共查询到20条相似文献,搜索用时 31 毫秒
1.
William Lowther Kevin Lorick Susan D. Lawrence Wen-Shuz Yeow 《Transgenic research》2012,21(6):1349-1357
Methods necessary for the successful transformation and regeneration of Aloe vera were developed and used to express the human protein, interferon alpha 2 (IFN??2). IFN??2 is a secreted cytokine that plays a vital role in regulating the cellular response to viral infection. Transgenic plants were regenerated from callus cultures initiated from zygotic embryos. Expression of the IFNA2 transgene in transformed plants was confirmed by RT-PCR and IFN??2 protein was detected by immunoblot analysis. Human A549 cells treated with transgenic aloe extracts for 6?h induced expression of the interferon stimulated gene 54, indicating activation of the IFN signaling pathway. The biological activity of the aloe produced IFN??2 was assessed using an antiviral assay with A549 cells treated with extracts from both the rind and pulp fractions of the shoot and subsequently infected with the lytic encephalomyocarditis virus. The highest level of activity attributable to recombinant IFN??2 was determined to be 625?IU/mg of total soluble protein (TSP) in the rind and 2,108?IU/mg TSP in the pulp. Two daughter plants that vegetatively budded during the course of this study were also confirmed to express IFN??2. These results confirm that Aloe vera is capable of expressing a human protein with biological activity, and that a secreted protein targeting the apoplast can be detected in the pulp fraction of the plant. 相似文献
2.
Abranches R Arcalis E Marcel S Altmann F Ribeiro-Pedro M Rodriguez J Stoger E 《Planta》2008,227(3):649-658
A number of recent reports suggest that the functional specialization of plant cells in storage organs can influence subcellular
protein sorting, so that the fate of a recombinant protein tends to differ between seeds and leaves. In order to test the
general applicability of this hypothesis, we investigated the fate of a model recombinant glycoprotein in the leaves and seeds
of a leguminous plant, Medicago truncatula. Detailed analysis of immature seeds by immunofluorescence and electron microscopy showed that recombinant phytase carrying
a signal peptide for entry into the endoplasmic reticulum was efficiently secreted from storage cotyledon cells. A second
version of the protein carrying a C-terminal KDEL tag for retention in the endoplasmic reticulum was predominantly retained
in the ER of seed cotyledon cells, but some of the protein was secreted to the apoplast and some was deposited in storage
vacuoles. Importantly, the fate of the recombinant protein in the leaves was nearly identical to that in the seeds from the
same plant. This shows that in M. truncatula, the unanticipated partial vacuolar delivery and secretion is not a special feature of seed cotyledon tissue, but are conserved
in different specialized tissues. Further investigation revealed that the unexpected fate of the tagged variant of phytase
likely resulted from partial loss of the KDEL tag in both leaves and seeds. Our results indicate that the previously observed
aberrant deposition of recombinant proteins into storage organelles of seed tissue is not a general reflection of functional
specialization, but also depends on the species of plant under investigation. This discovery will have an impact on the production
of recombinant pharmaceutical proteins in plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
Ga?tan Vanier Franziska Hempel Philippe Chan Michael Rodamer David Vaudry Uwe G. Maier Patrice Lerouge Muriel Bardor 《PloS one》2015,10(10)
Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans. 相似文献
4.
Kyung Il Kim Ha Young Chung Ki Hyun Yoo Jong-Hwa Park Hyun Ho Lee Yeon Ju Soek Ki Sung Ko Hyung Sik Kang Kyung Jin Lee Doo-Byoung Oh Young Hee Joung In Sik Chung 《Plant biotechnology reports》2012,6(3):233-242
We describe expression and characterization of recombinant human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Recombinant GA733-2-Fc/KDEL with a molecular mass of ~68?kDa was transiently expressed. The level of expression of GA733-2-Fc with ER retention signal KDEL (GA733-2-Fc/KDEL) in the expression vector system was 0.96% of total soluble proteins. Recombinant GA733-2-Fc/KDEL was purified using an affinity chromatography. Mice immunized with recombinant GA733-2-Fc/KDEL mounted a strong GA733-2-Fc/KDEL-specific serum antibody response. Vaccination of plant-derived recombinant GA733-2-Fc/KDEL regressed tumor volumes in BALB/c mice. The population of activated-T and NK-T cells increased notably in lymph node, spleen, and tumor-infiltrating lymphocytes derived from the tumor-regressed mice. Taken together, recombinant GA733-2-Fc/KDEL expressed in plants can be used as an effective experimental immunogen for research in cancer vaccine development. 相似文献
5.
Katarzyna Hnatuszko-Konka Piotr Łuchniak Aneta Wiktorek-Smagur Aneta Gerszberg Tomasz Kowalczyk Justyna Gatkowska Andrzej K. Kononowicz 《World journal of microbiology & biotechnology》2016,32(7):113
Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter. 相似文献
6.
Bellucci Michele Alpini Angelica Arcioni Sergio 《Plant Cell, Tissue and Organ Culture》2000,62(2):141-151
Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants
of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER
retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL
were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL
plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful
strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first
step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
7.
