Generating High-Quality Biotinylated Proteins

The interaction between avidin/streptavidin (SA) and biotin is exceptionally strong and specific, making it an outstanding tool in protein biochemistry. However, generating high-quality biotinylated proteins still poses a key challenge, and researchers frequently run into technical problems such as high batch-to-batch variation, insufficient labeling, and unpredictable performance. Additionally, the choice of biotinylation techniques has a huge impact on the ultimate outcome. Each target protein (assay) has its own unique biochemical characteristics, and thus requires a customized solution.

These confounding factors make the manufacturing and use of biotin conjugates too complex for individual labs. At the same time, there are also only a handful of products available at a commercial level.

In order to bridge this gap, ACROBiosystems has developed an exclusive range of pre-labeled and experimentally validated biotinylated recombinant proteins. The Mabsol® biotinylated protein collection features over a hundred commonly studied biomarker proteins and drug targets.

ACROBiosystems employs high-quality recombinant proteins that are closest to natural conformation and modification as starting materials. The company thoroughly inspects every option to realize the highest bioactivity and detection sensitivity. It also applies the most rigorous quality control measurement possible to guarantee minimal batch-to-batch variations.

Mabsol® biotinylated protein collection is comprised of two unique and complimentary product series—the UltraLys series, produced using the in-house developed chemical labeling method, and the PrecisionAvi series, which is built from AvitagTM technology. These products are manufactured with great attention to detail.

Products

PrecisionAvi Series

An Exclusive Collection of Ready-To-Use Avitagtm Biotinylated Proteins

The products in this series are exclusively produced using AvitagTM technology. In brief, the Avi tag—an exclusive 15 amino acid peptide—is introduced into the recombinant protein during expression vector construction. The E. Coli biotin ligase BirA enzymatically biotinylates the single lysine residue in the Avi tag.

This single-point enzymatic labeling method offers several advantages for commonly used binding assays, including:

  • No interference with the natural binding activities of the target protein
  • The protein orientation is uniform when immobilized on an avidin-coated surface
  • The biotinylation only occurs on the lysine residue of Avi tag

Commonly searched products:

. . . . . .
2B4 4 1BB Ligand ANGPTL7 APRIL B7-1 B7-2
B7-H3 B7-H4 B7-H5 B7-H6 B7-H7 BAFFR
BCMA BTLA BTN1A1 BTN3A1 CA125 CBLB
CD155 CD200 CD200 R1 CD23 CD27 CD27 Ligand
CD28 CD3 epsilon CD30 CD38 CD3E & CD3D CD4
CD40 CD40 Ligand CD47 CD52 CD84 CD96
CEACAM-1 CEACAM-5 CEACAM-6 CEACAM-8 CTLA-4 Dkk-1
EGF R EMMPRIN EpCAM Fc gamma RIII / CD16 FcRn (FCGRT & B2M) FOLR1
G-CSF R GDF-15 GITR GITR Ligand Glypican 3 GP120
HE4 Hemagglutinin (HA) Her2 HGF R HVEM ICOS
IGF-II IgG1 Fc IgG2a Fc IL-15 IL-17 RA IL-17A & IL-17F
IL-2 IL-21 IL-4 IL-4 R alpha IL-6 R alpha IL-7 R alpha
IL23A & IL12B Integrin alpha 2b beta 3 Integrin alpha 4 beta 1 Integrin alpha 4 beta 7 Integrin alpha 5 beta 1 Integrin alpha 6 beta 1
Integrin alpha D beta 2 Integrin alpha E beta 7 Integrin alpha L beta 2 Integrin alpha V beta 1 Integrin alpha V beta 3 Integrin alpha V beta 5
Integrin alpha V beta 6 Integrin alpha V beta 8 LAG-3 LAIR-1 LAIR-2 LAP (TGF-beta 1)
Latent TGF-beta 1 LDL R LIF LILRA2 LILRA3 LILRA5
LILRA6 LILRB1 LILRB2 LILRB3 LILRB5 M-CSF
M-CSF R Mesothelin Nectin-3 Neuropilin-1 NS1 OX40
OX40 Ligand PCSK9 PD-1 PD-L1 PD-L2 Prolactin
Protein L ROR1 ROR2 SCF Siglec-2 Siglec-3
SIRP alpha SLAMF1 SLAMF7 TACI TGF-beta 1 Thrombospondin-2
TIGIT TIM-3 TNF-alpha TNFSF11 TPBG TRAIL R2
TSLP VEGF R1 VEGF R2 VEGF R3 VEGF120 VEGF121
VEGF164 Vitronectin VSIG8      

