水分胁迫对小麦幼苗及悬浮培养细胞中诱导蛋白表达的影响

丰抗8号小麦幼苗及成熟胚诱导的悬浮培养细胞在水分胁迫（－1．0MPa PEG6000）下,可溶性蛋白含量与蛋白组分变化有差异,幼苗可溶性蛋白含量高于对照,并随生长的延长呈降低趋势;悬浮培养细胞可溶性蛋白含量低于对照,且略有上升;复水后均可恢复对照水平,SDS－PAGE电泳及薄层扫描分析结果表明,幼苗受水分胁迫诱导,出现44．2kD蛋白亚基,该蛋白亚基含量可随胁迫时间延长上升,复水后消失,在正常条件下悬浮培养细胞中含有44．2kD蛋白亚基表达,轻度胁迫处理时,该蛋白亚基含量上升,对悬浮培养细胞进行水分胁迫,该蛋白则表现下降趋势,复水后又可上升。     

Sulfation of Rat Apolipoprotein E

The synthesis of a 37-kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo-E) was increased after sciatic nerve injury of the rat. When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury. In this report it is shown that similar time-course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury. This level was maintained up to at least 18 weeks after crush. Furthermore, two-dimensional electrophoresis revealed that the characteristic "trailing" of the protein is due to its sialylation, because it was reduced after neuraminidase treatment. This treatment, however, detected a neuraminidase-resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue. The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media. Incorporation studies with 35SO4 revealed that apo-E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures. Because macrophages are likely to be the major source of apo-E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases. As a result, sialic acid residues may be more susceptible to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

鸡输卵管上皮细胞瞬时表达系统(英文)

在开发利用生物反应器生产特定蛋白的研究中,若先用特异组织作原代培养,建立瞬时表达系统,对特定调控元件和融合基因进行分析,可大大缩短研究进程。本文首次报道用鸡输卵管上皮细胞原代培养, 建立瞬时表达系统的方法。输卵管细胞体外培养(Fig.1~3),在二、三周时间内仍能保持分泌卵清蛋白的功能。当分泌功能减弱时,若在培液中添加激素,一般一周后大部分细胞又可恢复分泌功能(Fig.4)。由于卵清蛋白是一种分泌蛋白,通过斑点免疫渗滤法可迅速简便的从培液中检测到(Fig.4)。为了验证培养细胞能否表达外源基因,在原代培养细胞中转染绿色荧光蛋白基因,可在细胞浆中显示绿色荧光(Fig.5),说明这是一个有效而又方便的检测调控元件的瞬时表达系统。     

Overexpression of the Leishmania amazonensis Ca2+-ATPase gene lmaa1 enhances virulence

A gene for a Ca2+-transporting ATPase (lmaa1) from the trypanosomatid parasite Leishmania (mexicana) amazonensis was overexpressed in two clones of L. amazonensis differing in their virulence. RNA and protein expression of the gene was increased in transfectants, as was the infectivity of transfectants versus parental types in both mouse and in vitro macrophage infection experiments. The virulence of the almost avirulent clone was enhanced such that it was more virulent than the parental 'virulent' clone. Growth of the parasites in culture as promastigotes, after isolation from mouse lesions, indicated that transfection led to improved survival of promastigotes during the stationary phase of culture. As it is in this culture phase that infective metacyclic forms develop, the key role of the Lmaa1 protein may be in metacyclogenesis. The protein may be important in the synthesis and trafficking of new proteins through the secretory pathway, as we demonstrate, using a green fluorescent protein hybrid and by immunofluorescence, that the Lmaa1 protein is located in the endoplasmic reticulum in promastigotes and amastigotes of L. amazonensis.     

天然可降解生物材料在组织工程中的应用研究进展

细胞培养支架材料是组织工程学的重要研究内容之一 ,是实现产业化的关键。天然可降解生物材料是细胞培养支架材料中的重要组成部分 ,目前用于细胞培养支架的天然可降解生物材料主要有多糖类和蛋白质类。多糖类主要包括壳多糖、透明质酸 ;蛋白质类主要包括胶原纤维蛋白和血纤维蛋白。     

液体培养蛹虫草虫草素和腺苷的代谢量

为提高虫草素和腺苷的代谢量,用高效液相色谱法作检测手段,以二者含量为检测指标,对液体培养的氮源种类、氮源水平、碳源水平、动态变化、培养体系的虫草素及腺苷含量和总量进行研究,结果表明:不仅蚕蛹粉,豆粕和豆粉也是很好的氮源;培养液以3%氮源、4%碳源为佳;振荡培养8d,培养液中虫草素总量是菌丝体中虫草素总量的6倍多;振荡培养7~9d,可使每瓶培养物的虫草素和腺苷总量达到10mg以上。     

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

Changes in the Expression of a Neuronal Surface Protein During Development of Cerebellar Neurones In Vivo and in Culture

