Biotinylation extraction

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Purify the biotinylated DNA probe by ethanol precipitation, gel filtration, w-butanol extraction, or dialysis as discussed in previous sections. 

SPR systems also showed encouraging results with their ability to detect mycotoxins. The BIACORE was used to detect a mycotoxin, DON, produced by Fusarium species, from spiked wheat sample in a competitive inhibition assay (Schnerr et ah, 2002). Biotinylated DON was immobilized on the sensor chip which was previously coated with strep-tavidin. Mycotoxin extracts from wheat samples were first allowed to react with the antibody and then injected into the BIACORE. The detection range was established to be 0.13-10 pg/ml. In a slightly modified format, DON was also detected by SPR at a range of 2.5-30 ng/ml (Tudos et ah, 2003). 

Anti-anaphylactic activity. Water extract of the dried fruit, at a concentration of 1 pg/ mL, produced weak activity on the rat Leuk-RBL 2H3 vs biotinyl immunoglobulin E-avidin complex-induced degranulation of (3-hexosaminidase 5 Anti-androgenic effect. Ethanol (95%) extract of the aerial parts, administered intraperitoneally to castrated mice at a dose of 2 mg/animal, produced strong activity The dried leaf, smoked by 13 male adults for 21 days, was inactive . 

In addition to the attempts at unraveling the modification site, Dive and coworkers also constructed two biotinylated A/BPs, 22 and 23 (Scheme 3c), and used these to study the difference in affinity- and photoaffinity MMP enrichment from a complex proteome [57]. For this, tumor extracts were spiked with hMMP-12 and hMMP-8, after which compounds 22 and 23 were applied, followed by streptavidin-coated magnetic beads for MMP pull-down. Affinity-based labeling with 23 appeared superior to photoaffinity-based labeling with 22 in terms of quantity of captured MMPs and identification of the tryptic fragments by mass spectrometry. 

Yeast contains large concentrations of biotin or biotinyl derivatives. However, when yeast extracts are administered to symptomatic children with profound biotinidase deficiency, there has been no clinical improvement. What is a possible explanation for this observation ... 

The same authors also examined the S-nitrosylation of proteins by nNOS, in neuronal tissue from mice (Figure 11.10). Conversion of nitrosylated to biotinylated cysteines was done as above. Biotinylation was, in this case, used to extract the (formerly) nitrosylated proteins from the total mixture of cellular proteins by selective binding to solid-phase attached streptavidin. After retrieving the bound material by reduction with excess free thiol, the samples were run on a gel, blotted, and individual proteins detected using an-... 

Af-(D-Biotinyl)-0-(3,4,6-tri-0-acetyl-2-azido-2-deoxy-a-D-galactopyranosyl)-L-threonine te/t-Butyl Ester 23 [34], A mixture of D-biotin (150 mg, 0.6 mmol), l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC 580 mg, 3 mmol), and 1-hydroxy-benzotriazole (HOBt 540 mg, 4 mmol) in dimethylformamide (DMF 2 mL) is stirred under exclusion of moisture at 22 C. After 45 min, the biotin is dissolved, and a solution of freshly prepared glycosyl threonine ester 22 (0.3 mmol, preceding procedure) in dichloro-methane (2 mL) is added at 0°C. After stirring for 16 h at room temperature, the solvent is evaporated in vacuo, the remainder dissolved in dichloromethane (50 mL), extracted with ice-cold 0.2 N HCl (3 x 25 mL), water (25 mL), and saturated NaHCOj solution (2 x 25 mL), dried with MgSO, and concentrated in vacuo. Purification by flash chromatography on silica gel (20 g) in dichloromethane-ethanol (25 1) yields 23 200 mg (93%) [a] 96.5° (c 1, CHClj) Rf 0.29 (toluene-acetone 4 1). 

To monitor protease activities in plant extracts, Wang etal.li8 generated biotinylated peptides that contained a /3-lactone reactive group. The probes labeled several enzymes in leaf proteomes of Arabidopsis thaliana. Interestingly, these studies led to the identification of a papain-like protease called RD21 that has the unexpected ability to ligate donor molecules such as peptides or lactones, probably through a thioester intermediate, to unmodified N termini of acceptor molecules. 

A simple and economical way to obtain complexes of optimum activity consists of the use of small column of iminobiotin saturated with avidin according to Section 3.2.1.3 (crude extract from egg whites can be used directly). One of the binding sites of avidin is occupied by the iminobiotin while the other remains free for reaction with the subsequently added biotinylated POase. Avidin is then added to saturate additional free biotin groups on POase. Complexes are then eluted from the column with 50 mM ammonium acetate (pH 4.0). 

Phenol extraction of biotinylated or DIG-probes should always be avoided due to the possible partitioning of the probe into the phenol layer. Similarly, NACS resins should not be used for the purifica-... 

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