Expression and Identification of Mutated Osteoprotegerin in Culture Cells and Larvae of Silkworm

2004 ◽  
Vol 36 (5) ◽  
pp. 331-335 ◽  
Author(s):  
Hai-Long Xiao ◽  
Yong-Feng Jin ◽  
Yao-Zhou Zhang
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Recombinant Virus ◽  
Southern Blotting ◽  
Cultured Cells ◽  
Culture Cells ◽  
Sds Page ◽  
Baculovirus Transfer Vector ◽  
Hypocalcemic Effect ◽  
Bmn Cells

Abstract The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.

SEM and fluorescence imaging of callose deposition in root hair tips and suspension cultures of Streptanthus tortuosus

1990 ◽  
Vol 48 (3) ◽  
pp. 698-699
Author(s):  
K.S. Walters ◽  
R.D. Sjolund ◽  
K.C. Moore
Keyword(s):  
Cell Wall ◽  
Root Hair ◽  
Cultured Cells ◽  
Root Hairs ◽  
Callus Cultures ◽  
Callose Deposition ◽  
Culture Cells ◽  
Wall Structures ◽  
Ultraviolet Illumination ◽  
Callose Formation

Callose, B-1,3-glucan, a component of cell walls, is associated with phloem sieve plates, plasmodesmata, and other cell wall structures that are formed in response to wounding or infection. Callose reacts with aniline blue to form a fluorescent complex that can be recognized in the light microscope with ultraviolet illumination. We have identified callose in cell wall protuberances that are formed spontaneously in suspension-cultured cells of S. tortuosus and in the tips of root hairs formed in sterile callus cultures of S. tortuosus. Callose deposits in root hairs are restricted to root hair tips which appear to be damaged or deformed, while normal root hair tips lack callose deposits. The callose deposits found in suspension culture cells are restricted to regions where unusual outgrowths or protuberances are formed on the cell surfaces, specifically regions that are the sites of new cell wall formation.Callose formation has been shown to be regulated by intracellular calcium levels.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

scholarly journals Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

2006 ◽  
Vol 72 (8) ◽  
pp. 5225-5231 ◽  
Author(s):  
Emmanuel Frachon ◽  
Vincent Bondet ◽  
Hélène Munier-Lehmann ◽  
Jacques Bellalou
Keyword(s):  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Protein Production ◽  
Batch Cultures ◽  
E Coli ◽  
Working Volume ◽  
Versatile Tool ◽  
Labview Program ◽  
Medium Formulation ◽  
Conventional Medium

ABSTRACT A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2975-2986 ◽  
Author(s):  
Bisweswar Nandi ◽  
Ranjan K. Nandy ◽  
Amit Sarkar ◽  
Asoke C. Ghose
Keyword(s):  
Membrane Protein ◽  
Recombinant Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Protein ◽  
Secondary Structure Prediction ◽  
Sequence Data ◽  
Insertion Mutagenesis ◽  
Sds Page

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

2007 ◽  
Vol 4 (1) ◽  
pp. 63-67
Author(s):  
Yan Ke-Xia ◽  
Liu Jian-Jie ◽  
Wu Bin ◽  
Tang Xi-Biao ◽  
Cai Li-Jun ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Encoding ◽  
Lethal Challenge ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Protection Rate ◽  
Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

SCREENING OF CHLOROPLAST PROMOTERS FOR HEVEA BRASILIENSIS CHLOROPLAST TRANSFORMATION

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Anas Akmal Ag. Ismail ◽  
Zaima Azira Zainal Abidin ◽  
Zarina Zainuddin
Keyword(s):  
Recombinant Protein ◽  
Plant Species ◽  
Recombinant Proteins ◽  
Hevea Brasiliensis ◽  
Agronomic Traits ◽  
Chloroplast Transformation ◽  
Foreign Protein ◽  
Protein Accumulation ◽  
Nuclear Transformation ◽  
Single Chain

