scholarly journals Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

2020 ◽  
Vol 21 (17) ◽  
pp. 6324 ◽  
Author(s):  
Annamaria Sandomenico ◽  
Jwala P. Sivaccumar ◽  
Menotti Ruvo
Keyword(s):  
Expression System ◽  
Antibody Fragments ◽  
High Yield ◽  
E Coli ◽  
Mammalian Expression ◽  
Diagnostic Agents ◽  
Pros And Cons ◽  
Escherichia Coli Expression ◽  
Recombinant Fragments ◽  
Diagnostic Investigations

Antibodies and antibody-derived molecules are continuously developed as both therapeutic agents and key reagents for advanced diagnostic investigations. Their application in these fields has indeed greatly expanded the demand of these molecules and the need for their production in high yield and purity. While full-length antibodies require mammalian expression systems due to the occurrence of functionally and structurally important glycosylations, most antibody fragments and antibody-like molecules are non-glycosylated and can be more conveniently prepared in E. coli-based expression platforms. We propose here an updated survey of the most effective and appropriate methods of preparation of antibody fragments that exploit E. coli as an expression background and review the pros and cons of the different platforms available today. Around 250 references accompany and complete the review together with some lists of the most important new antibody-like molecules that are on the market or are being developed as new biotherapeutics or diagnostic agents.

Latent TGF-beta binding protein LTBP-1 contains three potential extracellular matrix interacting domains

2001 ◽  
Vol 114 (1) ◽  
pp. 187-197 ◽  
Author(s):  
C. Unsold ◽  
M. Hyytiainen ◽  
L. Bruckner-Tuderman ◽  
J. Keski-Oja
Keyword(s):  
Extracellular Matrix ◽  
Expression System ◽  
Covalent Binding ◽  
Sodium Deoxycholate ◽  
Current Data ◽  
Lung Fibroblasts ◽  
Tgf Beta ◽  
Mammalian Expression ◽  
The Matrix ◽  
Recombinant Fragments

Latent TGF-beta binding proteins (LTBPs) are components of the extracellular matrix (ECM). They belong to the fibrillin/LTBP-superfamily, and are high molecular weight glycoproteins characterized by EGF-like repeats and 8-Cys repeats. Most LTBPs associate with the small latent forms of TGF-beta. Their roles include to facilitate the secretion of latent TGF-beta and to target it to the ECM. In order to identify new matrix-binding domains of LTBP-1 and to characterize their association with the extracellular matrix, we have produced (in a mammalian expression system) partly overlapping recombinant fragments of its shorter form, LTBP-1S, and analyzed the binding of the purified fusion proteins to extracellular matrices of cultured human dermal and lung fibroblasts. Recombinant fragments from three different regions of the N- and C-termini showed affinity to the matrix. These interacting regions contain either the first (hybrid), second or fourth 8-Cys domains of the LTBP-1S molecule. They bound independently to the matrix. Each of them had an ability to inhibit the association of native exogenous LTBP-1 with fibroblast extracellular matrix. The interactions of the LTBP-1 fragments with the extracellular matrix resisted treatment with sodium deoxycholate, suggesting strong, possibly covalent binding. The binding occurred in a time- and dose-dependent fashion. The N-terminal fragments bound more readily to the matrices. With all fragments the binding took place both with intact fibroblast matrices and with matrices isolated by sodium deoxycholate. When using CHO cell layers, which form sparse matrices, only the N-terminal fragment of LTBP-1 was efficiently incorporated. The association of the binding fragments with isolated matrices was enhanced by soluble, cell-derived factors. The current data suggest that LTBP-1 contains three different domains with an ability to associate with the extracellular matrix.

scholarly journals A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Topoisomerase Ii ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Simple Expression ◽  
Amino Acid Sequences ◽  
Expression Plasmid ◽  
Iptg Induction ◽  
E Coli ◽  
Mammalian Expression ◽  
Effective Selection

AbstractWe introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

scholarly journals High Expression and Purification of Amino-Terminal Fragment of Human Amyloid Precursor Protein inPichia pastorisand Partial Analysis of Its Properties

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Wei Li ◽  
Xiang Gao ◽  
Junle Ren ◽  
Ting An ◽  
Yan Liu
Keyword(s):  
Amyloid Precursor Protein ◽  
Expression System ◽  
Precursor Protein ◽  
High Yield ◽  
Human Neuroblastoma ◽  
Amino Terminal ◽  
Mammalian Expression ◽  
Terminal Fragment ◽  
Mammalian Expression System ◽  
Amino Terminal Fragment

The cleaved amino-terminal fragment of human amyloid precursor protein (N-APP) binds death receptor 6 (DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer’s disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, obtaining abundant and purified N-APP is fundamental and critical. The recombinant N-APP has been produced in mammalian expression system. However, the cost and yield disadvantages of mammalian expression system make it less ideal for protein mass production. Here, we successfully expressed and purified recombinant N-terminal 18-285 amino acid residues of human amyloid precursor protein from the methylotrophic yeastPichia pastoriswith a high yield of 50 mg/L. Flow cytometry indicated the purified N-APP-induced obvious apoptosis of human neuroblastoma SHEP cells.

scholarly journals Investigation of a recombinant SMN protein delivery system to treat spinal muscular atrophy

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Ryan Anderton ◽  
Bruno Meloni ◽  
Frank Mastaglia ◽  
Sherif Boulos
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cortical Neurons ◽  
Muscular Atrophy ◽  
Expression System ◽  
Cell Penetrating Peptides ◽  
Full Length ◽  
Dependent Manner ◽  
E Coli ◽  
Mammalian Expression ◽  
Smn Protein

