A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies

Billy Bennett

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity.

Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution.

We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes.

We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.

The Aedes aegypti (Diptera: Culicidae) hsp83 Gene Promoter Drives Strong Ubiquitous DsRed and ZsGreen Marker Expression in Transgenic Mosquitoes

Transgenic strains of the mosquito disease vector Aedes aegypti (L.) are being developed for population suppression or modification. Transgenic mosquitoes are identified using fluorescent protein genes. Here we describe DsRed and ZsGreen marker genes driven by the constitutive Ae. aegypti heat shock protein 83 (hsp83) promoter in transgenic mosquitoes. Transgenic larvae and pupae show strong full body expression of the red and green fluorescent proteins. This greatly assists in screening for transgenic individuals while making new or maintaining already established lines. Transient marker gene expression after embryo microinjection was readily visible in developing larvae allowing the separation of individuals that are more likely to produce transgenic offspring. The strongly expressed marker genes developed in this study should facilitate the detection of transgenic Ae. aegypti larvae or pupae in the field.

A highly efficient identification of mutants generated by CRISPR/Cas9 using the non‑functional DsRed assisted selection in Aspergillus oryzae

The CRISPR/Cas9 system has become a great tool for target gene knock-out in filamentous fungi. It is laborious and time-consuming that identification mutants from a large number of transformants through PCR or enzyme-cut method. Here, we first developed a CRISPR/Cas9 system in Aspergillus oryzae using AMA1-based autonomously replicating plasmid and Cas9 under the control of the Aspergillus nidulans gpdA promoter. By the genome editing technique, we successfully obtained mutations within each target gene in Aspergillus oryzae.

Then, we put the protospacer sequence of a target gene and its protospacer adjacent motif (PAM) behind the start codon “ATG” of DsRed, yielding the non‑functional DsRed (nDsRed) reporter gene, and the nDsRed reporter gene could be rescued after successful targeted editing. Moreover, this method was also applied by targeting the kojic acid synthesis gene kojA, and the transformants with DsRed activity were found to harbor targeted mutations in kojA. These results suggest that the nDsRed can be used as a powerful tool to facilitate the identification of mutants generated by CRISPR/Cas9 in Aspergillus oryzae.

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses.

To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed42, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed42 accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis.

We show DsRed42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed42 signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed42 variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.

Rhizobium etli CE3-DsRed pMP604: a useful biological tool to study initial infection steps in Phaseolus vulgaris nodulation

Rhizobium etli CE3-DsRed pMP604 drives infection 12-24 h earlier than R. etli CE3-DsRed and it is an excellent tool in live-cell imaging studies of IT developement in P. vulgaris roots. The study of the cellular dynamics of nodulation has frequently been limited by the difficulty of performing live-cell imaging in nodule primordia and legume roots, which are constituted by multiple cell layers, such is the case of Phaseolus vulgaris.

RFP (dsRed) Antibody
3993-100	Biovision	each	516 EUR
RFP (dsRed) Antibody
3993-30T	Biovision	each	222 EUR
RFP Expressing Human Glioblastoma Cells
TR01-RFP	Neuromics	500,000 Cells	1624.8 EUR
dsRed Fluorescent protein
30R-2809	Fitzgerald	100 ug	589.2 EUR
DsRed- rab7 WT
PVT10355	Lifescience Market	2 ug	319.2 EUR
DsRed- Rab5 WT
PVT10499	Lifescience Market	2 ug	319.2 EUR
dsRed Fluorescent Protein
4997-100	Biovision	each	489.6 EUR
dsRed Fluorescent Protein
4997-1000	Biovision	each	3008.4 EUR
dsRed Fluorescent Protein
4997-5000	Biovision	each	8626.8 EUR
RFP Expressing Human Gastric Carcinoma N87 Cells
TR02-RFP	Neuromics	500,000 Cells	1624.8 EUR
RFP Expressing Human Renal Adenocarcinoma Cells (ACHN)
TR04-RFP	Neuromics	500,000 Cells	1624.8 EUR
RFP Expressing Human Prostate Carcinoma Cells (DU 145)
TR03-RFP	Neuromics	500,000 Cells	1624.8 EUR
Red Fluorescent Protein (dsRed) Antibody
3994-100	Biovision	each	451.2 EUR
pIRES2- DsRed- Express Plasmid
PVT1229	Lifescience Market	2 ug	289.2 EUR
pLVX- DsRed- Monomer- N1 Plasmid
PVT2312	Lifescience Market	2 ug	361.2 EUR
pBRPyCAG- Mettl3- dsRed- IRES- puro
PVT10449	Lifescience Market	2 ug	361.2 EUR
Recombinant purified Red Fluorescent Proteins (RFP/dsRed) protein control for Western blot
RFP12-C	Alpha Diagnostics	100 ul	343.2 EUR
PD1 ELISA Matched Antibody Pair (Risk Free)
RFP-0003	ProSci	1 Set	752.1 EUR

Seeking conditions to reduce the time it takes for rhizobia to infect P. vulgaris root, we decided to explore the nodulation properties of Rhizobium etli CE3 pMP604, a strain that constitutively produces Nod factors through a flavonoids-independent transcriptional activation which is often used to purify Nod factors. Even though the strain infects 12-24 h earlier than the parental R. etli CE3 strain, infection thread (IT) formation, nodule organogenesis processes and N₂-fixation activity are similar for both strains. Additionally, we have confirmed that R. etli CE3-DsRed pMP604 is an excellent tool to trace IT development in P. vulgaris roots.