000 subunit. The level of the beta-form mRNA activity in mouse liver was shown to increase following intraperitoneal transplantation of Ehrlich ascites tumor cells and the changes in the mRNA activity parallel those in the cellular level of S-adenosylmethionine synthetase beta.     

In vitro bioassay of erythropoietic activity in serum using mouse spleen cells. The effect of heat inactivation on serum erythropoietin

Untreated human serum is known to be toxic to in vitro assays for erythropoietin, including the mouse spleen cell assay system (MSCA). This phenomenon had previously been shown to be mediated by complement-dependent IgM heteroantibodies and can be overcome by heating the serum at 56 degrees C for 30 minutes. Using the MSCA, we have found that the toxic effect of serum could also be removed by treatment with a precipitating antibody against the C3c component of complement. The effects of the two methods of complement inactivation on the measurement of stimulatory activity in serum have been compared. For normal serum, the results after heat inactivation and antibody treatment were similar. In contrast, serum from a patient with aplastic anemia gave a result equivalent to 327 mU erythropoietin/ml after heat treatment, but after antibody treatment equivalent to 1,520 mU erythropoietin/ml. Gel permeation chromatography of unheated, heated, and antibody-treated sera showed that heating markedly reduced the activity of the erythropoietin peak. Seventy percent of the activity of partially purified urinary erythropoietin was lost during heating in the presence of normal serum. In addition, heating caused the appearance of high molecular weight compounds that are stimulatory in the MSCA. The level of this activity appeared to be directly related to the stimulatory activity of the unheated serum.     

Intracellular distribution and biosynthesis of lipoamide dehydrogenase in rat liver

Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-tenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S]methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase.     

Immunochemical Isolation of γ-Globulin mRNA and Estimation of Immunoglobulin Gene Reiteration

The polyribosomes synthesizing γ-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific γ-globulin-synthesizing activity (cpm of γ-globulin synthesized/μg RNA) of this RNA was 10-fold greater than that from total polysomes. γ-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis. This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the “half Cot value” is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains.     

Cell-free production of functional antibody fragments

Botulinum neurotoxin serotype B (BoNT/B)-specific Fab was expressed in a cell-free protein synthesis system derived from an E. coli extract. The cell-free synthesized antibody fragment was found to be effective in neutralizing the toxicity of BoNT/B in animal studies. Expression of functional Fab required an appropriately controlled and stably maintained redox potential. Under an optimized redox condition, the cell extract, whose disulfide reducing activity had been exhausted, could generate bio-functional Fab molecules. Use of a cell extract enriched with molecular chaperones (GroEL/ES) and disulfide bond isomerases were effective in obtaining larger quantities of functional Fab. Under the optimized reaction conditions, approximately 30 μg of functional Fab was obtained after purification from 1 mL reaction mixture.     

Immunologic and physicochemical properties of enhancing soluble factors for IgG and IgE antibody responses.

Rabbits were immunized with dinitrophenyl-coupled Ascaris antigen (DNP-Asc) or ragweed antigen (DNP-Rag) included in aluminum hydroxide gel and their mesenteric lymph node cells were cultured for 24 hr in vitro in the presence of free homologous carrier. The cell-free supernatant thus obtained enhanced both IgG and IgE antihapten antibody responses of DNP-primed cells to DNP-heterologous carrier conjugate (DNP-keyhole limpet hemocyanin). Since the cell-free supernatant obtained from Rag-specific cells enhanced antibody response of hapten-primed cells raised by immunization with DNP-Asc, no carrier specificity was involved in the enhancement. It was found that treatment of primed cells with 10-5 M pactamycin suppressed the formation of the enhancing soluble factor, whereas the factor was readily formed in the presence of 2 mug/mol of cytosine arabinoside in the culture. The results indicated that cell proliferation was not required but de novo synthesis of protein was essential for the formation of soluble factor(s). The enhancing factor was not absorbed by either carrier-coated or anti-carrier antibody-coated immunosorbent. It was also found that the enhancing factor was formed by incubating primed cells with carrier-coated Sepharose. The cell-free supernatant containing no free carrier enhanced both IgG and IgE anti-hapten antibody responses. The activities of the cell-free supernatant to enhance IgG and IgE antibody responses were not absorbed by anti-Fab, anti-gamma-or anti-mu-chain antibody immunosorbent, indicating that the nonspecific enhancing factor did not possess immunoglobulin determinant. The cell-free supernatant was fractionated by sucrose density gradient ultracentrifugation and by gel filtration with three radiolabeled proteins, i.e., IgG, ovalbumin, and cytochrome C as markers. Enhancing activity for IgG antibody response was recovered in a fraction between ovalbumin peak (40,000 m.w.) and cytochrome C peak (20,000 m.w.). The activity for IgE antibody response was recovered in a fraction containing IgG marker (150,000 m.w.). By block electrophoresis, both activities were detected in beta globulin fraction. The results suggested that different T cell factors are involved in the IgG and IgE antibody responses.     

