Production of recombinant endostatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED₅₀) for recombinant endostatin was approximately 0.35 g ml^–1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l^–1 at 6 days of cultivation.     

Optimal production and in vitro activity of recombinat endostatin from stably transformed Drosophila melanogaster S2 cells

Recombinant plasmids containing a complementary deoxyribonucleic acid coding mouse endostatin were transfected and stably expressed in Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing recombinant endostatin were isolated after 4 wk of selection with hygromycin B. Recombinant endostatin expressed in the stably transformed S2 cells under the influence of the Drosophila BiP protein signal sequence was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at maximum inhibition for recombinant endostatin was approximately 1.8 microg/ml. The stably transformed S2 cells produced 18 mg recombinant endostatin/L 7 d after induction with 5 microM CdCl2. Sodium butyrate supplementation (2.5 mM) increased recombinant endostatin production by 17%. These findings demonstrate optimal production and in vitro activity of recombinant endostatin from stably transformed D. melanogaster S2 cells.     

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 g ml^–1. A maximum production of 4.6 g recombinant tumstatin (10⁷ cells)^–1 was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO₄.     

Dimethylsulfoxide and sodium butyrate enhance the production of recombinant cyclooxygenase 2 in stably transformed Drosophila melanogaster S2 cells

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni²⁺-affinity fractionation to a specific activity of 24 800 U mg^–1 protein. The peak level of recombinant Cox 2 production was 1.6 g (10⁷ cells)^–1, seven days after induction with 0.5 mM CuSO₄. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.     

Functional expression of recombinant canstatin in stably transformed Drosophila melanogaster S2 cells

We describe the expression and in vitro activity of recombinant canstatin from stably transformed Drosophila melanogaster S2 cells. Southern blot analysis indicated that transformed S2 cells contained multiple copies of the canstatin gene in the genome. Recombinant canstatin with a molecular weight of 29kDa was secreted into the culture medium. Recombinant canstatin was purified to homogeneity using a simple one-step Ni(2+) affinity fractionation. Purified recombinant canstatin inhibited human umbilical vein endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED(50)) for recombinant canstatin expressed in stably transformed Drosophila S2 cells was approximately 0.37mug/ml. A maximum production level of 76mg/l of recombinant canstatin was obtained in a T-flask culture of Drosophila S2 cells 6 days after induction with 0.5mM CuSO(4).     

Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells

A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of ∼8 U μg⁻¹ for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ^®SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.     

Improved production of recombinant tumstatin in stably transformed Trichoplusia ni BTI Tn 5B1-4 cells

We describe the expression and in vitro activity of recombinant tumstatin from stably transformed Trichoplusia ni BTI Tn 5B1-4 cells. Recombinant tumstatin was secreted into a culture medium with a molecular weight of 29 kDa. Recombinant tumstatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant tumstatin expressed in stably transformed Tn 5B1-4 cells was approximately 0.76 microg/ml. A maximum production level of 4.0 mg/l recombinant tumstatin was obtained in a T-flask culture of Tn 5B1-4 cells, 6 days after cultivation. We also investigated the individual effects of both dimethyl sulfoxide (DMSO) and sodium butyrate on recombinant tumstatin production in stably transformed Tn 5B1-4 cells. Supplementing cultures with DMSO and sodium butyrate separately increased recombinant tumstatin production in stably transformed Tn 5B1-4 cells by 117 and 32%, respectively.     

Synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50–55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.     

Functional expression of Arabidopsis thaliana sterol glycosyltransferase from stably transformed Drosophila melanogaster S2 cells

Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production. Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. Radiometrical assay using uridine diphospho-d-[U-¹⁴C]glucose (UDP-¹⁴C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach ¹⁴C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-sitosterol, which was similar to the recombinant SGT produced by a bacterial expression system. The transfer of ¹⁴C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated ¹⁴C-glucosetransferred β-sitosterol and stigmasterol reactants.     

Expression of recombinant Atlantic salmon serum C-type lectin in Drosophila melanogaster Schneider 2 cells

The Atlantic salmon (Salmo salar) serum lectin (SSL) is a soluble C-type lectin that binds bacteria, including salmon pathogens. This lectin is a cysteine-rich oligomeric protein. Consequently, a Drosophila melanogaster expression system was evaluated for use in expressing SSL. A cDNA encoding SSL was cloned into a vector designed to express it as a fusion protein with a hexahistidine tag, under the control of the Drosophila methallothionein promoter. The resulting construct was stably transfected into Drosophila S2 cells. After CdCl₂ induction, transfected S2 cells secreted recombinant SSL into the cell culture medium. A cell line derived from stably transformed polyclonal cell populations expressing SSL was used for large-scale expression of SSL. Recombinant SSL was purified from the culture medium using a two-step purification scheme involving affinity binding to yeast cells and metal-affinity chromatography. Although yields of SSL were very low, correct folding and functionality of the recombinant SSL purified in this manner was demonstrated by its ability to bind to Aeromonas salmonicida. Therefore, Drosophila S2 cells may be an ideal system for the production of SSL if yields can be increased.     

