Oriented versus random protein immobilization

Immobilization of proteins on solid surfaces plays an important role in all the fields of modern biology. Two approaches are used in the immobilization of proteins: a random and an oriented mode of binding to solid matrices. In this note, we show that there is not much difference in using either mode of immobilization, since proteins usually bind to a matrix through only one or two bonds. This is demonstrated by the attachment of several proteins to CNBr-activated Sepharose through their lysines and the consequent conversion of those lysines to homoarginine upon treatment with ammonium hydroxide.     

Modulation of the distribution of small proteins within porous matrixes by smart-control of the immobilization rate

The distribution of enzymes attached to porous solid supports is a major concern in multienzymatic bioreactors. Herein, as proof of the concept that protein localization on porous surfaces can be controlled by tuning the protein immobilization rate. We study the distribution of two poly-histidine-tagged fluorescent proteins (His-GFP and His-mCherryFP) immobilized on different 4% crosslinked agarose-type carriers by confocal laser scanning microscopy. In this context, immobilization rate is easily modulated by controlling the (i) nature of physico-chemical interaction between protein and surface (reactive groups on surface), (ii) by controlling the reactive group density and (iii) by adding competitors to the immobilization process. His-GFP is 350-fold more rapid immobilized on agarose surfaces activated with either glyoxyl groups or chelates than the same matrix activated with primary amine groups instead. A similar effect is seen with agarose matrixes activated with lower glyoxyl densities that immobilize His-GFP roughly 350-fold slower than the corresponding highly activated matrix. When His-GFP is immobilized on agarose activated with chelates groups in presence of imidazol which competes with the protein for the reactive groups on the support, the immobilization rate is again 400-fold slower than when the same protein was immobilized on the same support but with no imidazol during the immobilization process. In all cases, it was observed that rapid immobilizations (quantitative immobilization in less than 10 min) located 100% of the loaded protein at the crown of the carrier beads, meaning that only the 10% of the bead radius was colonized by the protein. On the contrary, when immobilization is much slower, a homogeneous distribution is obtained, resulting in beads whose whole radius is occupied by the protein. Therefore, we set that the more rapid immobilization, the more heterogeneous distribution. All the knowledge gained in protein distribution by immobilization rate alteration of a single protein is applied to the co-immobilization of the two fluorescent proteins in order to develop four different co-immobilization patterns with an enormous applied potential to other multi-protein systems.     

Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.     

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.     

Novel surface display system for proteins on non-genetically modified gram-positive bacteria

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.     

Proteomic analysis of the peritrophic matrix from the gut of the caterpillar, Helicoverpa armigera

The peritrophic matrix from the midgut of the caterpillar, Helicovera armigera, was solubilized by treatment with anhydrous trifluoromethanesulfonic acid, apparently by depolymerisation of its chitin component. This allowed the efficient extraction of proteins in a technique that may be broadly applicable to the analysis of other structures containing chitin. Gel electrophoresis and mass spectrometry of tryptic peptides were used to identify the extracted proteins with gut-expressed cDNA sequences. The major proteins of this cohesive, digestion-resistant structure are chitin deacetylase-like and mucin-like proteins, the latter with multiple chitin-binding domains that may cross-link chitin fibrils to provide a barrier against abrasive food particles and parasites, one of the major functions of the matrix. Other proteins found in the H. armigera gut peritrophic matrix suggest that the matrix is a dynamic, complex structure that may participate in the immobilization of digestive enzymes, actively protect the gut from parasite invasion and intercept toxins such as lectins and Bacillus thuringiensis crystal proteins.     

Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.     

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.     

Application of different surface plasmon resonance biosensor chips to monitor the interaction of the CaM-binding site of nitric oxide synthase I and calmodulin.

Surface plasmon resonance biosensors depend on modified gold surfaces to allow immobilization of proteins or peptides for interaction analysis. We investigated sensor chip surfaces that differ in the geometry of the immobilization matrix: two contain a three-dimensional coupling matrix and two have a surface with immobilization sites on a two-dimensional plane. Properties of sensor chips were compared by studying the interaction of calmodulin with a peptide representing the calmodulin-binding site of nitric oxide synthase I. Apparent K(D) values were determined by three different procedures in order to apply tests for self-consistency. At low surface densities (5-8 fmol/mm(2)) on three of the four tested surfaces, estimated K(D) values were within one order of magnitude and similar to the value found in solution (K(D) = 1-3 nM). When immobilization densities were increased by one to two orders of magnitude, apparent association rate constants were less distorted on a flat carboxymethylated surface than on dextran-coated sensor chips.     

Oriented immobilization of proteins on hydroxyapatite surface using bifunctional bisphosphonates as linkers

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and β-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 × 10(-5) mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10-15 fold that of adsorbed EGFP, whereas the relative amount of β-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed β-lactamase. The enzymatic activity of HBP-immobilized β-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized β-lactamase improved 2-5 fold over adsorbed β-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.     

Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Comparative characteristic of activity of urease, incorporated into polysiloxane hydrogels and xerogels, obtained by sol-gel technique

Polysiloxane hydrogels with incorporated urease (degree of immobilization is 79-88%) that retains fermentative activity at the level of 56-84% were obtained by sol-gel technique. An influence of polysiloxane matrix functionalization on a degree of incorporation, activity retention and a factor of efficiency of urease immobilization was studied. Polysiloxane matrix functionalization with methyl groups causes decreasing a degree of ferment immobilization and a factor of immobilization efficiency. Functionalization of polysiloxane matrix with 3-aminopropyl groups leads to practically quantitative incorporation of the enzyme. And the highest degree of urease activity retention and maximal factor of its immobilization were observed at 3-aminopropyl groups content in polysiloxane matrix equals 2-5% (mol.). Transformation of hydrogels into xerogels via vacuum drying causes decreasing urease fermentative activity on nearly 2 orders.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin–avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

One step immobilization of peptides and proteins by using modified parylene with formyl groups

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.     

Thiophilic adsorption of immunoglobulins--analysis of conditions optimal for selective immobilization and purification

Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effects of water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.     

Enzyme immobilization on nylon-optimization and the steps used to prevent enzyme leakage from the support

Because of its low cost, chemical and mechanical properties and ready availability in a number of different forms (e.g. powders, beads, nets, tubes, film, sheets, etc.) Nylon is an attractive matrix for enzyme immobilization. We report here a thorough evaluation of a protocol for enzyme immobilization on nylon film with relatively inexpensive and non-toxic reagents, involving acid hydrolysis, glutaraldehyde coupling and spacer molecules and employing beta-glucosidase and trypsin as model enzymes. We also describe steps for virtually eliminating enzyme leakage and non-specific binding. Individual steps in the procedure are simple and conditions flexible so, whilst evaluated in terms of binding proteins to nylon film, they should be applicable to other forms of nylon and suitable for binding most enzymes and proteins, including antibodies, providing a method having potential in both affinity chromatography/adsorption and in bioreactor applications.     

Electrochemically controllable conjugation of proteins on surfaces

The rational design of surfaces for immobilization of proteins is essential to a variety of biological and medical applications ranging from molecular diagnostics to advanced platforms for fundamental studies of molecular and cell biology. We have developed an advanced electrochemically based approach for site-selective and reaction-controlled immobilization of proteins on surfaces. When a molecular monolayer of 4-nitrothiophenol on gold electrode surfaces is reduced electrochemically in a selective fashion at its nitro groups, to afford amino groups by potentiometric scans, the amine can be employed to orchestrate the immobilization of proteins to the surface. This protein immobilization strategy could allow one to fabricate intricate protein structures on surfaces for addressing fundamental and applied problems in biology and medicine.