Use of<Emphasis Type="SmallCaps">l</Emphasis>-buthionine sulfoximine for the efficient expression of disulfide-containing proteins in cell-free extracts of<Emphasis Type="Italic">Escherichia coli</Emphasis>

We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.     

Intracellular site of synthesis of microsomal heme oxygenase in pig spleen

In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.     

Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products.

Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.     

Isolation and characterization of an anti-peptide monoclonal antibody to human erythropoietin

A site-specific monoclonal antibody to human erythropoietin has been developed. It is secreted by a hybridoma cell line derived from the fusion of murine myeloma cells with the splenocytes of a mouse that had been immunized with a 26-residue synthetic peptide antigen homologous to the amino-terminal sequence of the hormone. The antibody binds specifically to peptide, 125I-erythropoietin, and biologically active erythropoietin. The equilibrium dissociation constants of the antibody-erythropoietin and the antibody-peptide interactions are identical, Kd = 6.7 X 10(-9) M, suggesting strong conformational similarity or identity of the epitope as expressed on the peptide and the hormone. Immune complexes formed between the antibody and either human or rat erythropoietin exhibit full biologic activity. However, the antibody does not recognize the baboon, sheep, or canine hormones, indicating antigenic differences or structural variation among these erythropoietins. These results indicate that the amino-terminal region of erythropoietin is not involved in receptor binding. Furthermore, they form a basis for the study of the structure and function of the hormone using anti-peptide antibodies.     

Insulin modulation of human apolipoprotein B mRNA translation: studies in an in vitro cell-free system from HepG2 cells.

Insulin modulation of apolipoprotein B gene expression was studied at the translational level by the use of a cell-free translation system from a hepatoma cell-line, HepG2. Extracts of HepG2 cells lysed with lysolecithin were found to have high in vitro protein synthesizing activity utilizing endogenous mRNA. The level of peptide chain initiation was high, as suggested by a significant inhibition of translation by edeine. The translation products of endogenous mRNA in HepG2 cell-free lysate were probed with anti-apolipoprotein B antibodies to investigate its synthesis. A 550 kilodalton (kDa) polypeptide was selected by a polyclonal antibody, as well as a monoclonal antibody, against the C-terminal end of apolipoprotein B molecule. This in vitro synthesized polypeptide was also found to compare well in size with the in vivo product. The HepG2 lysate was also shown to efficiently synthesize in vitro a number of other proteins including albumin, apolipoprotein E, apolipoprotein A1, and actin. The in vitro synthesis of polypeptides as large as 500 kDa was unexpected and has not previously been demonstrated in a cell-free system. The HepG2 translation system was used to investigate the effect of insulin on the in vitro translation of apolipoprotein B. Lysates prepared from HepG2 cells treated with insulin were found to have lower translational activity (by an average of 52.3%) for apolipoprotein B compared with lysates from control untreated cells. In vitro synthesis of actin and apolipoprotein E were unaffected under these conditions. The insulin-stimulated decline in in vitro apolipoprotein B synthesis was not due to a change in apolipoprotein B mRNA levels as determined by slot- and Northern-blot analyses, suggesting that the inhibitory effect of insulin may be exerted partly at the level of apolipoprotein B mRNA translation.     

Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Synthesis of fluorescent and photoaffinity-labeled derivatives of bisphenol A and their inhibitory activity toward hypoxic expression of erythropoietin

Bisphenol A derivatives, possessing a fluorescent dye and a photo-reactive group, were synthesized from bisphenol A, and the inhibitory activity of the derivatives was evaluated against hypoxic response. The synthesized derivatives were found to inhibit the hypoxic expression of erythropoietin in Hep3B cells as well as bisphenol A.     

Translation of glutamate dehydrogenase mRNA in a reticulocyte cell-free system

Messenger RNA template activity for glutamate dehydrogenase was detected in poly(A)-rich RNA extracted from rat liver polysomes. Enzyme synthesized in cell-free reticulocyte system was detected by measuring enzyme activity in the translation incubation mixture using dual wavelength spectrophotometric technique. The translation product was also identified by a partial purification of the labeled synthesized enzyme and by coelectrophoresis with the carrier enzyme preparation from mitochondrial matrix.     

Cell-free synthesis of Chironomus thummi (Diptera) globins

RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi.     

Translation of mouse interferon messenger RNA in a cell-free system

Crude mouse interferon mRNA extracted from poly (I) . poly (C)-induced L929 cells has been translated in a cell-free system from rabbit reticulocytes and in two-cell systems--L929 cells and chick embryo fibroblasts. Translational efficiency of interferon mRNA preparation was 100-fold higher in the cell-free system than in tissue culture cells. Interferon synthesized was species-specific and its antiviral activity was completely neutralized by mouse interferon antiserum.     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.