Single framework recombinant antibody fragments designed for protein chip applications

High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low abundance proteins. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused on recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragments that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact, a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified. Taken together, designed scFv fragments based on a single molecular scaffold, readily accessible in large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG₁) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.Key words: cell-free protein synthesis, Fab antibody, aglycosylated antibodies, HER2, trastuzumab     

单链抗体展示系统研究进展

单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

Efficient hydrolysis of hemicellulose by a Fusarium graminearum xylanase blend produced at high levels in Escherichia coli

A Fusarium graminearum-based enzyme blend for the efficient hydrolysis of hemicellulose, a crucial step for competitive bioethanol production, is described. The heretofore-uncharacterized endo-1,4-beta-xylanase (XylD), 1,4-beta-xylosidase (XyloA), and bifunctional xylosidase/arabinofuranosidase (Xylo/ArabA) were produced at high levels in Escherichia coli (10-38 mg/l). They displayed compatible pH and temperature-dependences, allowing their utilization for simultaneous substrate digestions. Monosaccharide analysis indicated a strong positive synergism between the enzymes during the degradation of oat spelt xylan. Two units of each protein catalyzed the release of 61% and 15% of the total amount of available d-xylose and l-arabinose, respectively, in only 4 h. The detailed cooperative mechanism of the three hydrolases was elucidated by polysaccharide analysis using carbohydrate gel electrophoresis (PACE) and the enzymes were shown to be suitable for the partial hydrolysis of pretreated crude plant biomass.     

Presence of an alpha-amylase isozyme with high temperature optima in the wheat variety tolerant to high temperature at juvenile plant stage

In the present study α-amylase was partially purified from detached grains of five day old seedlings of two wheat (Triticum aestivum L.) varieties, showing differential responses to high temperature stress at seedling stage. A three step purification via ammonium sulphate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150 was employed. A single α-amylase was detected in the high temperature sensitive PBW-175 variety, while two isozymes namely, α-amylase-1 and α-amylase-2 were obtained in the relatively tolerant WL-711 variety. The pH optima of the three α-amylases were in 5.0–5.5 range and comparable to the cereal amylases. The temperature optima of PBW-175 α-amylase and α-amylase-1 of WL-711, which appeared to be the major isozyme of the variety, were same at 45 °C and also comparable to cereal amylases. On the other hand the optimum temperature for α-amylase-2 was high at 70 °C, which is unusual and not reported earlier for cereal amylases. The Km of PBW-175 α-amylase was lower than the Km values of WL-711 isozymes, this was well co-related with an overall high α-amylase activity detected in the detached grains of five day old seedlings of PBW-175 compared to WL-711. However WL-711 variety showed a better inherent seedling growth, vigour and EUE than PBW-175, may be because it had two α-amylase isozymes which could compensate for the higher enzyme activity detected in PBW-175. Moreover, the presence of α-amylase-2 in the grain of WL-711 having temperature optima of 70 °C, possibly rendered its seedlings tolerant to HS of 50 °C, while the seedlings of PBW-175 succumbed to this temperature shock.     

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.     

提高小麦品种稳产性、适应性的后代选育策略研究

西农８７２７的成功选育为稳产性、适应性品种选育提供了借鉴,本试验以西农８７２７小麦品种选育过程中稳定的５个高代选系为材料,采用不同环境条件、不同试点的试验设计,运用Ｅberhart-Rusell分析方法,推算出参试品系的稳定性参数,分析和比较它们的产量稳定性及环境适应性,并据此筛选出适合性强、稳产性好的优良品系,在此基础上,提出了高代出圃选系后代选育的探讨性见解,为今后高产、稳产、广适性小麦新品种的选育提供了一定的方法参考。     

Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris

Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1). Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.     

Test of semiochemicals and a resistant wheat variety for Russian wheat aphid management in South Africa

Abstract:  Aphid behaviour-modifying semiochemicals were tested against Russian wheat aphid Diuraphis noxia in South African wheat. Volatile substances from plant essential oils, methyl salicylate, 1,8-cineole and menthol were tested in the laboratory and field in combination with the D. noxia -susceptible wheat variety Betta and the resistant variety Elands. All three substances were repellent to D. noxia in olfactometric tests. Diuraphis noxia settled less on Elands plants that had been exposed to the volatiles, whereas the effect of the volatiles on D. noxia settling on Betta was less obvious. A slow-release pellet formulation was used to apply semiochemicals in wheat in replicated plot field trials in 2004 and 2005. In 2004, semiochemicals reduced aphid populations in Elands, but led to increased aphid populations in Betta. Further, the impact of the chemicals on aphid numbers and grain quality (thousand grain weight) varied according to plant variety, indicating an interaction between semiochemicals and plant resistance/variety.     

Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris

The potential of recombinant antibody fragments is likely to be fulfilled only if they can be produced routinely at high concentrations. We have compared the ability of Escherichia coli and Pichia pastoris to produce functional recombinant single chain antibody (scAb) fragments. Two scAb fragments were expressed, an antihuman type V acid phosphatase (TRAP) and an anti-Pseudomonas aeruginosa lipoprotein I. We report here that, while expression from P. pastoris resulted in a significantly increased level of expression of the anti-TRAP scAb compared to E. coli, neither fragment was able to bind its target antigen as well as the bacterial product.     

冬小麦高产高效群体的年际间稳产性能

以冬小麦品种‘泰农18’为试验材料,设置传统农民管理习惯的农户模式(T₁)、不计肥水和人工等投入以获得高产的高产模式(T₂)和优化种植密度、播期、肥水管理运筹的高产高效模式(T₃)3种种植模式,于2012年10月—2016年7月连续4个小麦生育季,研究了不同种植模式小麦主茎与分蘖成穗特性、不同年份的光合有效辐射截获、利用、光合产物分配等光能利用特性和产量差异,结合年际间的光、温、水等生态因子分布差异,分析了高产高效群体的稳产性能.结果表明: 冬小麦不同生育时期内的光、温、降雨等生态因子的总量和各生育时期分布存在较大变异.T₁、T₂、T₃模式主茎穗的比例分别为38.9%、58.7%、66.9%,表明T₁、T₂、T₃模式在群体结构构建上分别是以分蘖穗为主、主茎穗和分蘖穗并重、主茎穗为主.T₂模式的生物量和产量最高,T₁最低,T₃模式自孕穗期之后,尤其是在开花14 d后仍能维持较高的叶面积指数、光能截获率和截获量、群体光合速率,并具有较高的干物质分配能力,使其在干物质积累相对偏低的情况下仍可获得高产.T₃模式花后的叶面积指数、光能截获率和截获量以及光合速率等的年际间极差、标准差、变异系数均小于T₁和T₂模式,由此也保证了T₃模式生物产量的稳定.相关分析表明,以主茎成穗为主的T₃种植模式能够有效应对年际间气候变化并实现稳产,关键是具有年际间稳定的生物产量.     

Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments

A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen-binding specificity of the parental mAb.     

Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures

An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the culture supernatant (1 mg l^–1) and in cells (5 mg kg^–1). A simple one-step scFv purification was developed using immobilized metal ion affinity chromatography.     

Arbuscular mycorrhizas enhance nutrient uptake in different wheat genotypes at high salinity levels under field and greenhouse conditions

Since most experiments regarding the symbiosis between arbuscular mycorrhizal (AM) fungi and their host plants under salinity stress have been performed only under greenhouse conditions, this research work was also conducted under field conditions. The effects of three AM species including Glomus mosseae, G.?etunicatum and G.?intraradices on the nutrient uptake of different wheat cultivars (including Roshan, Kavir and Tabasi) under field and greenhouse (including Chamran and Line 9) conditions were determined. At field harvest, the concentrations of N, Ca, Mg, Fe, Cu, and Mn, and at greenhouse harvest, plant growth, root colonization and concentrations of different nutrients including N, K, P, Ca, Mg, Mn, Cu, Fe, Zn, Na and Cl were determined. The effects of wheat cultivars on the concentrations of N, Ca, and Mn, and of all nutrients were significant at field and greenhouse conditions, respectively. In both experiments, AM fungi significantly enhanced the concentrations of all nutrients including N, K, P, Ca, Mg, Mn, Cu, Fe, Zn, Na and Cl. The synergistic and enhancing effects of co-inoculation of AM species on plant growth and the inhibiting effect of AM species on Na(+) rather than on Cl(-) uptake under salinity are also among the important findings of this research work.