A nonradioactive biochemical characterization of membrane proteins using enhanced chemiluminescence.

Here we demonstrate a nonradioactive immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique which replaces the standard practice of isotopic protein labeling by iodination or metabolic tagging in the analysis of membrane proteins. The technique has proved extremely valuable in the biochemical analysis of small quantities of frozen, pathological tissue. Membranes were prepared from Dx3 (a human melanoma cell line), C6 (a rat glial cell line), and osteoclastoma (a human giant cell tumor of bone). The membranes were labeled with biotin and immunoprecipitated with a variety of antibodies to the vitronectin receptor (VNR). The VNR proteins were resolved by SDS-PAGE and immunoblotted onto nitrocellulose paper. The biotinylated protein was visualized using streptavidin horseradish peroxidase and enhanced chemiluminescence (ECL). Film exposures ranged from 15 min to 16 h. Good visualization of the VNR, yielding the typical heterodimeric receptor of 90 and 150 kDa, was given. Signals generated were high and background noise low with a 30-min film exposure. An overnight exposure increased the detection of weaker bands. In conclusion, biotinylation of membrane proteins proved a satisfactory label for immunoprecipitation and SDS-PAGE analysis. The ECL development stage was extremely flexible with visualization of strong and weak signals. The method has several advantages over a conventional radioactive immunoprecipitation in that it is relatively inexpensive, simple, quick and nonhazardous.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

Quantum-dye labeled proteins for glycobiology: a viable nonradioactive alternative tracer

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology, and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin - mannose- binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 ( Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.      

Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein

Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.     

Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.     

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.     

Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution

The interaction between streptavidin and biotin is one of the most widely used tools in chemistry and biology. However, the release of biotinylated proteins from streptavidin resins remains a major problem, due to the extraordinary stability of this complex. We present a new protocol for the quantitative elution of biotinylated proteins from streptavidin Sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method was demonstrated by the quantitative recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester derivative of biotin.     

The amino acid composition of proteins: a method of analysis.

Four quasiloglinear models are proposed for describing relationships between the amino acid composition of proteins and the structure of the genetic code. The models allow estimation of base frequencies in all three codon positions and can be used to investigate “interactions” between any two codon positions. The estimation procedure proposed by Ohta and Kimura (Genetics64 (1970), 387–395) is discussed and using two of the proposed quasiloglinear models an analysis of the amino acid composition of human cytochrome c is presented. The analysis suggests that of the six codons which code for leucine (CUU, CUC, CUA and CUG) do not occur in human cytochrome c.     

ESIPT-exhibiting protein probes: a sensitive method for rice proteins detection during starch extraction.

The 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzoxazole dye was successfully applied as label of rice proteins during the alkaline extraction of starch. Direct fluorescence measurements were used to observe the presence of proteins labelled in different steps of rice starch extraction. The results were compared to those obtained with the well-known biuret colorimetric test. Whereas the colorimetric test indicates the absence of protein after the third extraction step, the fluorescence emission of the conjugate could be observed in all extraction steps. The separation of different rice proteins could also be observed.     

Construction of biotinylated peptide nanotubes for arranging proteins

Three kinds of biotinylated peptides with different linkers between biotin and beta-sheet peptide were designed and synthesized. The transmission electron microscopy revealed that the biotinylated peptides self-assembled to form a tubular structure with external diameter of ca. 60 nm and inner diameter of ca. 30 nm in an aqueous solution. The anti-biotin antibody effectively bound to biotin groups in the peptide nanotubes. The binding of antibody was regulated by not only the concentration of the protein in the solution but also the properties of biotinylated peptides forming the tubes. The antibody preferentially bound to the biotinylated peptide tubes assembled from the peptide with the most hydrophilic linker, suggesting that the surface properties and functions of the tubular structure were modulated and engineered by the design of the peptides.     

A proteomic approach for the identification of cell-surface proteins shed by metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins.     

Design and synthesis of a novel biotinylated photoreactive insulin for receptor analysis.

B1-(4-Azido-salicyloyl)-[B1-biocytin,B2-lysine]insulin was synthesized by double Edman degradation of A1,B29-Msc2-insulin and stepwise acylation at the N-terminus of the B-chain. This derivative is homogeneous in RP-HPLC and has a biological in vitro activity of 20% and receptor binding of 15%, relative to insulin. Radioiodination and HPLC gave the B1-labelled 125I-derivative (I) as well as the 4 isomers with 125I-labelled tyrosine (A14, A19, B16, B26). UV-induced crosslinking of I with insulin receptors led to specific labelling of the alpha-subunit (Mr 130,000). The peptide bond LysB2-AspB3 is completely cleavable by trypsin (EC 3.4.21.4). I is thus a new tool for the analysis of the hormone-binding region by making possible the isolation of tryptic, biotinylated receptor fragments labelled by the dipeptide 125I-4-azidosalicyloyl-biocytinyl-Lys.     

Partial characterization of cell-surface protein coded for by human chromosome 7

An antigen reactive with antisera to a human chromosome 7-coded cell-surface antigen can be extracted from human cells with the nonionic detergent NP-40. Immune complexes of radiolabeled cell extracts analyzed on SDS polyacrylamide gels showed a single protein with an apparent M.W. of approximately 165,000. This protein was immunoprecipitated from extracts of hybrid cells containing chromosome 7 and human cell extracts, but not from mouse cell extracts or from extracts of a hybrid clone without human chromosome 7. The data presented here indicate that the protein identified on the gels is a cell-surface glycoprotein coded for by human chromosome 7.     

Hybridization of horseradish peroxidase-labeled probes and detection by enhanced chemiluminescence

This article describes the use of probes directly labeled with horseradish peroxidase in conjunction with enhanced chemiluminescence, which allows a flexible approach to hybridizations and detections. This system may be used with the following applications: Southern blots, Northern blots, colony and plaque screening for positive clones, YAC clone screening, and PCR products detection. The major steps required for the use of directly labeled HRP probes are hybridization, stringent washes, and detection.