Biolayer Interferometry and its Applications
Published Date: August 20, 2018
Biolayer Interferometry and its Applications
Shivani Bhagwat and Anil Kumar*
School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore- 452001, India
*Corresponding author: Anil Kumar, School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore- 452001, India, E-mail: firstname.lastname@example.org
Citation: Bhagwat S, Kumar A (2018) Biolayer Interferometry and its Applications. J Mole Biol Tech 2(1):106.
Protein-protein interaction is the center point in predicting the functionality of target proteins and their drug abilities. Interaction of a protein with other protein(s) can modulate its function(s). Elucidation of biomolecular interaction networks so far is the method to analyze signal transduction pathways. Recent studies have used in silico methods for construction of protein networks for signaling pathways and consequently for identification of complex disease pathways. This paper is intended to emphasize on a basic but important light scattering technique, Bio-layer interferometry and its applicability. It is a label free method for measuring biomolecular interactions. This is based on a change in the path of white light reflected from a layer of immobilized protein placed on a bio-sensor tip and an internal reference surface.
Keywords: Protein-protein interaction; Signaling network; Biosensor; Surface plasmon resonance
Protein-protein interactions (PPIs) mainly deal with the biological processes viz. cell-cell interactions, developmental control and metabolic controls . This is an important research area in system biology also. The non-covalent bonding between amino acid residues is the basis of folding in the proteins and their assembly. The protein folding helps in making the proteins functional which includes cell growth, gene expression, nutrient uptake, cell proliferation, morphology, intercellular communications, motility and apoptosis. Depending upon various functional and structural characters, PPIs can be divided into several ways. On the basis of stability, proteins can be obligate or non-obligate; on the basis of persistence, protein can be transient or permanent; and on the basis of interaction of the surface, these can be homo- or hetero- oligomeric. A transient interaction leads to signaling pathways while permanent interaction plays a role in forming stable protein complexes. Molecular interaction investigation is necessary for digging deep into how molecular complexes are formed and break down when coming in proximity. This information can lead to new areas of drug discovery and life science research. For that matter, a label free interaction method can prove to be hassle free technique with lesser expenses. This technique is easier and faster compared to conventional methods viz. radioligand or fluorescence based methods. A biosensor technology can be used for analysis of kinetic affinity and protein concentration analysis. The analysis is based on any change in the number of molecules which are bound to biosensor tip and causes a shift in the interference pattern. This can be measured in real time and provides detailed information about the kinetics of association and dissociation of the bio-molecular complexes. It has a very sensitive sensor which provides information about both purified and crude samples as well as high throughput sample size. Bio-layer interferometry (BLI) was first introduced by ForteBio.
Basis of the Technique
In a basic optical biosensor experiment, one protein type is tethered on the sensor surface and other is injected across the surface. The results are generated according to the association and dissociation of the complexes. During association phase, analytes are exposed to the sensor surface, where the optical detection system measures the change in the refractive index of buffer which is placed near the sensor surface. This change is due to the presence of mass of the analyte which is accumulated on the surface. As the reaction proceeds for a longer period, equilibrium may be reached. For dissociation phase, the sensor surface is washed with the buffer and signals can be monitored because protein complex will break down with time. The major advantage of biosensor technology is that it does not require any washing of samples before quantitation. This does not cause hindrance in reaction equilibrium and characterization of much weaker and transient interactions can be recorded. There is a possibility of changing parameters of buffer, temperature, analyte concentration or pH and that of changing analyte family viz. protein mutants or related proteins for characterizing interaction in details.
The sensor chips are of various types depending upon capacity and material of the chip. For example, ProteOn system of Bio-rad using varying length of alginate layer on the chip, dextran based chips of Biacore have varying percentage of carboxylation of dextran. On the other hand, XanTech chips are controlled by three major factors: i) spatial distance between hydrogel chains (brush structured), ii) hydrogel thickness which can be changed via chain length and iii) the charge density which can be changed by the composition of the polycarbonates . Any change in the biosensor chip causes a shift in the interference pattern. The binding of immobilized ligand (on biosensor tip surface) and an analyte (in solution) develops an increase in optical thickness causing wavelength shift. This wavelength shift is a measure of change in thickness of biological layers. On the other hand, the unbound molecules can change the refractive index of surrounding medium but do not affect the interference pattern. This property is useful to perform BLI on crude samples which are required for quantitation affinity and kinetics study of bio-molecular interaction .
The basic aim of the immobilization is attachment of interactant to the sensor surface. This is a kind of permanent covalent bonding achieved by means of capturing. With the help of surface plasmon resonance (SPR) one of the molecules is attached to sensor surface which is opposed by Iso-thermal calorimetry (ITC) and microscale thermophoresis (MST). However, major drawback of immobilization is loss of biological activity of ligand because active sites have covalent bonds. Besides, covalent bonds also restrict movement of the ligand . The satisfactory immobilization method can be chosen based on the type of ligand (protein, DNA, sugars or low molecular weight substances), types of analyte (large or small interactants) or on purpose of the study (concentration, specificity , kinetics or affinity). The foremost requirement is that ligand should retain its biological activity after immobilization [5,6]. The method of immobilization can be determined using some information about the pI of the ligand, size, amino acid composition and probable sites for oriented coupling.
