Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Comparative evaluation of recombinant protein production in different biofactories: the green perspective.

ABSTRACT: In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms.

SUBMITTER: Merlin M

PROVIDER: S-EPMC3972949 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Comparative evaluation of recombinant protein production in different biofactories: the green perspective.

Merlin Matilde M Gecchele Elisa E Capaldi Stefano S Pezzotti Mario M Avesani Linda L

BioMed research international 20140312

In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of fou ...[more]

PMID: 24745008

Similar Datasets

Comparative analysis of different biofactories for the production of a major diabetes autoantigen.

Project description:The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 μg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 μg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.

| S-EPMC3951962 | biostudies-literature

Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites.

Project description:Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription-translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

| S-EPMC8832058 | biostudies-literature

Multiplex secretome engineering enhances recombinant protein production and purity

Project description:Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

2020-03-19 | GSE144624 | GEO

Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in <i>E. coli</i>.

Project description:Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.

| S-EPMC11230288 | biostudies-literature

Generation of recombinant vaccinia viruses via green fluorescent protein selection.

Project description:We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase-deficient (Tk(-)) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk(-) rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.

| S-EPMC2925039 | biostudies-literature

Amino acid supplementation for enhancing recombinant protein production in E. coli

Project description:Amino acid starvation during recombinant protein production (RPP) induces metabolic stress to cellular host which results in reduced productivity of the recombinant bioprocess. In present study, supplementation of amino acids in a chemically defined medium showed several folds increase in recombinant product titers in E. coli BL21 DE3 strain. To understand, the effect of amino acid supplementation in alleviating cellular stress during RPP a deep insight of cellular physiology must be studied. Here, we performed transcriptomic analysis of E. coli expressing a recombinant protein in two different conditions: with (5 mM of all 20 amino acids) as test and without supplementation of amino acids as control. RNA-seq data revealed downregulation of several genes associated with stress in test culture confirming the critical role of amino acids supplementation in improving RPP.

2020-05-18 | GSE138044 | GEO

Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid.

Project description:Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus. However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA.

| S-EPMC8334870 | biostudies-literature

Effects of recombinant protein expression on green fluorescent protein diffusion in Escherichia coli.

Project description:Fluorescence recovery after photobleaching was used to measure the diffusion coefficient of green fluorescent protein (GFP, 27 kDa) in Escherichia coli in the presence or absence of four coexpressed proteins: cytoplasmic maltose binding protein (42 kDa), tau-40 (45 kDa), alpha-synuclein (14 kDa), or calmodulin (17 kDa). The GFP diffusion coefficient remains constant regardless of the type of coexpresseed protein and whether or not the coexpressed protein was induced. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP. These results have implications for the utility of in-cell nuclear magnetic resonance spectroscopy.

| S-EPMC4529995 | biostudies-literature

Comparative phylogenetic analyses of recombinant noroviruses based on different protein-encoding regions show the recombination-associated evolution pattern.

Project description:Noroviruses are the major cause of acute gastroenteritis worldwide, and recombination is recognized as the important mechanism for its continuous emergence. In this study, for the common GII.P12 and GII.3 recombinants, phylogenetic relationships based on different proteins in three ORFs were comparatively analyzed, focusing on the influence of intergenic recombination. By using newly designed primers, genomes of two GII.P12/GII.3 Guangzhou recombinants were firstly amplified. Combined with other reported sequences of GII.P12_ORF1 (n?=?20), GII.3_ORF2 (n?=?131), GII.3_ORF3 (n?=?36), all GII.P12 and GII.3 strains could be divided into 6, 8, and 7 clusters based on different ORFs, which showed an obvious recombination-associated and temporally sequential evolution pattern (with the exception of GII.P12/GII.13 recombinants). Based on multiple alignments, 126 informative sites were identified in three ORFs (44, 54, and 28), and four proteins (p48, p22, VP1, and VP2) were found under positive selection. Furthermore, by using homology modeling, predicted epitopes were mapped on the P proteins of seven GII.3 representative strains, without one (Epi: 353-361) specific to the GII.4 VA387 strain. In summary, via the genome analyses, phylogenetic relationships of GII.P12 and GII.3 recombinants based on the different proteins presented a special temporally sequential evolution process associated with their recombinant types.

| S-EPMC5504017 | biostudies-literature

Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.

Project description:Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and ?-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and ?-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (µmol/L) of IP was found to be higher than that of ?-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.

| S-EPMC4128558 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data