Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments

The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jemalong during the 8 weeks of culture.     

A proteome study of the proliferation of cultured Medicago truncatula protoplasts

A proteome study of the first five days of Medicago truncatula protoplast cultures was done to investigate molecular changes taking place during protoplast proliferation. A total of 1556 protein spots were analysed, of which 886 protein spots showed significant (p<0.005) changes in abundance at some time during the first five days of protoplast culture. Of the 886 significantly changing protein spots, 89 proteins were identified by MALDI-TOF MS. The majority of the identified proteins were part of four main cellular processes that may be involved in protoplast proliferation: energy metabolism, defence or stress response, secondary metabolism and protein synthesis and folding. The accumulation pattern of these proteins indicates extensive changes in the energy metabolism of the cells, accompanied by the activation of stress response pathways and modifications of the cell wall. In addition, seven PR10-like (pathogenesis related) proteins were identified. The accumulation pattern of these seven PR10-like proteins suggests that they could have a developmental role during protoplast proliferation.     

Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.     

Comparative proteomic analysis of matched primary and metastatic melanoma cell lines

Identification of the biochemical pathways involved in the transformation from primary to metastatic melanoma is an area under intense investigation. A 2DE proteomics approach has been applied herein to the matched patient primary and metastatic melanoma cell lines WM-115 and WM-266-4, respectively, to better understand the processes that underlie tumor progression. Image analysis between samples aligned 470 common gel spots. Quantitative gel analysis indicated 115 gel spots of greater intensity in the metastatic line compared with the primary one, leading to the identification of 131 proteins via database searching of nano-LC-ESI-Q-TOF-MS/MS data. This more than tripled the number of proteins previously shown to be of higher abundance during melanoma progression. Also observed were 22 gel spots to be of lesser intensity in the metastatic line with respect to the primary one. Of these gel spots 15 proteins could be identified. Numerous proteins from both groups had not been reported previously to participate in melanoma progression. Further analysis of one protein, cyclophilin A, confirmed that this protein is expressed at higher levels in metastatic melanoma compared with primary melanoma and normal fibroblasts. Overall, this study expands our knowledge of protein modulation during melanoma stages, and suggests new targets for inhibitor development.     

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

Fourier transform infrared (FT‐IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non‐secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody‐producing and non‐producing cell lines, and analyzed by FT‐IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC‐DFA), were applied to normalized FT‐IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C–O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT‐IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on‐line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432–442. © 2010 Wiley Periodicals, Inc.     

Combinatorial engineering of <Emphasis Type="Italic">ldh-a</Emphasis> and <Emphasis Type="Italic">bcl-2</Emphasis> for reducing lactate production and improving cell growth in dihydrofolate reductase-deficient Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating LDH-A in a dhfr ⁻ CHO cell line. When the dhfr ⁻ CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were reduced to 21–65% and 37–78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing rCHO cells for producing therapeutic proteins.     

Antisera inhibiting mammalian cell spreading and possible cell surface antigens involved

Antiserum against a rat neuronal tumor cell line (B103) has been prepared in rabbit by intravenous injection of live cells. This immune serum (anti-B103) precipitates a few cell surface proteins recognizable by two-dimensional gel electrophoresis as common radioiodinatable spots in 15 different rat neural cell lines and in mouse and rat fibroblast cell lines. The apparent molecular weight of one major common protein (II4) is estimated by SDS gel electrophoresis to be somewhere between 80,000 and 90,000 and another protein (I3) to be 120,000. These two proteins are consistently recognized in various cell lines by this antiserum. Furthermore, at a 1:20 dilution, this serum causes monolayer cells to round up usually within 0.5 h and detach from the plate within 3 h. It also inhibits spreading of freshly plated cells. These effects of the antiserum are reversible. Upon absorption of the antiserum with cells (e.g., absorbed with a glial cell line, B27), the serum no longer causes the rounding up of the monolayer cells, it does not inhibit cell spreading, and it does not immune-precipitate the two common proteins from the cell surface of various cell lines. Antisera against several other rat cell lines also precipitate the same common proteins (II4 and I3) from the cell surface and prevent cell spreading. These data suggest that the antibody acts first at the cell surface and then inhibits cell spreading or rounding up of spread cells. The consistent pattern of the immunoprecipitated cell surface proteins on the two- dimensional gel electrophoresis makes these two common surface proteins (II4 or I3 or both) possible candidates for target proteins to which the antibody binds. Thus, they may play a critical role in cell spreading.     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Proteome analysis of male gametophyte development in rice anthers

