Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures

The effects of culture media on the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) and intracellular protein expression patterns were investigated in transgenic rice cell suspension cultures. Using comparative proteomic analysis, changes in the intracellular proteome in different culture media were identified. Culture media were found to be an important factor for the production of the recombinant target protein in this expression system, which was under the control of the rice α-amylase 3D (RAmy3D) promoter. In terms of hCTLA4Ig production, the N6 medium produced a 3.7-fold higher level of protein than the AA medium. In addition, the N6 medium provided better protein stability and cell viability. In the intracellular proteome analysis, we identified eight proteomes that were differentially expressed. These results could provide valuable information for the improvement of cell growth and target protein production.     

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.     

Fed-batch cultivation of transgenic rice cells for the production of hCTLA4Ig using concentrated amino acids

RAmy3D promoter is capable of expressing high levels of recombinant proteins in response to the depletion of sugar in transgenic rice cell suspension cultures. For this reason, it is necessary to change the growth medium into sugar-free production medium to produce the target protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), using the inducible RAmy3D promoter. Since the two-stage culture is a complex process to perform in large-scale, a fed-batch method was evaluated with the addition of concentrated amino acids before the depletion of sugar to induce hCTLA4Ig production. This fed-batch culture was found to be effective and the production of hCTLA4Ig was enhanced up to 1.2-fold compared to that of two-stage cultures with medium exchange. In addition, when this fed-batch culture was performed in a 15-l stirred-tank bioreactor, maximum hCTLA4Ig level was 76.5 mg l⁻¹ at day 10.     

Assessment of Recovery Medium for Production of hCTLA4Ig after Cryopreservation in Transgenic Rice Cells

A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.     

Skin-specifically transgenic expression of biologically active human cytoxic T-lymphocyte associated Antigen4-Immunoglobulin (hCTLA4Ig) in mice using lentiviral vector

Xenogeneic skin, especially porcine skin, has already been used to cover large wounds in clinic practice of wound care. Our previous data showed that transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic burn wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong its survival in xenogeneic wounds for coverage. Lentiviral transgenesis provides an extremely efficient and cost-effective method to produce transgenic animals. However, tissue-targeted transgenic expression of biologically functional protein by lentiviral transgenesis is rarely reported. In this work, a recombinant lentiviral vector (LV), named FKCW in this article, was constructed by inserting a skin-specific hCTLA4Ig expression cassette consisting of keratin 14 (K14) promoter, hCTLA4Ig coding sequence and an intronic fragment. Its efficacy for transgenesis and skin-specific expression of bio-active hCTLA4Ig protein was tested using mice as models. The LV FKCW was readily to be packaged and concentrated to high titres (1.287-6.254 × 10(9) TU/ml) by conventional lentivirus package system. Using eggs collected from only five mated females having been subjected to conventional super-ovulation treatment, 8 hCTLA4Ig transgenic founder mice were generated with the concentrated FKCW vector, and transgenic founder per injected and transferred egg was 6.3%, which was nearly 9-fold higher than that for DNA micro-injection with a similar transgene construct in our previous work. The lentiviral transgenic hCTLA4Ig exhibited strictly skin-specific expression at a level comparable to or even slightly higher than that of transgenic hCTLA4Ig delivered by micro-injection in a similar cassette. Lentiviral transgenic hCTLA4Ig protein remarkably suppressed human lymphocyte proliferation in vitro to a degree comparable to that of commercially purchased purified hCTLA4Ig protein with defined activity at similar concentrations. Besides, lentiviral hCTLA4Ig transgenic mouse skin grafted into rat burn wounds exhibited remarkably extended survival compared to wild-type skin of the same strain (13.8 ± 3.8 vs. 6.8 ± 3.0 days), indicating that lentiviral transgenic hCTLA4Ig did inhibit immune rejection against xenogeneic skin graft in vivo. These results laid down the foundation to further efficiently generate transgenic pigs skin-specifically expressing bio-active hCTLA4Ig by lentiviral transgenesis, and provided a demonstration that transgenic animals with tissue-targeted expression of biologically functional protein can be efficiently produced using LV.     

Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four high-abundant protein removal methods were observed. Antibody-based albumin removal and high-abundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum.     

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig^P) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig^M), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig^P and hCTLA4Ig^M had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig^P and hCTLA4Ig^M had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ, but did not suppress the expression of macrophage-derived cytokines, including TNF-α and IL-1β, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig^P is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig^M.     

Application of anoxia with glucose addition for the enhanced production of hCTLA4Ig in transgenic rice suspension cell cultures

To enhance the production of hCTLA4Ig in transgenic rice suspension cell cultures, anoxic conditions were applied during the production phase. Under the anoxic conditions in sugar-depleted media, cell viability was reduced rapidly and protease activity increased compared to aerobic conditions. However, the maximum production level of hCTLA4Ig with sugar-depleted anoxic conditions was the same as that in aerobic conditions. In addition, the production of hCTLA4Ig under anoxic conditions reached a peak 2 days earlier than that in aerobic conditions. Addition of 30 mM glucose at the production phase under anoxic conditions markedly improved cell viability. A viability level over 65% could be maintained for more than 30 days. Repression of the RAmy3D promoter by residual sugar in the production of hCTLA4Ig was not observed under anoxic conditions with 30 mM glucose. In addition, the production periods of hCTLA4Ig was extended up to 30 days and the maximum production level of hCTLA4Ig under anoxic conditions was 2.1-fold higher. Therefore, anoxic conditions could be used for the enhanced production of hCTLA4Ig in transgenic rice cell cultures.     