 

For the following items, a pre-order option is available: Besides the regular catalog products, ACROBiosystems also accepts pre-orders for the following molecules, which are currently being developed:

. . . . . .
BACE CRP/C-Reactive ErbB3 IL-5 IL-6 IL-1a
IL-10 IL-13 IL2R IL20 R beta IL5R INSR
Tiki 1 TRAILR1 TRALR4      

 

UltraLys Series

A Unique Series of Chemically Labeled Biotinylated Proteins With Ultra Sensitivity

The products in this series are created using the company’s in-house developed chemical labeling method. The N-terminus of the protein and the primary amines in the side chains of lysine residues are conjugated with biotins, usually leading to multiple biotin attachments on a single protein molecule.

  • In-house developed chemical labeling approach
  • Higher detection sensitivity

Commonly searched products:

. . . . . .
B7-H4 CD19 CD3 epsilon CD3E & CD3D CX3CL1 EpCAM
EphB4 ErbB3 FcRn (FCGRT & B2M) FGF basic GM-CSF GPA33
Growth Hormone R Her2 IL-6 Mesothelin SOST TFPI
TNF-alpha Transferrin R VEGF165      

 

Applications

Key Features

Closest to Natural Conformation and Modification

ACROBiosystems’ proprietary HEKMax® expression platform is used to produce the recombinant proteins including the biotinylated proteins. As expression hosts, the human HEK293 cells have a range of advantages over other cell types, as summarized in the following table.

Expression Systems Folding Phosphorylation Proteolytic Processing Glycosylation
E.Coli + N/A N/A N/A
Insect Cell ++ ++ ++ Poor
Plant Cell +++ +++ +++ Poor
CHO Cell ++++ ++++ ++++ Non-Human Like
Human Cell +++++ +++++ +++++ Human Authentic

 

High Bioactivity and Detection Sensitivity

Both the structure of the protein and the way biotinylation is performed determine the bioactivity of biotinylated proteins. For every single protein, multiple tag options and biotinylation methods are tested, and the products are evaluated in a wide range of binding assays. Only those with the best performance are then chosen for production. An example of ACROBiosystems’s internal evaluation experiments is demonstrated in Figure 1:

 

Binding activity of different forms of biotinylated PD1 evaluated in a functional ELISA against rhPD-L1 (Cat. No. PD1-H5258).

Figure 1. Binding activity of different forms of biotinylated PD1 evaluated in a functional ELISA against rhPD-L1 (Cat. No. PD1-H5258).

Biotinylated proteins from ACROBiosystems exhibit high bioactivity and excellent detection sensitivity in various applications. In functional ELISA, the biotinylated human BCMA shows high binding activity with BAFF, as represented in Figure 2. Interestingly, oriented immobilization of biotinylated CD155 provides much higher sensitivity when compared to random immobilization of unconjugated CD155, as illustrated in Figure 3.

Figures 4 and 5 demonstrate that the biotinylated PD1 can be applied to AlphaLISA to detect the binding activity with its ligand PD-L1 and PD-L2. Figures 6 and 7 demonstrate that the affinity between protein and therapeutic antibodies in SPR assay can be determined using the biotinylated protein. Furthermore, the biotinylated proteins are frequently used in the cell-based assay, for example, in cytotoxicity assay and during evaluation of CAR expression (Figure 8 and Figure 9).

ELISA

Immobilized Human BAFF, Fc Tag (Cat. No. BAF-H4268) at 5 μg/mL (100 μL/well) can bind Biotinylated Human BCMA, Fc Tag (Cat. No. BC7-H82F0) with a linear range of 0.12–1.95 ng/mL.

Figure 2. Immobilized Human BAFF, Fc Tag (Cat. No. BAF-H4268) at 5 μg/mL (100 μL/well) can bind Biotinylated Human BCMA, Fc Tag (Cat. No. BC7-H82F0) with a linear range of 0.121.95 ng/mL.

