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.     

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.     

Expression and characterization of recombinant beta-secretase from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Gal beta1,4-GlcNAc alpha 2,6-sialytransferase

Beta-Secretase (betaSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Gal beta1,4-GlcNAc alpha 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 57 to 59 k Da. A lectin blot analysis indicated that recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant beta-secretase from Tn5B1-4 beta SEC/GalT-ST cells had a lower isoelectric point than beta-secretase from control Tn5B1-4 betaSEC cells (Tn5B1-4 cells transformed only with beta-secretase cDNA). The enzyme activity of recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4 betaSEC cells. The concentrations at half-maximum inhibition (IC(50)) values estimated from inhibition analyses using purified beta-secretases from Tn5B1-4/betaSEC and Tn5B1-4/betaSEC/GalT-ST cells were 32 and 290 nM, respectively.     

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.     

Effect of Trichoplusia ni BTI-Tn 5B1-4 cell density on human secreted alkaline phosphatase production

Summary The effect of Tn 5B1-4 cell density at infection on the production of human secreted alkaline phosphatase (SEAP) was investigated using a split-flow air-lift bioreactor. Volumetric productivities of SEAP were highest when Tn 5B1-4 cells were infected at about 3 × 10⁵ cells/cm². Cell-to-cell contact inhibition is an important factor responsible for the reduced protein production at high cell densities. Other factors such as oxygen or nutrient limitation, or build up of metabolic inhibitors do not appear to be as important.     

Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates.

The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.     

印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理,本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性,处理后3 d,其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL,印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现,印楝素A和印楝素B处理可导致细胞变形,贴壁能力下降,并出现明显空泡,印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明,印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大,印楝素A处理细胞体积增大程度显著高于印楝素B;印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位,1.25 μg/mL印楝素A和印楝素B处理后3 d,细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%,印楝素A的影响显著高于印楝素B。荧光显微镜观察发现,印楝素对BTI-Tn-5B1-4细胞核具有明显影响,印楝素B的影响明显高于印楝素A,印楝素B处理后,细胞核受损细胞数更多,受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异,本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。     

DnaK/DnaJ-assisted recombinant protein production in Trichoplusia ni larvae

The DnaK/DnaJ Escherichia coli chaperone pair, co-produced along with recombinant proteins, has been widely used to assist protein folding in bacterial cells, although with poor consensus about the ultimate effect on protein quality and its general applicability. Here, we have evaluated for the first time these bacterial proteins as folding modulators in a highly promising recombinant protein platform based on insect larvae. Intriguingly, the bacterial chaperones enhanced the solubility of a reporter, misfolding-prone GFP, doubling the yield of recombinant protein that can be recovered from the larvae extracts in a production process. This occurs without negative effects on the yield of total protein (extractable plus insoluble), indicative of a proteolytic stability of the chaperone substrate. It is in contrast with what has been observed in bacteria for the same reporter protein, which is dramatically degraded in a DnaK-dependent manner. The reported data are discussed in the context of the biotechnological potential and applicability of prokaryotic chaperones in complex, eukaryotic factories for recombinant protein production.     

粉纹夜蛾细胞中Bt Cry1Ac选择抗性的研究及离体品系与毒素结合的比较

采用Bt Cry1Ac活性毒素对粉纹夜蛾BTI-Tn-5B1细胞进行56代筛选后获得了抗性比为1280倍的抗性细胞。ELISA检测表明抗性细胞总蛋白和膜蛋白结合的Cry1Ac数量都少于敏感细胞。配体结合Western杂交实验显示：抗性细胞和敏感细胞的膜蛋白与总蛋白都有5条电泳迁移率相同的毒素结合多肽带,其分子量分别为207,158．5,118.8,72,38．5 kD;抗性细胞的118．8和72kD的阳性带比敏感细胞的略弱,这可能与抗性的形成相关。     

Rapid initiation of suspension cultures of Trichoplusia ni insect cells (TN 5B-1-4) using heparin

The High Five or Trichoplusia ni 5B-1-4 cell line is more productive for heterologous proteins on a per cell basis than other commonly used insect cells lines. Adapting the TN 5B-1-4 cells to suspension culture and maintaining them as suspensions, which is usually difficult due to severe aggregation, but could be achieved by adding heparin at 100IU/mL. Unfortunately, the heparin inhibited the infection process even at very high values of multiplicity of infection (10 to 20 infectious particles per cell). This was reflected by the continued growth of the cells after virus addition. Since considerable cell growth was obtained after infection in the presence of 100IU/mL of heparin, final cell densities were higher and per cell production was lower most likely due to nutrient limitation.     

Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.     

Production of recombinant human osteoprotegrin from Trichoplusia ni cells and Bombyx mori larvae

Human osteoprotegrin (OPG) and its truncated mutant OPG-280 and lengthened mutant OPG-Fc were constructed and successfully expressed in Trichoplusia ni cells and Bombyx mori larvae. Native SDS-PAGE and Western blot analysis revealed that OPG-Fc is present as a homodimer in Tn cells or B. mori larvae compared with OPG and OPG-280. Furthermore, the hypocalcemic effect assay showed that truncation of the C-terminal 100 residues OPG does not abolish the biological activity and Fc can be helpful in forming the OPG homodimer with improved biological activity.     

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.     

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).     

Production of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Drosophila melanogaster S2 cells. Recombinant endostatin expressed in the stably transformed S2 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni²⁺ affinity fractionation method. The purification yield was approximately 4 g from the medium fraction of 8 ml cultures of stably transformed S2 cells. In a T-flask, the stably transformed S2 cells produced 24 mg recombinant endostatin/l at 7 days post-induction by 0.5 mM CuSO₄. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the S2 cells produced up to 13 mg recombinant endostatin/l.     

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.     

Aflatoxin B1: Toxicity,bioactivation and detoxification in the polyphagous caterpillar Trichoplusia ni

Trichoplusia ni caterpillars are polyphagous foliage‐feeders and rarely likely to encounter aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB1, we evaluated the toxicity of AFB1 to T. ni caterpillars at different developmental stages and found that AFB1 tolerance significantly increases with larval development. Diet incorporation of AFB1 at 1 μg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3 μg/g AFB1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases (P450s), reduced the toxicity of AFB1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as flavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFB1 toxicity, suggesting that P450s are also involved in AFB1 detoxification in T. ni.     

Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009