采用定量蛋白质组学技术筛选小鼠肝癌淋巴道转移相关蛋白

淋巴道转移是上皮来源恶性肿瘤转移的早期阶段,其发生机制不清,一直是肿瘤学研究面临的难题,为寻找淋巴道转移相关蛋白,以一对来源于同一亲本细胞,且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株为研究对象,其中Hca-F为高淋巴道转移力细胞株,Hca-P为低淋巴道转移力细胞株,采用定量蛋白质组学技术——荧光差异双向凝胶电泳,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白质.经DeCyde软件分析,共得到163个有统计学差异的蛋白质点,选择2倍以上的差异性蛋白质点23个,经质谱鉴定得到17个蛋白质,在Hca-F中高表达的蛋白质有7个:转羟乙醛酶、波形蛋白、肌酸激酶(脑)、膜联蛋白7、膜联蛋白5、烯酰辅酶A水合酶1(过氧化物酶体)、核内异质核糖核蛋白A2/B1异构体1.而在Hca-F中低表达的蛋白质有10个:真核翻译延长因子2、Ero1样蛋白、乙醛脱氢酶2(线粒体)、苹果酸盐脱氢酶2(NAD)、β-内酰胺酶2、谷胱甘肽S转移酶"1、泛素C末端水解酶同工酶L3、内质网蛋白29(前体)、溶血磷脂酶1、微管不稳定蛋白.这些差异性蛋白质的功能涉及到代谢、蛋白质分泌、蛋白质结合、核苷酸结合,钙离子结合、凋亡和调节生长等过程.对这些蛋白质功能的进一步验证,将有助于解析肿瘤淋巴道转移的分子机制.     

2-D DIGE analysis of the budding yeast pH 6-11 proteome in meiosis

Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Highly sensitive saturation labeling reveals changes in abundance of cell cycle‐associated proteins and redox enzyme variants during oocyte maturation in vitro

Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2‐D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4–7 images and 1291 spots in pH 6–9 images. Thirty‐eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano‐LC‐MS/MS analysis. The ten spots which could be unambiguously identified include the Ca²⁺‐binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14‐3‐3 ?, elongation factor‐1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin‐3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.     

Two-dimensional fluorescence difference gel electrophoresis analysis of spermatogenesis in the rat

The molecular mechanisms underlying normal and pathological spermatogenesis remain poorly understood. We compared protein concentrations in different germ cell types to identify those proteins specifically or preferentially expressed at each stage of rat spermatogenesis. Crude cytosolic protein extracts and reversed-phase HPLC prefractionated cytosolic extracts from spermatogonia, pachytene spermatocytes, and early spermatids were subjected to two-dimensional difference gel electrophoresis (2-D DIGE). By comparing gels and carrying out statistical analyses, we were able to identify 1274 protein spots with relative abundances differing significantly between the three cell types. We found that 265 of these spots displaying highly differential expression (ratio > or = 2.5 between two cell types), identified by mass fingerprinting, corresponded to 123 nonredundant proteins. The proteins clustered into three clades, corresponding to mitotic, meiotic, and post-meiotic cell types. The differentially expressed proteins identified by 2-D DIGE were confirmed and validated by western blotting and immunohistochemistry, in the few cases in which antibodies were available. 2-D DIGE appears a relevant proteomics approach for studying rat germ cell differentiation, allowing the establishment of the precise expression profiles for a relatively large number of proteins during normal spermatogenesis.     

Adsorptive loss of secreted recombinant proteins in transgenic rice cell suspension cultures

Adsorptive loss of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic rice cell suspension cultures was investigated using glass flasks, plastic flasks, disposable vessels, and stainless steel vessels. When hCTLA4Ig was added to the glass flasks containing sterile AA medium, a rapid decrease in the concentration of hCTLA4Ig, independent on pH, was observed resulting in more than 90% of the protein loss within 1 h due to the surface adsorption. When the same experiments were performed on four different types of culture equipments mentioned above, the lowest adsorption level was observed in the plastic flasks and the highest level was observed in the glass flasks. The use of the plastic flasks retarded the adsorptive loss of hCTLA4Ig at the early stage of the protein production. There was a significant increase in the production of hCTLA4Ig when the flasks were coated with bovine serum albumin. However, the spike test of purified hCTLA4Ig at two different concentrations of 15 and 100 mg L⁻¹ in 500-mL spinner flasks confirmed that the amount of hCTLA4Ig adsorbed was dependent on the surface area of the flasks but not on the concentrations. In conclusion, although the protein adsorption affected the total amount of the protein yielded to some extent, it could be regarded as a minor factor in transgenic plant cell cultures with higher titer.     

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.     

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.     

AspC基因导入前后E.coli BL21蛋白质组的解析

为了考察表达天冬氨酸转氨酶工程菌在转基因前后蛋白质水平的差异变化,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对转基因前后的大肠杆菌（E．coli BL21）的总蛋白进行分离,银染、显色后,使用2D蛋白质图象分析系统Image Master 2D Platinum 5．0和SWISS-2D PAGE蛋白质组数据库对双向电泳图谱进行分析,识别了近600个蛋白点,比较分析了与苯丙氨酸合成途径相关的关键蛋白的差异,初步探讨了AspC基因的导入后大肠杆菌蛋白质水平的精细调控。