AbstractSpinal muscular atrophy (SMA), the most common genetic cause of infant death, is a neurodegenerative disorder affecting motor neurons. SMA results from a loss in full-length survival of motor neuron (SMN) protein due to deletions/mutations in the SMN1 gene. In this study, we assessed the ability of cell-penetrating peptides (CPP) to deliver recombinant SMN protein to cultured neurons as a prelude for a potential therapeutic to treat SMA. Firstly, we confirmed that E. coli produced recombinant GFP protein fused to TAT (YGRKKRRQRRR; TAT-GFP) transduced rat cortical neurons in a concentration dependent manner. However, due to low yields of recombinant TATSMN protein obtainable from E. coli, we investigated the potential of a modified TAT (TATκ: YARKAARQARA) or R9 (RRRRRRRRR) peptide downstream of the fibronectin (FIB) secretory signal peptide to generate recombinant CPP-fused SMN protein. While U251 cells transduced with an adenoviral vector expressing CMV-FIB-TATκ-SMN secreted recombinant TATκ-SMN protein, we did not detect TATκ-SMN protein transduction of cortical neurons. Further, purified TATκ-SMN was unable to transduce SH-SY5Y cells, nor block apoptosis following LY294002 treatment of these cells. Our findings indicate that TATκ is not a suitable CPP to deliver SMN protein to neurons. Nonetheless, we have developed a novel method to generate full-length recombinant SMN protein using a mammalian expression system, which can be used to explore the application of other CPPs to deliver SMN protein as a treatment for SMA.

scholarly journals Multimerization through Pegylation Improves Pharmacokinetic Properties of scFv Fragments of GD2-Specific Antibodies

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3835 ◽  
Author(s):  
Irina V. Kholodenko ◽  
Daniel V. Kalinovsky ◽  
Elena V. Svirshchevskaya ◽  
Igor I. Doronin ◽  
Maria V. Konovalova ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Expression System ◽  
Cross Reactivity ◽  
Antibody Fragments ◽  
The Body ◽  
Radioactive Isotopes ◽  
Cancer Therapies ◽  
Specific Antibodies ◽  
Ganglioside Gd2 ◽  
Mammalian Expression

Antigen-binding fragments of antibodies specific to the tumor-associated ganglioside GD2 are well poised to play a substantial role in modern GD2-targeted cancer therapies, however, rapid elimination from the body and reduced affinity compared to full-length antibodies limit their therapeutic potential. In this study, scFv fragments of GD2-specific antibodies 14.18 were produced in a mammalian expression system that specifically bind to ganglioside GD2, followed by site-directed pegylation to generate mono-, di-, and tetra-scFv fragments. Fractionated pegylated dimers and tetramers of scFv fragments showed significant increase of the binding to GD2 which was not accompanied by cross-reactivity with other gangliosides. Pegylated multimeric di-scFvs and tetra-scFvs exhibited cytotoxic effects in GD2-positive tumor cells, while their circulation time in blood significantly increased compared with monomeric antibody fragments. We also demonstrated a more efficient tumor uptake of the multimers in a syngeneic GD2-positive mouse cancer model. The findings of this study provide the rationale for improving therapeutic characteristics of GD2-specific antibody fragments by multimerization and propose a strategy to generate such molecules. On the basis of multimeric antibody fragments, bispecific antibodies and conjugates with cytotoxic drugs or radioactive isotopes may be developed that will possess improved pharmacokinetic and pharmacodynamic properties.

scholarly journals Baculovirus-free insect cell expression system for high yield antibody and antigen production

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Janin Korn ◽  
Dorina Schäckermann ◽  
Toni Kirmann ◽  
Federico Bertoglio ◽  
Stephan Steinke ◽  
...  
Keyword(s):  
Insect Cell ◽  
Expression System ◽  
High Yield ◽  
Human Immune System ◽  
Mammalian Expression ◽  
Insect Cell Expression System ◽  
Human Immune ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

AbstractAntibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

Escherichia colias an expression system for K+transport systems from plants

2001 ◽  
Vol 281 (3) ◽  
pp. C733-C739 ◽  
Author(s):  
Nobuyuki Uozumi
Keyword(s):  
Membrane Proteins ◽  
Expression System ◽  
Functional Expression ◽  
Host Cells ◽  
Transport Systems ◽  
Membrane Structures ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Experimental Findings ◽  
And Function

The value of the Escherichia coli expression system has long been established because of its effectiveness in characterizing the structure and function of exogenously expressed proteins. When eukaryotic membrane proteins are functionally expressed in E. coli, this organism can serve as an alternative to eukaryotic host cells. A few examples have been reported of functional expression of animal and plant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologous K+transporters exist in prokaryotic cells and in eukaryotic cells; 2) plant K+transporters can functionally complement mutant K+transporter genes in E. coli; and 3) membrane structures of plant K+transporters can be elucidated in an E. colisystem. These experimental findings suggest the possibility of utilizing the E. coli bacterium as an expression system for other eukaryotic membrane transport proteins.

scholarly journals A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

2019 ◽  
Vol 103 (21-22) ◽  
pp. 8875-8888 ◽  
Author(s):  
Marloes L. C. Petrus ◽  
Lukas A. Kiefer ◽  
Pranav Puri ◽  
Evert Heemskerk ◽  
Michael S. Seaman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Antibody Fragment ◽  
Green Fluorescence Protein ◽  
Antibody Fragments ◽  
Single Chain ◽  
Mammalian Expression ◽  
Anti Hiv

Abstract Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

scholarly journals High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures

BioTechniques ◽  
2021 ◽  
Author(s):  
Yoshiro Hanyu ◽  
Mieko Kato
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Recombinant Antibody ◽  
Antibody Fragments ◽  
High Yield ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Antibody Fragments ◽  
Single Chain Variable Fragments ◽  
High Yields

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

Sign in / Sign up

Export Citation Format

Share Document