Analysis of Heparin-Binding Sites in Human Lipoprotein Lipase Using Synthetic Peptides

Synthetic nonbasic peptides based on the type I repeats of thrombospondin (TSP) and four peptides corresponding to the predicted basic clusters in lipoprotein lipase (LPL) have been analyzed for heparin binding. In the present report we examine the structural requirement for the binding of these peptides to heparin-Sepharose column. The peptide containing the sequence Phe-Ser-Trp-Ser-Asp-Trp-Trp-Ser (residues 388–395 in lipoprotein lipase, which include the consensus TSP type I sequence) showed strong binding to heparin. Both the first and second Trp residues in this sequence were essential for tight heparin binding. Substitution of either of the Trp residues by an Ala resulted in the complete loss of heparin binding. The peptides representing the four basic cluster regions of lipoprotein lipase showed variable heparin binding. Strong retention was observed for peptides representing cluster 1 (residues 261–287) and cluster 3 (residues 147–151) peptides followed by cluster 2 (residues 290–302) peptide. A peptide corresponding to LPL cluster 4 (residues 405–414) did not show binding to heparin column. The present study confirms the presence of specific heparin-binding sites in LPL. Furthermore, this study also demonstrates the potential use of synthetic peptides to investigate the interaction between peptides and heparin as an alternative approach to site-directed mutagenesis in selected regions of large protein molecules. The affinity of these peptides toward heparin can be explored to block molecular interactions at these specific sites or to carry and deliver other coupled molecules at the site(s) of attachment of these peptides for therapeutic applications.     

Utilization of Arg-elution method for FLAG-tag based chromatography

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein–protein or protein–ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5–1 M arginine at pH 3.5–4.4, as reported in this paper.     

Automated LC–LC–MS–MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses

A simple multidimensional liquid chromatography system utilizing an isocratic pump and a HPLC system is described for the comprehensive proteomic analysis of complex peptide digest mixtures by coupled LC–LC–MS–MS techniques. A binary ion-exchange separation was achieved through the use of a strong cation-exchange column followed by a reversed-phase column for data-dependent LC–MS–MS analysis of the unbound analytes, and following salt elution (and concomitant column reequilibration), the bound analytes. Off-line validation of the platform showed near quantitative recovery of fractionated peptides and essentially complete ion-exchange partitioning. In comparative analyses of a highly complex peptide digest mixture a >40% increase in the number of peptide and protein identifications was achieved using this multidimensional platform compared to an unfractionated control.     

Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F

The receptor binding to interleukin (IL)-13 is composed of the IL-13 receptor α1 chain (IL-13Rα1) and the IL-4 receptor α chain (IL-4Rα). In order to investigate the interaction of IL-13 with IL-13Rα1 and IL-4Rα, the DNA fragments coding the extracellular regions of human IL-13Rα1 and the IL-4Rα (containing a cytokine receptor homologous region) were fused with mouse Fc and expressed by a silkworm–baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. After further purification with anion-exchange chromatography, these receptors were used to investigate the ligand–receptor interaction. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13Rα1 showed predominant affinity to IL-4Rα, although neither detectable affinity of IL-13 nor IL-13Rα1 was observed against IL-4Rα. Combining these data with the moderate affinity of IL-13 to IL-13Rα1, this indicates that IL-13 first binds to IL-13Rα1 and recruits consequently to IL-4R.     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Three-step purification of a fragment of the large immunophilin FKBP52

PPIases catalyze the interconversion of cis and trans isomers of peptidyl–prolyl (Xaa–Pro) bonds in peptide and protein substrates. The PPIase family comprises three subfamilies, two of which interact with immunosuppressant drugs and are therefore termed immunophilins. One subgroup of the immunophilins are the FK506 binding proteins (FKBPs). FKBPs of a relative molecular mass higher than 40 000 also display chaperone activity and are part of the multichaperone complex that Hsp90 forms with substrate proteins. Their function in this chaperone complex is still enigmatic. To further characterize the function of FKBP52 we want to analyze constructs of FKBP52-fragments. Here we describe a fast and effective three-step purification procedure for a fragment of FKBP52 with a relative molecular mass of 48 000, termed FKBP52–123, consisting of affinity chromatography, anion-exchange column and gel-permeation chromatography. A yield of 1 mg pure protein per gram of cells was achieved.     

