1,720,981 research outputs found
Characteristics of an immunoglobulin-binding protein based on a single domain of protein G from Streptococcus group G
No full textStudies on the binding interaction between peptostreptococcal protein L and a recombinant Fv
No full textProtein L is a multidomain cell wall protein from Peptostreptococcus magnus that is one of a group of proteins capable of binding to antibodies without producing an immune response. In contrast to other immunoglobulin (Ig) binding proteins, such as protein A from Staphylococcus aureus and protein G from group C and G streptococci, which bind at the CH2–CH3 interface, protein L binds exclusively to the VL domain of k-chains. It has previously been shown that a single Ig binding domain of protein L (PpL) has two sites of interaction with the VL domain, with the affinity of the second site up to 50-fold less than that of the first, depending on the nature of the k-chain. The two sites on PpL are distinct, with only one common residue implicated in both interactions. In contrast, the binding sites on the VL domain appear to have 10 residues common to both PpL interactions.The binding interaction between PpL and a 12.6 kDa variable light chain fragment (Fv) has been investigated. Production of a recombinant PpL and recombinant Fv fragment allows the structure of each species and the specific nature of the binding interaction to be examined using a range of techniques, including stopped-flow fluorimetry, circular dichrosism, isothermal titration calorimetry and NMR. To further understand the interaction between PpL and Fv, in particular at the second binding site, mutagenesis of both species has been undertaken and here we discuss the effect of those changes on the affinity of PpL for Fv and vice versa
Construction and characterization of protein rLG, a novel 16.5 kDa hybrid protein with a large binding repertoire for immunoglobulin fragments
No full textSeveral proteins isolated from the surface of Gram-positive pathogenic bacteria have been shown to bind immunoglobulin (Ig) in a non-antigenic manner. The most widely studied of these proteins are protein A from Staphylococcus aureus, protein G from groups C and G streptococci, and protein L from Peptostreptococcus magnus. Although very useful reagents these all have limitations to their general applicability. Attempts have previously been made to broaden the binding spectra of individual Ig-binding molecules through the production of multidomain fusion proteins. The binding repertoires of proteins A and G restricts binding of a construct to specific Ig isotypes only. The ability of protein L to uniquely bind the variable domain of k-chains makes this protein a potential tool for the purification of Ig regardless of isotype. In the present study, a single k-chain binding domain of protein L was linked to a single Fc-binding domain of protein G to yield the novel, recombinant protein rLG gene. The rLG gene was cloned into the expression vector pKK223-3 allowing it to be over-expressed in E.coli JM103 cells. These studies show that despite its small size (Mr = 16.5 kDa), protein rLG exhibits simultaneous binding of ligands to both moieties, thereby making it a more versatile tool for Ig purification. A program of site directed mutagenesis (SDM) has been employed to characterize the binding properties and structural stability of protein rLG by equilibrium and stopped-flow fluorimetry, isothermal titration calorimetry, immuno-diffusion assay, affinity chromatography, circular dichroism and chemical denaturation studies
Studies on a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus: the role of tyrosine-53 in the reaction with human IgG
No full textChemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P, magnus strain 3316) and the kappa light chain (?-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr(51) and Tyr(53)) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr(53). Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG, Modification of ?-chain by TNM resulted in the nitration of 3.1 +/- 0.09 tyrosine residues. When the PpL-?-chain complex was incubated with TNM, 4.1+/-0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The K-d for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112 +/- 20 nM. A similar value (153 +/- 33 nM) was obtained for the complex formed between IgG and the Tyr(64) ? Trp mutant (Y64W). However, the K-d values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2+/-0.2 and 4.6+/-1 muM respectively. These suggest that the phenol group of Tyr(53) in PpL is important to the stability of the PpL-?-chain complex
Recombinant production of a V-L single domain antibody in Escherichia coli and analysis of its interaction with peptostreptococcal protein L
No full textA ?-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin binding domains from peptostreptococcal protein L (PpL) have been shown to bind specifically to ?-light chains. We have produced recombinant PpL, at high yield, and this was used to custom-produce PpL–Sepharose affinity columns. Here, we show that the affinity purification of VL dAb by this method is simple and efficient with no apparent loss in protein at any stage. The truncated dAb protein product was confirmed by electrospray mass spectrometry and N-terminal sequencing. When analyzed by SDS–PAGE it was shown to be over 95% pure and produced at yields of 35–65 mg/L of culture medium. The dAb protein produced was shown by NMR and CD to be a folded ?-sheet domain. This domain is bound by PpL with a Kd of not, vert, similar50 nM as determined by stopped-flow fluorimetry
Protein patterning on silicon (100)
No full textSilicon is an ideal substrate for the development of micro and nano bio-molecular sensor devices; the fabrication technology developed for microelectronic industries is now finding extensive applications in the creation of microfluidic devices. To realise these devices an approach for the creation of precise, highly stable motifs of biomolecules on the silicon surface is needed. Covalent attachment of proteins to crystalline silicon (100) surfaces has recently been achieved in patterns with micron sized features, using a straightforward two-step process, by us. UV light exposure through a mask was used to covalently attach a monolayer of alkene, functionalized with N-Hydroxysuccinimide ester groups, onto a hydrogen-terminated silicon (100) surface. The N-Hydroxysuccinimide ester surface is a template for the subsequent bioconjugation of a single lg-binding domaion of Protein L via the protein amino group present in external lysine residues of the protein. The patterned surfaces were revealed using epifluorescence microscopy; fluorescently labelled Mouse lgG was associated with the surface attached protein L domain. This method is to be developed for the creation of novel microfluidic sensing technology in silicon for proteomic applications
Stereochemistry at phosphorus of the reaction catalyzed by myo-inositol monophosphatase
No full textmyo-Inositol monophosphatase (IMPase), the proposed target for lithium therapy for manic depression, is an important enzyme in the biosynthesis of second messengers. Earlier studies have shown that the IMPase-catalyzed hydrolysis of myo-inositol monophosphates to inorganic phosphate and myo-inositol proceeds by direct attack of water at phosphorus. However, research groups have independently proposed either an in-line displacement (with inversion of stereochemistry at phosphorus) or an adjacent attack with a pseudorotation (with retention of stereochemistry at phosphorus). Here, the elucidation of the stereochemical pathway is presented. The IMPase-catalyzed hydrolysis of D-1-Sp-myo-inositol [17O]-thiophosphate in the presence of H218O gave inorganic Rp-[16O,17O,18O]-thiophosphate, with inversion of configuration at phosphorus. This is only consistent with an in-line displacement, and it rules out the controversial adjacent/pseudorotation mechanism. This result will assist in the design of alternative inhibitors of IMPase
Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor f chain by the protein tyrosine kinase Lck
No full textWe report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor f chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the f chain. Preliminary 1H-NMR studies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six f chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C >2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor f chain. After in vitro phosphorylation by Lck, the partially phosphorylated f chain was digested with trypsin. Separation and identification of the f chain fragments using LC–MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six f chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo
Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin
No full textDomain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug-protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process
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