Earlier we talked about the structure of biotin, which is composed of lipophilic heterocycles and hydrophilic carboxylic acid chains.
Therefore, the carboxyl group on the hydrophilic carboxylic acid chain of biotin can react with many groups. Biotinylated peptides are also based on this principle. During Fmoc solid-phase synthesis, biotin can be attached to the N-terminus of the peptide or the side chain of lysine (Lys) or glutamic acid (Glu). The synthesized biotinylated peptide can use the side chain thiol of cysteine (Cys) residues for other reactions.
Because it has a high affinity between biotin and avidin and multi-stage amplification effect, and is organically combined with luciferin, enzyme, isotope and other immunolabeling technologies, the specificity and sensitivity of various tracer immunoassays are further improved. There are mainly biotin N-hydroxysuccinimide ester (BNHS) and biotin p-nitrophenol ester (pBNP), which are used to label the amino groups of polypeptides. Among them, BNHS is the most commonly used.
Biotinylated peptides are usually combined with avidin to be detected or purified, and can be used in multiple ways in combination with immunoblotting, ELISA, immunoprecipitation, peptide affinity purification, immunohistochemistry, cell surface labeling, flow cytometry/fluorescence activated cell classification, etc.
Application in quantitative detection
It is mainly through the biotin-streptavidin system, combined with ELISA or immunochromatography and other methods for detection. Fang et al. pre-coated with streptavidin (SA), solidified biotinylated anti-cyclic citrullinated peptide (bio-CCP), and explored the optimal experimental conditions for detecting anti-CCP antibodies, and conducted methodological assessment and clinical application evaluation. The experiment finally successfully applied the biotin-streptavidin ELISA system to the detection of anti-CCP antibodies. The method was sensitive, specific, and reliable, and can be used for routine laboratory diagnosis of rheumatoid arthritis. Sun et al. introduced a biotin-streptavidin system, combined with immunochromatographic methods, and established a new method for rapid quantitative detection of NT-proBNP (an inactive N-terminal polypeptide fragment) (see Figure 1). NT-proBNP, as the main indicator of heart failure diagnostic test, plays an important role in the stage of disease diagnosis and prognosis evaluation.
Application in preparation and purification of antigen peptide complex monomer
Lu et al. bound biotin to the BSP sequence at the C-terminus of the H chain in the HLA-A*2402 antigen peptide complex to form a biotinylated HLA-A*2402 antigen peptide complex monomer. Western blot with specific monoclonal clonal and streptavidin was used to detect the folded products of diluted renaturation and biotinylation. This method successfully prepared biotinylated HLA-A*2402 antigen peptide complex monomers, which laid the foundation for the further construction of tetramers and the preparation of artificial antigen-presenting cells. Song et al. used prokaryotically expressed H-2Kd-BSP as the heavy chain and β2m as the light chain. After purification, they were co-folded and refolded with H-2Kd restricted 9 peptides (HBc131～139) in vitro by dilution. Then, under the action of BirA enzyme, the folded product was biotinylated to form a biotinylated H-2Kd-HBc complex [4]. The biotinylated complex monomer was further purified by gel filtration chromatography. The obtained monomers laid the foundation for the further construction of sH-2Kd antigen peptide tetramer and the preparation of artificial antigen-presenting cells in vitro, and the in-depth study of specific CTL responses and effect mechanisms during HBV infection.
Application in the regulation of biological activity
Bradykinin is a polypeptide containing 9 amino acid residues. Many pathogenesis, such as inflammation, pain, and asthma, are related to bradykinin. It can interact with the receptors on the surface of PC12 cells to cause the release of calcium ions in the organelles. Under a confocal microscope, the biological activity of bradykinin can be monitored by observing the increase in fluorescence of the calcium indicator Fluo-3. In this study, a solid-phase peptide synthesis method was used to synthesize a biotin-linked bradykinin. Studies have shown that the peptide connected with biotin can maintain the biological activity of natural bradykinin, and through steric hindrance and competitive interaction, it can regulate the biotin and avidin to control the biological activity of biotin-linked bradykinin. This was of great significance for studying the structural relevance of biological activities in biological systems.
