scholarly journals Transient Expression of a Recombinant Monoclonal Antibody in HEK293T Cells

2018 ◽  
Vol 24 (3) ◽  
pp. 207-212 ◽  
Author(s):  
Omid Mohammadian ◽  
Masoumeh Rajabibazl ◽  
Hadi Bayat ◽  
Azam Rahimpour
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Expression System ◽  
Cell Culture Supernatant ◽  
Egfp Expression ◽  
Enzyme Analysis ◽  
Bicistronic Expression

Background: Monoclonal antibodies (mAbs) are considered the most important and financially successful category of the biopharmaceuticals. Extensive optimization of the expression vector, host system and culture parameters are required for the successful production of active monoclonal antibodies in mammalian cells. In this regards, transient expression enables rapid and cost-effective production of recombinant proteins for initial characterization. Methods: In the present study, an internal ribosome entry site (IRES) based bicistronic expression system has been evaluated for the transient expression of an anti-CD52 monoclonal antibody in mammalian cells. The IRES based bicistronic vector was generated through sequential cloning of the Light chain (LC), IRES, and Heavy chain (HC) in an intermediate vector and transfer of the resulting fragment to the expression vector. Transfection of the HEK293T cells was performed and antibody expression was analyzed in cell culture supernatant. Results: Restriction enzyme analysis indicated successful cloning of the antibody coding unit in the expression vector. Analysis of EGFP expression indicated successful transfection of the HEK293T cells. Production levels of 220 µg/L of antibody were achieved in HEK293T cells during three days of culture. Conclusion: Our results show the convenience and efficiency of the bicistronic expression system for transient expression of the whole monoclonal antibodies in mammalian cells.

Transient expression of oatp organic anion transporter in mammalian cells: identification of candidate substrates

1996 ◽  
Vol 270 (2) ◽  
pp. F319-F325 ◽  
Author(s):  
N. Kanai ◽  
R. Lu ◽  
Y. Bao ◽  
A. W. Wolkoff ◽  
V. L. Schuster
Keyword(s):  
Hela Cell ◽  
Transient Expression ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Organic Anion ◽  
Expression System ◽  
Indigo Carmine ◽  
Organic Anions ◽  
Phenol Red ◽  
Cell Monolayers

The cDNA for the rat liver organic anion-transporting polypeptide "oatp" has been shown to encode transport of bromosulfophthalein (BSP) and bile salts in Xenopus oocytes (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). Because oatp mRNA is expressed strongly in the kidney, we sought to determine whether renal oatp might play a role in the known secretion of a large variety of organic anions by the kidney. We transiently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa cell monolayers using the recombinant vaccinia virus vtf7-3. We tested an array of organic anions as candidate substrates by determining their ability to compete with tracer BSP for transport. HeLa cell monolayers transfected with the oatp cDNA transported tracer BSP and taurocholate at rates substantially higher than monolayers transfected with a control plasmid. Thus good expression can be obtained with the vaccinia-HeLa system using a standard plasmid cloning vector. BSP transport varied as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions known to be secreted by the classic secretory pathway, including p-aminohippurate (PAH), phenol red, and indigo carmine (10 microM) failed to inhibit oatp-mediated BSP transport. Direct testing using tracers revealed no oatp-mediated transport of sulfate, urate, PAH, several eicosanoids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by approximately 50% by 10 microM corticosterone sulfate, spironolactone, and several other steroids. We conclude that the functional properties of oatp expressed in the HeLa cell/vaccinia transient expression system are comparable to those following expression in Xenopus oocytes and that steroids are likely to represent high-affinity endogenous oatp substrates. The latter hypothesis is addressed in greater detail in a companion paper.

scholarly journals Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

2021 ◽  
Vol 12 ◽  
Author(s):  
Tanapati Phakham ◽  
Christine Joy I. Bulaon ◽  
Narach Khorattanakulchai ◽  
Balamurugan Shanmugaraj ◽  
Supranee Buranapraditkun ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Expression System ◽  
Sodium Dodecyl ◽  
Production Costs ◽  
Plant Production ◽  
Production Platform

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

EXPRESSION OF HUMAN FACTOR IX IN MAMMALIAN CELLS

1987 ◽  
Author(s):  
Shu Wha Lin ◽  
J Ware ◽  
H Roberts ◽  
N McGraw ◽  
W McAllister ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vitamin K ◽  
Mammalian Cells ◽  
Human Factor ◽  
Expression System ◽  
Consensus Sequence ◽  
Factor Ix ◽  
In Vitro Mutagenesis ◽  
Translational Initiation ◽  
Human Factor Ix

Human factor IX has been expressed in mammalian cells. A cloned factor IX cDNA missing the first 15 nucleotides of the 5’ end was modified by in vitro mutagenesis to restore the missing codons and add the translation consensus sequence, CCACC, proposed by Kozak to be optimal for translational initiation. Additionally, Bgl II and BamHI sites were added immediately upstream of the CCACC sequence for ease of portability of the fragment. This modified cDNA was inserted into a bovine papillomavirus (BPV) vector under the control of a mouse met alio thionein promoter. The constructed plasmid pBPV-IX was used to transfect a mouse fibroblast cell line C127. After 3 weeks, the transformed foci were isolated and the established cell lines were grown in the presence or absence of vitamin K. Media was collected at 3 day intervals and assayed for factor IX activity in a one stage clotting assay. A standard curve was constructed using purified human factor IX. Cells grown in the presence of vitamin K (3 mg/L) exhibited an activity equivalent to 350 ng/ml of factor IX in the cell media; no (less than 3 ng/ml) activity was detectable in the absence of vitamin K. A monoclonal antibody column specific for the Ca++ dependent form of human factor IX allowed the isolation of approximately 7 ug of purified factor IX from approximately 100 ml of culture medium. Western blot analysis of the purified factor IX revealed 2 protein bands which reacted with a goat anti-human factor IX antibody as well as a human specific monoclonal antibody. One of the immunoreactive bands migrates with authentic human factor IX and the other migrates slower. This expression system provides a convenient way to produce suitable amounts of factor IX and mutated factor IX protein for functional analyses.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

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