The Effect of Selenite Ions on Growth and Selenium Accumulation in Spirulina platensis

Selenium accumulation and the growth of cyanobacterium Spirulina platensis (Nordst.) Geitl. were studied in a culture with sodium selenite-supplemented nutritional medium. Selenite concentrations below 20 mg/l did not inhibit the growth of S. platensis. The addition of 30 mg/l of this salt somewhat decreased the growth rate during the linear growth phase, induced the earlier suspension transition to the steady-state phase, and substantially lowered the highest optical density of the suspension. However, even at 170 mg/l Na₂SeO₃, the culture still demonstrated a capacity for growth. The content of selenium in the cells depended directly on its concentration in the medium, up to the lethal level. At high selenium concentrations (100–170 mg/l), S. platensis reduced Se(IV) up to Se(0). The latter was secreted onto the cell surface and into the cultural medium. The high concentrations of Na₂SeO₃ acidified the cytoplasmic pH as was measured by ³¹P-NMR spectroscopy. At the same time, the content of protein on a dry weight basis decreased and that of carbohydrates and lipids somewhat increased, just as was observed in S. platensis cells under other stress factors. In the presence of 20 mg/l Na₂SeO₃, the selenium content in the biomass increased by 20000 times as compared to that in the control cells, whereas the biochemical composition of biomass did not change. In this case, the selenium was incorporated almost completely in the protein fraction. The selenium concentration in this fraction increased more significantly when the sulfur content was lowered in the medium.     

Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO₂, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations4 mg l^-1, but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of¹⁴C-ring-LAS to ¹⁴CO₂ by P. chrysosporium was <1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain-length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition to-oxidation in the chain-shortening process.     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

Biosynthesis and secretion of recombinant human growth hormone in Pichia pastoris

Mature human growth hormone (hGH) cDNA was cloned by homologous recombination into the yeast Pichia pastoris genome. The hGH gene expression was placed under the control of the methanol-inducible alcohol oxidase 1 (AOX1) gene promoter and the Saccharomyces cerevisiae -factor signal sequence to direct the secretion of recombinant human growth hormone (rhGH) into the growth medium. O₂-limited induction of recombinant yeast strains in shake tubes with 3 ml of culture medium produced up to 11 mg rhGH l^–1, while high cell density cultures using a 2-l bioreactor produced about 49 mg rhGH l^–1 achieving 40% of total protein of the culture medium supernatant.     

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Response of Intact Cyanobacterial Cells and their Photosynthetic Apparatus to Cd2+ Ion Treatment

Intact cells of Synechococcus elongatus were treated with different concentrations (0.1 and 1.0 mM = Cd_0.1, Cd_1.0) of CdCl₂ for 24 h. Cd_0.1 treatment stimulated growth of the cell culture and chlorophyll (Chl) a concentration in the culture. Cd_1.0 inhibited both the above mentioned parameters. The oxygen evolving activity of intact cells (H₂O BQ) as well as of isolated thylakoid membranes, TM (H₂O DCPIP; H₂O PBQ + FeCy) decreased after 24 h of Cd_1.0 cultivation to 7 %. Photosystem 1 (PS1) activity was less sensitive to the effect of Cd²⁺ than PS2 activity. CdCl₂ concentration in cultivation media after 24 h of cultivation proved that the cyanobacterium cells take up these ions to a large extent from the cultivation medium. After 24 h of the Cd_1.0 treatment only 12 % of the amount of Cd²⁺ originally added to the cultivation medium was found. The ratio of external-antenna pigments, phycocyanin, and allophycocyanin to Chl increased approximately twofold with growing Cd²⁺ concentration in the cultivation medium. This ratio was found in both TM and dodecylmaltoside extracts.     

Enhancement of tryptophan production by Escherichia coli as an application of genetic engineering

Summary A feedback resistant trp operon plasmid that transformed a multiple mutant (trpR tnaA) of Escherichia coli was found to enhance remarkably the production of tryptophan in a bench-scale fermentor. 5.5 g of tryptophan was accumulated per litre of culture medium at 24th hr in batch. The productivity was 0.229 g/l/hr. This productivity is the highest among those ever reported by other workers. The recombinant plasmid (Tc^r Trp⁺ I^-) used was completely stable in each run when tetracycline was added by 10 g/l into the medium.     

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT₁) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT₁ receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT₁ receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT₁ receptor in a pure cell system, the recombinant human AT₁ receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT₁-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [K_D=0.15±0.02 nM] for [¹²⁵I][SAR¹, IIe⁸] angiotensin II (SIA). The pharmacological profile of ligands competing for [¹²⁵I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5-(-thio) triphosphate [GTPS]. Angiotensin II evoked a rapid efflux of⁴⁵Ca²⁺ from LhAT₁-D6 cells that was blocked by AT₁ receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT₁-D6 cell line provides a powerful tool to explore the human AT₁ receptor regulation.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG₁ which is reported stable, and IgG₁ production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM ₁ chain and cultured at nonpermissive temperature to enhance production of ₁. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6 recombinant human interleukin-6 - TGF- recombinant human TGF-₁ - X63.653-P3X63 Ag8.653 myeloma     

Drosophila melanogaster S2 cells are more suitable for the production of recombinant COX-1 than Trichoplusia ni BTI TN-5B1-4 cells

Recombinant human cyclooxygenase-1 (COX-1) was expressed from stably transfected Trichoplusia ni BTI TN-5B1-4 (TN-5B1-4) and Drosophila melanogaster S2 cells. Two kinds of recombinant COX-1 with molecular weights (MWs) of 68 and 74 kDa were expressed in the intracellular fractions of stably transfected TN-5B1-4/ COX-1 and S2/COX-1 cells, due to glycosylation. The recombinant COX-1 secreted to medium fractions has a MW of 72 kDa. Recombinant COX-1 in the intracellular fractions was purified to homogeneity using a one-step Ni-NTA affinity fractionation method. Recombinant COX-1 purified from TN-5B1-4/COX-1 and S2/COX-1 cells contained 11,389 and 33,850 Unit/mg of specific peroxidase activity, respectively. The maximum productions of intracellular recombinant COX-1 were 1.7 and 5.6 μg/10⁷ cells in the T-flask cultures of TN-5B1-4/COX-1 and S2/COX-1 cells, respectively. Taken together, our findings indicate that S2 cells can be more suitable system to produce recombinant COX-1, compared to TN-5B1-4 cells.