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.     

Effect of Bcl‐xL overexpression on lactate metabolism in chinese hamster ovary cells producing antibody

Previously, overexpression of anti‐apoptotic proteins, such as E1B‐19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl‐x_L, a well‐known anti‐apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody‐producing rCHO cell lines with regulated Bcl‐x_L overexpression (CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L) were established using the Tet‐off system. When cells were cultivated without Bcl‐x_L overexpression, the specific lactate production rate (q_Lac) of CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl‐x_L overexpression, in the absence of doxycycline, did not affect the q_Lac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose‐6‐phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl‐x_L overexpression either. Taken together, Bcl‐x_L overexpression showed no significant effect on the lactate metabolism of rCHO cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1594–1598, 2013     

Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level.     

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.Institut National Polytechnique de Lorraine, ENSAIA BP 172, 2 avenue de la forêt de Haye, 54505, Vandoeuvre Cedex France     

Functional expression of human pyruvate carboxylase for reduced lactic acid formation of Chinese hamster ovary cells (DG44)

To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21–39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.     

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.     

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology

Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5×10⁷ cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.     

Carbohydrate and amino acid metabolism during batch culture of a human lymphoblastoid cell line,BTSN6

This work presents data on the carbohydrate and amino acid metabolism of a lymphoblastoid cell line producing an IgG1 antibody. In static culture, it was observed that lactate levels were significantly lowered when the cells were cultured on galactose as a carbon source. The use of carbohydrate substitution may be useful in lowering lactate levels, if it is established that this component is toxic to the cells. In addition, carbohydrate substitution may be used to modify glycosylation patterns and hence pharmacokinetic properties of glycoproteins.The amino acids glutamine and tryptophan were shown to be limiting in batch culture on this medium (DR, a 1:1 mixture of DMEM and RPMI, with 4mM glutamine). Amino acids produced included alanine, proline and glutamate. Serine was consumed to exhaustion, which was followed by a depletion of extracellular glycine. Amino acid metabolism, specific antibody productivity and specific growth rate were shown to be functions of the inoculation density in stirred flask culture. The results have implications for the design of media for both low and high density antibody manufacture by these cell lines.     

Effect of Bcl-2 Expression on Hybridoma Cell Growth in Serum-Supplemented, Protein-Free and Diluted Media

Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher membrane intact index at increasing dilution steps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative proteomic analysis for hCTLA4Ig production in transgenic rice suspension cultures using two-dimensional difference gel electrophoresis

The new technology, two-dimensional difference gel electrophoresis (2D DIGE), uses fluorescent dyes to simplify the process of detecting and matching proteins between multiple gels by allowing for the separation of up to three separate protein samples within the same gel. In this study, recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4lg) was produced in transgenic rice suspension cell cultures and the intracellular proteins were analyzed by 2D DIGE. The highest level of hCTLA4Ig (25.4 mg/L) was obtained five days after induction. The intracellular proteins expressed at both the growth and induction culture stages were separated and analyzed using DeCyder software. At least 2,218 spots were detected with two-fold thresholds and 95% confidence. We found that 29 spots increased and 20 spots decreased in their intensities during the production of recombinant hCTLA4Ig. In addition, the 2D Western blot of hCTLA4Ig revealed that this fusion protein was expressed in a variety of isoforms.