Application in immunodiagnostics
The biotinylated enzyme and avidin are heated in advance in a certain proportion to form an ABC complex, and then the ABC complex is reacted with the biotinylated antibody. This method not only reduces the reaction steps, in addition, because the ABC grid can contain a considerable number of enzyme molecules, it can also greatly increase the sensitivity of the biotin-avidin system (BAS). The researchers used this method to determine the peptide hormones by enzyme labeling, and found that the antibody titer can be increased by 20-40 times. In addition, by comparing seven different enzyme labeling methods on tissue sections such as tonsil, skin and thyroid, it was found that the ABC method has the best effect, not only the sensitivity was the highest, but the specific staining was also the lowest [6]. Because the ABC complex has the characteristics of multivalent binding and high affinity, high sensitivity, strong specificity, simple operation, economical and time-saving, wide application range, etc., it is generally concerned and favored by the medical and biological circles.
Applications in the study of the interaction between peptides and proteins
With the principle of interaction between biotinylated peptide and streptavidin, the interaction between protein and target peptide is studied. Wu et al. obtained the polypeptide BRBP1 that binds to human breast cancer metastatic brain metastasis cell line 231-BR in the early stage. This peptide specifically bound to human breast invasive ductal carcinoma tissue specimens and tended to bind to tumor cells with high metastatic potential. Therefore, looking for a target protein that binds to BRBP1 peptide was of great significance for exploring the mechanism of breast cancer brain metastasis, deep understanding and broadening the application value of BRBP1 peptide. The researchers connected the carboxyl end of the BRBP1 peptide to lysine and attached biotin to its side chain to synthesize BRBP1-BIOTIN. The membrane proteins of 231-BR, MDA-MA-231, and MCF-7 cells were incubated with BRBP1-BIOTIN, and streptavidin magnetic beads were used to obtain proteins bound to biotin peptides. According to the results of mass spectrometry and bioinformatics analysis, the target proteins that may bind to BRBP1 peptides were analyzed and identified to find the target proteins that specifically bind to BRBP1.
References:
1. Fang GX, Wu YJ, Wei SX, Yu W, Li XJ. Detection of anti-cyclic citrullinated peptide antibodies with biotin-streptavidin amplification ELISA system [J]. Journal of Clinical Laboratory Science, 2009, 27(1). 20-23.
2. Sun HH, Liang YF, Zhao XS, Huang L, Liao TZ. Biotin streptavidin combined with immunochromatography for rapid detection of NT-proBNP [J]. Journal of South-Central University for Nationalities: Natural Science Edition, 2018, 37 (2): 6-9+15.
3. Lu XL, Wu XW, Weng XF, Li Q, Liang ZH, Gong FL. Preparation of biotinylated HLA-A*2402-antigen peptide complex [J]. Journal of Immunology, 2006, 22(1): 78 -81.
4. Song N, Hao YH, Zhang ZM, Wu J, Yang XX, Ding HH, Yang DL. Construction and purification of biotinylated sH-2Kd-HBc complex monomer [J]. Journal of Medical Molecular Biology, 2006, 3(3): 176-180.
5. Zhang HQ, Shinohara H, Gu N, Sayuki YJ, Agamoto Masahiko. Using biotin-neutral avidin binding caused by steric hindrance to control the biotin-linked bradykinin biological activity [J]. Progress in Biochemistry and Biophysics, 2002, 29(1): 60-63.
6. Wang SL, Zhang M. The application of avidin-biotin-enzyme complex (ABC) method in immunodiagnostics [J]. Heilongjiang Animal Science and Veterinary Medicine, 1989(5): 29-30.
7. Wu WY. Identification of BRBP1 binding related protein molecules targeting breast cancer brain metastasis cells [D]. Jiangsu: Southeast University, 2018.