Biotinylated Proteins for Research Use

Biotin and avidin or streptavidin (SA) have a strong specific interaction, making it an ideal tool for protein biochemistry. However, generating high-quality biotinylated proteins poses a substantial hurdle.

Scientists frequently encounter technical issues, such as excessive batch-to-batch variance, poor labeling, and unpredictable performance. The choice of biotinylation procedures also significantly influences the final output. Every target protein (assay) has unique biochemical properties and requires a specific solution.

These challenging characteristics make it difficult for laboratories to create and use biotin conjugates, and only a few solutions are available on the market.

ACROBiosystems has developed a unique library of pre-labeled and experimentally verified biotinylated recombinant proteins. The Mabsol^® biotinylated protein collection contains over a hundred commonly studied drug targets and biomarker proteins.

The company starts with high-quality recombinant proteins that closely resemble natural conformation and modification. It exhausts all possibilities to achieve maximum bioactivity and detection sensitivity. To ensure the fewest batch-to-batch changes, the company implements the strictest quality control techniques.

The Mabsol^® biotinylated protein collection is comprised of two unique and complementary product series: the UltraLys series, which utilizes an in-house-designed chemical labeling approach, and the PrecisionAvi series, which is built using Avitag^™ technology. Every aspect is carefully considered when creating these products.

Products

PrecisionAvi series

An Exclusive Collection of Ready-to-Use Avitag^™ Biotinylated Proteins

This product line utilizes Avitag^™ technology exclusively. In short, the Avi tag, a 15 amino acid peptide, is attached to the recombinant protein during expression vector assembly. The Avi tag’s single lysine residue is enzymatically biotinylated by BirA, an E. Coli biotin ligase.

This single-point enzymatic labeling approach has various advantages for binding assays that are often used.

• The biotinylation only occurs on the lysine residue of the Avi tag
• Even when immobilized on an avidin-coated surface, the protein orientation remains consistent
• There is no disruption to the target protein’s inherent binding properties

All Products

Table 1. Source: ACROBiosystems

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UltraLys Series

A Special Series of Chemically Labeled Biotinylated Proteins with Ultra-Sensitivity

This series of products is manufactured using an in-house-designed chemical labeling procedure. The primary amines in the side chains of lysine residues and the N-terminus of proteins are conjugated with biotins, resulting in many biotin attachments on a single protein molecule.

• Higher detection sensitivity can be obtained
• Includes an in-house designed chemical labeling process

All Products

Table 2. Source: ACROBiosystems

Applications

• FACS
• Enrichment
• Biopanning
• SPR
• ELISA
• BLI

Key Features

Closest to Natural Conformation and Modification

Recombinant proteins, such as biotinylated proteins, are produced using the patented HEKMax^® expression platform. As expression hosts, human HEK293 cells have several advantages over other cell types, as shown in the table below.

Table 3. Source: ACROBiosystems

High Bioactivity and Detection Sensitivity

The protein's structure determines the bioactivity of biotinylated proteins and how biotinylation is carried out. The company evaluates several tag and biotinylation techniques for each protein while evaluating the results in various binding experiments. However, only those with the best performances are chosen for production. Figure 1 shows the internal evaluation experiments.

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

ACROBiosystems’ biotinylated proteins provide high bioactivity and great detection sensitivity in various applications. In functional ELISA, biotinylated human TACI binds strongly to BAFF (Figure 2).

Figure 2. Immobilized Human BAFF, Fc Tag, active trimer (Cat. No. BAF-H5261) at 1 μg/mL (100 μL/well) can bind Biotinylated Human TACI, Fc, Avitag (Cat. No. TAI-H82F6) with a linear range of 0.002–0.05 μg/mL. Image Credit: ACROBiosystems

Interestingly, compared to random immobilization of unconjugated CD155, oriented immobilization of biotinylated CD155 offers much higher sensitivity (see Figure 3). Figures 4 and 5 show that the biotinylated PD1 on AlphaLISA can be utilized to measure the binding activity with its ligands, PD-L1 and PD-L2.

ELISA

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. Image Credit: ACROBiosystems

AlphaLISA

Figure 4. 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. Image Credit: ACROBiosystems

Biotinylated proteins can easily determine the affinity between a protein and a therapeutic antibody in an SPR test, as shown in Figures 6 and 7. Biotinylated proteins are also frequently used in cell-based assays, such as cytotoxicity assays and CAR expression evaluations (see Figure 8).

Figure 5. 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. Image Credit: ACROBiosystems

SPR

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

BLI

Figure 7. Loaded Biotinylated Human PD-1, Avitag, His Tag (recommended for biopanning) (Cat. No. PD1-H82E4) on SA Biosensor, can bind Human PD-L1, His Tag (HPLC verified) (Cat. No. PD1-H5229) with an affinity constant of 2.4 μM as determined in BLI assay (ForteBio Octet Red96e). Image Credit: ACROBiosystems

Cell-Based Assay

Figure 8. 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. Image Credit: ACROBiosystems

Low Batch-to-Batch Variation

ACROBiosystems routinely implements strict quality control methods to maintain the products' functioning. Recently released products are compared side by side with the internal standard in various tests.

Products can only be released if there is a reasonable margin of variance. Three batches of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were examined and compared using a conventional ELISA assay against Adalimumab, as shown in Figure 9. The findings show barely any batch variation among the examined samples. As demonstrated by the cell-based assay (see Figure 10), there is high batch-to-batch conformity.

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. Image Credit: ACROBiosystems

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 0.029–0.052 μg/mL. Image Credit: ACROBiosystems