Comparison of oriented immobilization (biotinylated CD155 on precoated streptavidin plate, Cat. No. CD5-H82E3) and random immobilization (unconjugated CD155 direct coating on 96-well plate, Cat. No. CD5-H5223) binding to TIGIT (Cat. No. TIT-H5254). Oriented immobilization of biotinylated CD155 offers much higher sensitivity than random immobilization of unconjugated CD155.

Figure 3. Comparison of oriented immobilization (biotinylated CD155 on precoated streptavidin plate, Cat. No. CD5-H82E3) and random immobilization (unconjugated CD155 direct coating on 96-well plate, Cat. No. CD5-H5223) binding to TIGIT (Cat. No. TIT-H5254). Oriented immobilization of biotinylated CD155 offers much higher sensitivity than random immobilization of unconjugated CD155.

AlphaLISA

Biotinylated Human PD-1(Cat. No. PD1-H82F1)at 1 μg/mL (5 μL/well) can bind Human PDL1 (Cat. No. PD1-H5229) with a linear range of 0.02–0.625 μg/mL.

Figure 4. Biotinylated Human PD-1Cat. No. PD1-H82F1at 1 μg/mL (5 μL/well) can bind Human PDL1 (Cat. No. PD1-H5229) with a linear range of 0.020.625 μg/mL.

Biotinylated Human PD-1(Cat. No. PD1-H82F1)at 1 μg/mL (5 μL/well) can bind Human PDL2 (Cat. No. PD2-H5220) with a linear range of 0.02–0.625 μg/mL

Figure 5. Biotinylated Human PD-1Cat. No. PD1-H82F1at 1 μg/mL (5 μL/well) can bind Human PDL2 (Cat. No. PD2-H5220) with a linear range of 0.020.625 μg/mL

Immobilized biotinylated human VEGF165 (Cat. No. VE5-H82Q0) on CM5 Chip via streptavidin, can bind Avastin with an affinity constant of 0.417nM as determined in SPR assay (Biacore T200).

Figure 6. Immobilized biotinylated human VEGF165 (Cat. No. VE5-H82Q0) on CM5 Chip via streptavidin, can bind Avastin with an affinity constant of 0.417nM as determined in SPR assay (Biacore T200).

Cell-Based Assay

Recombinant human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.014–0.029 ng/ml. The result shows that the biotinylated human TNF-alpha is consistent with naked TNF-alpha in cytotoxicity assay.

Figure 7. Recombinant human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.0140.029 ng/ml. The result shows that the biotinylated human TNF-alpha is consistent with naked TNF-alpha in cytotoxicity assay.

293 cells were transfected with FCM63-scFv and RFP tag. 2x105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml). C. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml) and FMC63 (Mouse anti-CD19 antibody). FITC Streptavidin was used to analyze with FACS. RFP was used to evaluate CAR (FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat. No. CD9-H8259).

Figure 8. 293 cells were transfected with FCM63-scFv and RFP tag. 2x105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml). C. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml) and FMC63 (Mouse anti-CD19 antibody). FITC Streptavidin was used to analyze with FACS. RFP was used to evaluate CAR (FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat. No. CD9-H8259).

Low Batch-to-Batch Variation

In order to ensure consistent performance of products, ACROBiosystems routinely applies stringent quality control measures. Newly manufactured products are subjected to a side-by-side comparison with the company’s internal standard in various assays. Only those within an acceptable margin of difference are permitted to be released. As illustrated in Figure 9, three different batches of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were tested and compared using a standard ELISA analysis against Adalimumab, and the results show that the batch variation among the tested samples is insignificant. The batch-to-batch conformance is also good, as shown in the cell-based assay (Figure 10).

In the above ELISA analysis, three different lots of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were used to detect immobilized Adalimumab (0.5 µg/ml). The result showed that the batch variation among the tested samples is negligible

Figure 9. In the above ELISA analysis, three different lots of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were used to detect immobilized Adalimumab (0.5 µg/ml). The result showed that the batch variation among the tested samples is negligible

Recombinant biotinylated human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.029–0.052 ng/ml

Figure 10. Recombinant biotinylated human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.0290.052 ng/ml

ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Nov 21, 2018 at 9:37 AM

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