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.     

Isolation and study of a fragment of human serum albumin containing one of the antigenic sites of the whole molecule

1. A fragment of human serum albumin called `inhibitor' has been degraded by trypsin, and one of the degradation products, designated fragment F1, has been isolated. Fragment F1 has a molecular weight of 6600. It contains neither tyrosine nor tryptophan. It is not precipitated with rabbit anti-sera to human serum albumin. 2. Fragment F1 was coupled to p-aminobenzylcellulose to form an insoluble conjugate. Rabbit anti-(human serum albumin) antibodies reacting with fragment F1 were specifically adsorbed on this conjugate and were desorbed by glycine–hydrochloric acid buffer. The isolated antibodies are composed of γ-globulin and β₂-macroglobulin. 3. Human serum albumin and fragment F1 formed with 7s anti-(fragment F1) antibodies soluble complexes that were studied by passive haemagglutination, ultracentrifugation and electrophoresis. Fragment F1 was shown to contain only one of the antigenic sites of albumin molecule. The 7s anti-(fragment F1) antibodies were shown to be bivalent and monospecific.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Directed co-evolution of interacting protein–peptide pairs by compartmentalized two-hybrid replication (C2HR)

Directed evolution methodologies benefit from read-outs quantitatively linking genotype to phenotype. We therefore devised a method that couples protein–peptide interactions to the dynamic read-out provided by an engineered DNA polymerase. Fusion of a processivity clamp protein to a thermostable nucleic acid polymerase enables polymerase activity and DNA amplification in otherwise prohibitive high-salt buffers. Here, we recapitulate this phenotype by indirectly coupling the Sso7d processivity clamp to Taq DNA polymerase via respective fusion to a high affinity and thermostable interacting protein–peptide pair. Escherichia coli cells co-expressing protein–peptide pairs can directly be used in polymerase chain reactions to determine relative interaction strengths by the measurement of amplicon yields. Conditional polymerase activity is further used to link genotype to phenotype of interacting protein–peptide pairs co-expressed in E. coli using the compartmentalized self-replication directed evolution platform. We validate this approach, termed compartmentalized two-hybrid replication, by selecting for high-affinity peptides that bind two model protein partners: SpyCatcher and the large fragment of NanoLuc luciferase. We further demonstrate directed co-evolution by randomizing both protein and peptide components of the SpyCatcher–SpyTag pair and co-selecting for functionally interacting variants.     

Binding of laminin and fibronectin by the trypsin-resistant major structural domain of the crystalline virulence surface array protein of Aeromonas salmonicida.

The surface of Aeromonas salmonicida is covered by a tetragonal paracrystalline array (A-layer) composed of a single protein (A-protein, Mr = 50,778). This array is a virulence factor. Cells containing A-layer and isolated A-layer sheets specifically bound laminin and fibronectin with high affinity. Binding by cells was inactivated by selective removal of A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor tetragonal paracrystalline array producing Aeromonas hydrophila and Aeromonas sobria strains bound either matrix protein. Laminin binding was by a single class of high affinity interactions (cell Kd = 1.52 nM), whereas fibronectin bound via two classes of interactions, one being similar to that of laminin (cell Class 2 interaction Kd = 6.6 nM). This interaction with both proteins was partly hydrophobic. The Class 1 fibronectin interaction was of lower affinity (cell Kd = 218 nM) and distinct. Purified A-protein inhibited binding of both matrix proteins to A-layer, and trypsin cleavage localized the matrix-protein binding region to the N-terminal major trypsin-resistant structural domain of A-protein. Monoclonal antibody inhibition studies showed that A-protein was folded such that Fabs of only one of two antibodies with epitopes mapping C-terminal to this trypsin-resistant peptide was capable of blocking binding.     

mRNA display selection and solid‐phase synthesis of Fc‐binding cyclic peptide affinity ligands