N. A. Matvieieva Yu. I. Kudryavets A. A. Likhova A. M. Shakhovskij N. A. Bezdenezhnykh E. Yu. Kvasko 《Cytology and Genetics》2012,46(5):285-290
This paper studies the biological activity of protein extracts of the Cichorium intybus L. and Lactuca sativa L. transgenic plants with the human interferon ??2b gene againsf vesicular stomatitis virus. Extracts from transgenic lettuce and chicory roots, which were obtained after A. rhizogenes-mediated transformation, had antiviral activity in the range 1620?C5400 IU/g of weight; extracts from leaves of chicory plants transformed by A. tumefaciens, up to 9375 IU/g. The dependence of the antiviral activity of plant extracts from roots or leaves on the vector used for plant transformation is shown. The extracts of plant roots obtained by A. rhizogenes-mediated transformation had antiviral activity; at the same time, such activity was absent in the extracts from leaves. 相似文献
8.
9.
Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins
or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus
like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection
also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein
production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph
of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant
protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative
western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability
and productivity of recombinant protein in BEVS/IC system. 相似文献
10.
Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times. 相似文献
11.
Zhijin Chen Dongmei Wang Yanguang Cong Jing Wang Junmin Zhu Jie Yang Zhen Hu Xiaomei Hu Yinling Tan Fuquan Hu Xiancai Rao 《Applied microbiology and biotechnology》2011,89(2):281-291
As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-??, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-?? products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9?Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of ??-factor by the STE13 protease. Expressed hPAB-?? was secreted into the culture medium at a level of 241.2?±?29.5?mg/L. Purified hPAB-?? with 95% homogeneity was obtained by 10?kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose? XL column. The two major protein species separated through a SOURCE? 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-?? with minimal inhibitory concentrations of 8?C64???g/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-??, and that expressed hPAB-?? in P. pastoris may be a potential antimicrobial agent against MRSA infection. 相似文献
12.
13.
Barbara Manconi Tiziana Cabras Alberto Vitali Chiara Fanali Antonella Fiorita Rosanna Inzitari Massimo Castagnola Irene Messana Maria Teresa Sanna 《Protein expression and purification》2010,69(2):219-225
This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase. 相似文献
14.
Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems 总被引:7,自引:0,他引:7
Wirth Sonia Calamante Gabriela Mentaberry Alejandro Bussmann Leonardo Lattanzi Mariano Barañao Lino Bravo-Almonacid Fernando 《Molecular breeding : new strategies in plant improvement》2004,13(1):23-35
15.
16.
The localization of soluble endoplasmic reticulum (ER) chaperones in the cell organelle is mediated by the C‐terminal KDEL (lysine, aspartic acid, glutamic acid and leucine) motif. This motif is recognized by the KDEL receptor, a seven‐transmembrane protein that cycles between the ER and cis‐Golgi to capture missorted KDEL chaperones from post‐ER compartments in a pH‐dependent manner. The KDEL receptor's target chaperones have a substantial role in protein folding and assembly. In this study, the gene expression level of KDEL receptor 1 shows a moderate upregulation during either ER stress or growth of Chinese hamster ovary (CHO) cells in batch culture, while the ER chaperones show higher upregulation. This might indicate the possibility of saturation of the ER retention machinery or at least hindered retention during late stage batch culture in recombinant CHO cells. KDELR1 is overexpressed in a monoclonal antibody‐producing CHO cell line to improve the intracellular chaperone retention rate in the ER. An increase in the specific productivity of IgG1 by 13.2% during the exponential phase, and 23.8% in the deceleration phase of batch culture is observed. This is the first study to focus on the ER retention system as a cell engineering target for enhancing recombinant protein production. 相似文献
17.
Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol
fungus Trichoderma harzianum. Calli from four primary transformants had high levels of endochitinase activity, like the plants from which they were derived.
Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions.
Western blots demonstrated the presence of the expected 40-kDa T. harzianum protein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the
fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration
was increased up to 34-fold by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions, probably
due to the secretion and concentration of endogenous enzymes.
Received: 6 May 2000 / Revision received: 6 September 2000 · Accepted: 14 September 2000 相似文献
18.
A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted
into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative
lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic
activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed
and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage
site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases
stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains
a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with
a synthetic substrate revealed a K
m of 10.55 ± 0.9 μM, a k
cat of 0.6 ± 0.01 s−1 and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAα1), a biopharmaceutical protein, that has proven difficult to produce in tobacco
BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced
in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical
proteins in plants and plant cells. 相似文献
19.
The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l−1, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1. 相似文献
20.
Sandra Luz Martínez-Hernández Daniel Cervantes-García Martín Muñoz-Ortega Liseth R. Aldaba-Muruato Victor M. Loera-Muro Jorge A. Ascacio-Martínez María de Jesús Loera-Arias Roberto Montes de Oca-Luna Javier Ventura-Juárez 《Biotechnology letters》2017,39(8):1149-1157