Diverse covalent coupling chemistry is available these days, depending upon available reactive groups of ligand in question. There are three well developed methods namely amine (-NH2), aldehyde (-CHO) and thiol (-SH) coupling. In general, covalent coupling is the most stable method but is prone to random orientation of ligand on sensor surface. The random orientation, in turn, reduces the number of binding sites and reactive groups near binding sites are hindered and reduce the affinity of analyte . In some cases during immobilization, the use of blocking agent or low pH conditions can inactivate the ligand. The drawbacks of random orientation immobilization can be overcome by unidirectional immobilization and affinity capturing.
Unidirectional immobilization is used to eliminate/reduce induced heterogeneity. For example, carbohydrate groups of the antibodies can be biotinylated which provides unidirectional immobilized molecules . The antibodies are digested with papain for separating two Fab fragments connected by disulfide bonds (Figure 1). Thereafter, separated Fab fragment is reduced (conversion of –S-S- bridge to –SH groups) and the reduced fragment is immobilized unidirectionally on the sensor chip using thiol chemistry .
Affinity capturing system can also be used to have sufficient affinity to ligand for making it stable on the chip. This process does not require high purity ligand because capturing is comparable to affinity purification. Most of the affinity capturing use immobilized antibody with amine coupling. In this procedure, many antibodies become non-functional. However, it is not of great concern because of enough functional antibodies present on the chip. For special tags, system involves some modification of capturing technique. This includes introduction of six histidine residues on nickel-nitrilotriacetic acid (NTA) sensor chip . Hydrophobic compounds can be used to modify sensor chip which allows capture of vesicles. The immobilized vesicles can be used as vehicles into which hydrophobic proteins can be captured for example in L1 sensor chips. There are some commercially available sensors that measure cellular activations .
The design of the experiment is a crucial step to increase the sensitivity of the platform and robustness of the experiment. To start with, a flow buffer is required which is a carrier of an analyte. The osmolarity, pH and composition are necessary for proper binding of the ligand and analyte. Some commonly used buffers are: HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% P20) ; TBS-P buffer (50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 0.005% P20) and PBS-P buffer (10.1 mM Na2PO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4, 0.005% P20). In these buffers, P20 is a surfactant.
Addition of certain compounds apart from basic composition of the buffer can increase or decrease the interaction between ligand and the analyte. For example, addition of 0.1% BSA to the running buffer decreases the analyte adsorption to stationary system. Increasing detergent or salt concentrations is helpful to decrease non-specific binding. Addition of metal ions like Ca2+ or Zn2+, increases the interaction (12, 13). Flow buffer should be made with special care and its degassing is necessary to avoid micro- bubble formation in flow system. The temperature of the system also plays a key role in the interaction of ligand and analyte. The machine temperature should be pre-set before the flow buffer arrives at the interaction point.
The experimental workflow can be defined for the purpose of the experiment and proposed interaction kinetics. Basically, the experimental strategy determines the concentration of a ligand which is immobilized on the biosensor chip. The level of immobilization also determines the sensitivity of the instrument. Whereas, interaction kinetics between an analyte and ligand depends upon the injection strategy. For various experimental setups, multi cycle kinetics is used. In this kinetics, an analyte and blank is injected separately in each cycle. For analysis, all the single runs are put together in one sensorgram at the end of the experiment. The single cycle kinetics (Kinetic titration) is applied when the interactions are very difficult to regenerate or regeneration of the ligand is damaging to the ligand [14,15]. In this technique, the analyte is injected using low to high concentrations with short pulse of dissociations in between and long pulses of dissociations at the end. The system often limits the kinetic titration upto five analytes per injection. While, all the injections are analysed on one sensorgram with dedicated equation for the same.
When the dissociation phase is low, short and long experiments are carried out. The analytes having low concentrations are injected and they preferably take long time to dissociate and could be analysed properly. To speed up the process, the lesser concentrated analyte injections are regenerated after brief dissociation period. On the other hand, if dissociation rate is very high that is when kd is faster than 10-3s-1, steady state experiments can be carried out. When dissociation rate is very slow, it takes much time in one analyte injection volume to reach steady state. Therefore, small compounds having faster dissociation rate are taken for this experiment. When, the dissociation rate is smaller than 10-1, the association and dissociation rates are difficult to reach and therefore steady state experiment can be taken into consideration to get at least the dissociation constant. In addition, a steady state experiment is generally used to confirm the dissociation constant.
Bio-layer Interferometry is a competitive technique to decipher the biomolecular interactions and allosteric ligand effects. The instrument with BLI allows a high throughput experimental design and flexibility in assays for interaction studies. The interaction between a sensor-immobilized molecule and its analyte in solution can be examined under different conditions with the help of multiple sensors in parallel. For robust results of association and dissociation rates, multiple concentrations of binding molecules can be used and the data traces should be fit for global analysis. Bio-layer interferometry can be used on unprocessed samples for their interaction, affinity and kinetic studies.
Conflict of Interest
Nothing to disclose.
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Copyright: © 2018 Kumar A, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.