In recent years, the growth in the use of recombinant proteins has grown tremendously. With the aid of the advances in DNA recombinant biotechnology, molecular farming in plants has been applied to meet this increasing demand where plants have emerged as one of the most promising general production platforms for recombinant proteins. Hevea brasiliensis is one of the main commodities in Malaysia and widely cultivated species for commercial production of latex. This important plant has been used to express recombinant proteins such as a single-chain variable fragment (scFv) antibody against the coat protein of Streptococcus gordonii (an oral dental bacterium), human serum albumin and human atrial natriuretic. The genes that encodes for the recombinant proteins were targeted into the nucleus genome of Hevea but the proteins were expressed in low concentration. Generating transgenic plant using chloroplast transformation offers many advantages in comparison to nuclear transformation and many researches have been made to apply this strategy to enhance agronomic traits or produce recombinant protein in several plant species. Since chloroplast is highly polyploidy, it allows high-level foreign protein expression. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts, the aim of this study is to screen a number of chosen endogenous Hevea chloroplast promoters to drive the expression of the reporter gene, uidA for Hevea specific chloroplast transformation vector. Three promoters were chosen for this experiment which are; rbcL, psbA and rrn16 promoters. The putative regions of these promoters were derived from the chloroplast genome sequence of Hevea. Analyses of the three putative promoter regions using multiple sequence alignment with comparable regions from other plant species show significant sequence homology. Further analyses of the putative regions using in-vitro transcription are planned for future study. It is hoped that with the development of an optimized expression vector will allow high expression of valuable recombinant protein in the chloroplast of Hevea.

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

scholarly journals Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells.

1996 ◽  
Vol 7 (2) ◽  
pp. 225-232 ◽  
Author(s):  
L G Cao ◽  
Y L Wang
Keyword(s):  
Epithelial Cells ◽  
Mitotic Spindle ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Critical Role ◽  
Equatorial Region ◽  
Culture Cells ◽  
Cultured Epithelial Cells ◽  
Early Anaphase ◽  
Spindle Midzone

The interaction between the mitotic spindle and the cellular cortex is thought to play a critical role in stimulating cell cleavage. However, little is understood about the nature of such interactions, particularly in tissue culture cells. We have investigated the role of the spindle midzone in signaling cytokinesis by creating a barrier in cultured epithelial cells with a blunted needle, to block signals that may emanate from this region. When the barrier was created during metaphase or early anaphase, cleavage took place only on the sides of the cortex facing the mitotic spindle. Microtubules on the cleaving side showed organization typical of that in normal dividing cells. On the noncleaving side, most microtubules passed from one side of the equator into the other without any apparent organization, and actin filaments failed to organize in the equatorial region. When the barrier was created after the first minute of anaphase, cells showed successful cytokinesis, with normal organization of microtubules and actin filaments on both sides of the barrier. Our study suggests that transient signals from the midzone of early anaphase spindles are required for equatorial contraction in cultured cells and that such signaling may involve the organization of microtubules near the equator.

scholarly journals Purification and partial sequence analysis of plant annexins

1990 ◽  
Vol 270 (1) ◽  
pp. 157-161 ◽  
Author(s):  
M Smallwood ◽  
J N Keen ◽  
D J Bowles
Keyword(s):  
Higher Plants ◽  
Sequence Similarity ◽  
Exchange Chromatography ◽  
Plant Proteins ◽  
Peptide Fragments ◽  
Pectic Polysaccharide ◽  
Culture Cells ◽  
Fractionation Procedure ◽  
Sds Page ◽  
Tomato Suspension Culture

A fractionation procedure for annexins involving Ca2(+)-dependent binding to exogenous phospholipid was applied to tomato suspension culture cells. Two polypeptides (34 kDa and 35.5 kDa) were purified and separated from each other and from contaminant pectic polysaccharide by ion-exchange chromatography. After proteolytic digestion of SDS/PAGE-purified products, N-terminal sequencing of the peptide fragments revealed substantial similarity to sequences of known members of the annexin family characterized from a range of animal tissues. In particular, sequence similarity to the 70-amino acid-residue repeat region found in all annexins sequenced to date was present in both of the plant proteins. The data are discussed within the context of annexin involvement in Ca2(+)-mediated events in higher plants.

scholarly journals Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways

2006 ◽  
Vol 80 (19) ◽  
pp. 9608-9618 ◽  
Author(s):  
Harald Wodrich ◽  
Aurelia Cassany ◽  
Maximiliano A. D'Angelo ◽  
Tinglu Guan ◽  
Glen Nemerow ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Nuclear Import ◽  
Cultured Cells ◽  
Core Protein ◽  
Deletion Mapping ◽  
Nuclear Entry ◽  
Permeabilized Cells ◽  
Importin Α ◽  
Import Receptors

ABSTRACT Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

Sign in / Sign up

Export Citation Format

Share Document