Cyclic peptides are attractive candidates for synthetic affinity ligands due to their favorable properties, such as resistance to proteolysis, and higher affinity and specificity relative to linear peptides. Here we describe the discovery, synthesis and characterization of novel cyclic peptide affinity ligands that bind the Fc portion of human Immunoglobulin G (IgG; hFc). We generated an mRNA display library of cyclic pentapeptides wherein peptide cyclization was achieved with high yield and selectivity, using a solid‐phase crosslinking reaction between two primary amine groups, mediated by a homobifunctional linker. Subsequently, a pool of cyclic peptide binders to hFc was isolated from this library and chromatographic resins incorporating the selected cyclic peptides were prepared by on‐resin solid‐phase peptide synthesis and cyclization. Significantly, this approach results in resins that are resistant to harsh basic conditions of column cleaning and regeneration. Further studies identified a specific cyclic peptide—cyclo[Link‐M‐WFRHY‐K]—as a robust affinity ligand for purification of IgG from complex mixtures. The cyclo[Link‐M‐WFRHY‐K] resin bound selectively to the Fc fragment of IgG, with no binding to the Fab fragment, and also bound immunoglobulins from a variety of mammalian species. Notably, while the recovery of IgG using the cyclo[Link‐M‐WFRHY‐K] resin was comparable to a Protein A resin, elution of IgG could be achieved under milder conditions (pH 4 vs. pH 2.5). Thus, cyclo[Link‐M‐WFRHY‐K] is an attractive candidate for developing a cost‐effective and robust chromatographic resin to purify monoclonal antibodies (mAbs). Finally, our approach can be extended to efficiently generate and evaluate cyclic peptide affinity ligands for other targets of interest. Biotechnol. Bioeng. 2013; 110: 857–870. © 2012 Wiley Periodicals, Inc.     

Identification and characterization of an anti-isoaspartic acid monoclonal antibody

The deamidation and rearrangement of protein-bound asparagine residues occurs when peptides and proteins are exposed to acidic or alkaline aqueous media. Asn⁹⁹ of bovine growth hormone (bGH) is readily modified via these mechanisms. We have generated a monoclonal antibody (MAb) that interacts with a bGH fragment that contains an isoaspartyl residue. To obtain this antibody, CAF₁/J mice were immunized with [isoaspartyl⁹⁹]-bGH(96–112) conjugated to BSA. Using a competitive ELISA assay, the interaction of this MAb to [isoaspartyl⁹⁹]-bGH(96–112) has been observed to have an apparentK _m of 150 nM. The corresponding native peptide and other bGH fragments do not bind to this antibody with high affinity. For example, the binding affinities of [Asp⁹⁹]-bGH(96–112) and [Glu⁹⁹]-bGH(96–112) to this antibody are 54- and 78-fold lower than the corresponding isoaspartyl peptide. The antibody also binds to bGH that is enriched in isoaspartic acid at position 99, but not to the unmodified protein. The binding epitope of the peptide has been further characterized by comparing the binding of bGH(96–112) analogues to the MAb. Alanine substitution at residues 99, 100, 101, and 103 reduce binding affinity to the antibody by more than 10³-fold. Replacement of valine with alanine at position 102 has much less impact on antibody affinity. Further experiments suggest that the relative insensitivity to this substitution is due to the structural similarity of these sidechains. Other isoaspartic acid-containing peptides not derived from the bGH sequence do not bind to the antibody. We conclude that the epitope binding site of this MAb is highly specific for 99–103 of [isoaspartyl⁹⁹]-bGH(96–112). This strategy can be used to obtain monoclonal antibodies that selectively interact with other proteins that have been similarly modified, or that contain other chemical modifications. The potential for generating antibodies that universally recognize protein- and peptide-bound isoaspartic acid residues is discussed.     

Isolation of α6β1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OVCAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the α6β1 integrin by immunoprecipitation with specific antibodies against either the α6 or the β1 subunit. The α6β1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the αIIbβ3 integrin from platelets. Furthermore, in immunofluorescence staining, the α6β1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the α6β1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin α3β1. When α3β1 was removed from lysates of OVCAR-4 cells by preclearing with an α3-specific monoclonal antibody, α6β1 was able to bind to human laminin as well. Integrin α6β1 on platelets which do not express α3β1 binds directly to human laminin. These results indicate that both α3β1 and α6β1 can act as receptors for human laminin and may interfere by steric hindrance. The α6β4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.