Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.     

Improving the baculovirus expression vector system with vankyrin‐enhanced technology

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex‐type N‐glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P‐vank‐1), which encodes an anti‐apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five^TM cells, and SfSWT‐4 cells, which can produce glycoproteins with complex‐type N‐glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N‐glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P‐vank‐1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT‐4 cells were infected with a vankyrin‐encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin‐expressing cells were combined with a vankyrin‐encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin‐encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1496–1507, 2017     

昆虫杆状病毒系统表达外源蛋白的糖基化

昆虫表达系统作为一类应用广泛的真核表达系统 ,具有与多数高等真核生物相类似的翻译后修饰的过程。但其生产的重组糖蛋白一般仅具有高甘露糖或寡甘露糖型糖链 ,难以生成复杂构型糖链成为该系统的缺陷之一。综述了目前昆虫杆状病毒系统表达外源蛋白的糖基化研究进展。     

糖组学研究技术及其进展

多细胞生物机体内,蛋白质糖基化是一个重要后修饰事件 . 蛋白质的糖链不仅仅是区别细胞种类的标志,且与众多的生物现象有关,如细胞发育、分化、形态、肿瘤转移、微生物感染等 . 糖组学的内容主要涉及单个个体的全部糖蛋白结构分析,确定编码糖蛋白的基因和蛋白质糖基化的机制 . 综述了糖组学的分离和结构鉴定技术及其最新进展 .     

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.     

Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.     

Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.     

贴壁培养方式中昆虫细胞的生长限制性因素研究

以昆虫细胞为宿主生产病毒杀虫剂或进行基因工程产品的开发,是动物细胞培养领域十分有吸引力的研究方向。近年来,昆虫细胞的体外培养尽管在培养条件的优化⑴、培养基的开发⑵以及工艺癍程的设计⑶等诸多方面取得了令人鼓舞的进展,但由于对影响细胞生长及代谢的各类限制性因素尚缺乏全面系统的了解,因而目前的技术水平还远不能设计出优化的培养系统以满足产业化的需要。众所周知,细胞的生长和产物的形成是多种环境因素和细胞内复杂代谢反应的综合结果,因此。对生长限制性因素的考察也应有全局观念,即：在重视生化环境(外因)对细胞培养过程影响的同时,也不能忽视细胞株自身性能(内因)对最终培养结果的影响。以往的研究〔4.5〕大多仅就个别营养限制性因素进行孤立地探讨,缺乏从内外因两方面对昆虫细胞培养过程的认识。最近,以微载体技术或堆积床技术贴壁培养昆虫细胞业已证明具有很大的发展前途〔6.7〕。为此．本文以贴壁培养的秋黏虫细胞(IPLB-Sf2l—AE)和粉蚊夜蛾细胞(BTI—Tn一5Bl-4)为研究对象,在考察环境限制性因素的同时,亦从细胞自身特点出发考察了不同细胞株的生长及代谢性能。研究结果可望为贴壁培养技术的深入应用以及培养过程的合理设计提供参考。     

Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins

The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis. The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide. The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase. These terminal sugars are linked to the alpha-mannose residues of the glycan cores. The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.     

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site‐Specific Glycan Structures

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site‐specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi‐5 cells. The data indicate that three of four potential N‐linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site‐dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation*

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.Androgen is important for the development, function, and proliferation of both normal and cancerous prostate cells (1). At the earliest stage of prostate cancer, prostate cancer cells are dependent on the presence of androgen, and androgen-deprivation therapy (ADT)¹ is used to treat prostate cancer (2). However, cells become androgen-independent as a result of androgen deprivation therapy, and they become more aggressive. This results in androgen-independent remission of prostate cancer (3). LNCaP and PC3 cell lines have been widely used as models of prostate cancer. LNCaP is an androgen-dependent cancer cell line, whereas PC3 is an androgen-independent cell line. The LNCaP cell line is less aggressive as compared with PC3 cells that have a high metastatic potential. LNCaP and PC3 cells have been previously studied by genomics and proteomics approaches to understand the mechanism(s) responsible for the aggressive and metastatic nature of prostate cancer (4–8).Post-translational modifications (PTMs) such as phosphorylation are important in the function of the androgen-dependent pathway. Androgen receptors bind to androgen and are then phosphorylated before translocating into the nucleus (3). However, protein PTMs cannot be directly inferred from gene expression. Glycosylation is an abundant PTM and most cell surface or secreted proteins are expected to be glycosylated (9). Glycosylation is one of the more complex PTMs because of the fact that different glycosylation machineries are present in different cells, multiple glycosylation sites exist on many glycoproteins and each glycosylation site can be modified by several different glycans (10, 11). Such microheterogeneity of glycan structures at each glycosylation site with different site occupancy significantly increases the structural diversity of each glycoprotein that is specific to the microenvironment of the cells where each glycoprotein is produced. Although these characteristics of protein glycosylation pose considerable challenges to the structural and functional analyses of glycoproteins, we expect that cell and cell microenvironment-specific glycoproteins differ according to the physiological and pathological states of the cells. Aberrant glycosylation is the result of alterations in glycosylation genes that may lead to the development of cancer. A systematic approach to analyze proteins, glycoproteins, and glycosylation is expected to permit the identification of the glycoprotein alterations that are specific to each cell state and aid the understanding of the functions of glycosylation because alterations in glycosylation can affect glycoprotein abundance or function (12, 13). A detailed analysis of glycoproteins in cancer cells with different functions is needed to understand tumor biology and how glycoproteins can function as therapeutic targets or diagnostic biomarkers (14, 15).In this study, a comprehensive proteomic and glycoproteomic platform was designed to investigate the differences in proteins, glycoproteins, and site-specific glycosylation forms of glycoproteins between LNCaP and PC3 cells (Fig. 1). To our knowledge, this is the first report to characterize glycoproteins with respect to protein abundance, glycosylation occupancy, and glycosylation heterogeneity at specific glycosites. These altered glycosylation patterns among proteins between LNCaP and PC3 cell lines have a significant potential to aid our understanding of the altered glycoprotein expression in prostate cancer cells, thus leading to novel specific methods to detect aggressive prostate cancer.Open in a separate windowFig. 1.Schematic representation of the workflow for the integrated analysis of glycosite-containing peptides, global protein expression, and intact glycopeptides. Proteins were obtained from LNCaP and PC3 cell lines followed by tryptic digestion and iTRAQ labeling. Labeled peptide samples were then combined and separated into two aliquots. One aliquot was enriched for glycosite-containing peptides using Solid Phase Extraction of Glycopeptides (SPEG) and the other aliquot was used for bRPLC separation followed by the analysis of global proteins and intact glycopeptides. Finally, peptides were analyzed using LC-MS/MS.     

Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins

Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.     

Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.     

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.