The expression of the neurone-specific D2 protein changes both quantitatively and qualitatively during development in vivo and in cultures of cerebellar nerve cells. The total D2 content per unit protein shows a two-fold increase in vivo from birth to postnatal day 6, after which it declines progressively to about 50% of the maximal value. This increase can be accounted for by an immature form of the protein anodic D2 being preferentially expressed at the early stages of cerebellar development. After postnatal day 9 this form gradually switches to a mature form cathodic D2. This switch can be mimicked by neuraminidase treatment, suggesting a developmental loss of sialic acid from the D2 protein. In freshly isolated cells the total D2 content per unit protein is only 30% of that in the corresponding intact tissue from 8-day-old cerebella, but it increases rapidly during the first 8 days of culture to levels similar to those of the equivalent age in vivo. The switch from anodic D2 to cathodic D2 also occurs at a faster rate in culture, probably reflecting the culture conditions that favour differentiation. The changes in the expression of D2 during development of cerebellar nerve cells in culture suggest that anodic D2 is preferentially expressed on nerve cells that are proliferating, migrating, or in the initial stages of differentiation, whereas cathodic D2 is associated with differentiated neurones. The transition between the two forms appears to occur during the formation of interneuronal contacts.     

Characterization of proteins from human synovium and mononuclear leucocytes that induce resorption of cartilage proteoglycan in vitro

Both human synovial tissue in culture and lectin-stimulated mononuclear leucocytes produced a protein that induced proteoglycan resorption in explants of bovine nasal cartilage and human articular cartilage. On gel filtration the protein had Mr 16000-20000 and on isoelectric focusing its pI was 5.2-5.3. The protein corresponded to catabolin, which has previously been identified as a product of cultured porcine synovial tissue and mononuclear leucocytes. The action of partially purified human catabolin was not inhibited by cortisol, although the activity of the leucocyte supernatants from which it had been isolated was inhibited. For this reason it is not possible to be sure that the active factor detected in the bioassay of the crude leucocyte culture supernatants is in fact catabolin.     

Hypoxia Induces Synthesis of a Novel 22-kDa Protein in Neonatal Rat Oligodendrocytes

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of cryparin, a fungal hydrophobin

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.     

Secretion of Cryparin, a Fungal Hydrophobin

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.     

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.     

SPEX, a System for the Expression of Recombinant Proteins from Gram-Positive Bacterial Vectors

Using a conserved pathway for surface protein extrusion, a system has been developed for the expression and secretion of proteins from gram-positive bacteria. As proof-of-concept, theStreptococcus gordoniiChallis strain has been engineered to express a series of recombinant proteins fused to the conserved region of the M6 protein ofStreptococcus pyogenes.In the prototype surface protein expression system, the recombinant M6 protein is anchored to the surface ofS. gordoniicells expressing it. In order to overexpress the protein and easily purify it away from the bacteria, the protein was modified to enable it to be secreted into the medium. To accomplish this, a stop codon was introduced into the gene just prior to the anchor region using site-directed mutagenesis. Using enzyme-linked immunosorbent assays, it was possible to quantitate the amount of protein expressed using this system. With little or no optimization, 3 mg of protein per liter of culture was expressed and secreted into the medium of a bacterial culture grown to an OD₆₀₀equal to 1.0. This system should be broadly applicable for the expression and secretion of a variety of proteins (antigens, hormones, and enzymes) directly into the medium.     

Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli

Abstract Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi . This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD . The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains ix histidyl residues in its N-terminus.     

哺乳动物细胞灌流培养工艺研究进展

当前生物制药领域,由于成本压力、市场需求急剧波动以及生物仿制药的竞争日益激烈,现有的生物制造技术受到诸多挑战,生物技术公司越来越倾向于开发灵活、高效的创新型生产制造工艺。灌流培养作为当前哺乳动物细胞培养的重要工艺之一,不仅可以通过不断移出副产物和添加营养物来提供有利于细胞的稳定环境,以解决蛋白质量不稳定或者表达量偏低等问题,还可以通过提高单位体积产率来优化产能利用率并提高生产效率。通过系统介绍灌流培养用于哺乳动物细胞培养的研究进展,为其进一步开发与应用提供参考。     

Thialysine utilization by thialysine resistant CHO cells

Thialysine resistant CHO cells utilize thialysine added to the culture medium to a lesser extent than the parental cells. Thialysine is utilized in protein synthesis and it is incorporated into proteins in place of lysine. The parental strain substitutes up to 11% of protein lysine by thialysine, while variant cells substitute a maximum of 5% of protein lysine.     

Production of extracellular nitrogen-containing components by Azotobacter vinelandii fixing dinitrogen in oxygen-controlled continuous culture

Up to 5% of the totally fixed nitrogen and up to 11% of the totally formed protein were detected in cell-free culture fluids of diazotrophic Azotobacter vinelandii growing in continuous culture. The actual amounts of nitrogen and protein changed with ambient oxygen concentrations in the growth medium. While with whole cells the ratio of nitrogen per protein remained constant it varied with the extracellular moiety with changes of the oxygen concentration. Analyses of the cell-free culture fluid revealed the presence of a typical polypeptide pattern with a predominant 60 K polypeptide, significant amounts of ammonia at low oxygen concentrations as well as glutamic acid in both monomeric and polymeric form. Steady state levels of these extracellular components varied independently of each other with changes of the ambient oxygen concentration.Dedicated to Prof. G. Drews on occasion of his 60th birthday