Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.     

A versatile non-radioactive technique for restriction analysis of recombinant DNA

Restriction analysis of recombinant DNA is most frequently performed according to Smith and Birnstiel by labeling 5'-termini with 32P, followed by partial digestion, separation, and autoradiographic detection of labeled fragments. We describe a rapid, non-radioactive technique for restriction analysis of recombinant DNA which combines Southern blotting of partial restriction digests and hybridization with a vector-specific probe labeled with the steroid-hapten Digoxigenin for immunological detection. This technique has several advantages compared to conventional methods. Labeling with 32P is not necessary and as the labeled DNA-fragment used as probe is vector-specific, it can be applied for numerous constructs using the particular cloning vector (e.g.pBR322). Furthermore, the probe can be stored for several months and can be reused many times.     

Criteria for the selection of the most desirable radionuclide for radiolabeling monoclonal antibodies

Efficient labeling of monoclonal antibodies depends on a number of key factors, mostly related to the characteristics of the radionuclide itself and to the manner of its incorporation into the protein. Such factors include the physical half-life, the photon or particle energy of the radionuclide and its selective deposition of energy in tissues, the method of labeling used (covalent binding or chelation), and the effect that the chemical changes inherent in the labeling process may have on the properties of the protein or of its fragments. The major biological factor in determining the radionuclide of choice for labeling is the projected use of the labeled antibody. When the intended use is diagnostic, then what is required is high-photon density for achieving the high resolution needed for imaging, whereas therapeutic use requires radionuclides with high energy deposition at the target sites, i.e. β or α emitters. A further consideration is to be given to the mode of administration of the radiolabeled monoclonal antibody: determination of the radiopharmacokinetic parameters of compartmental models of biodistribution of the labeled monoclonal antibody and/or its fragments may also assist in selecting which radionuclide may be best to use for radiolabeling a given monoclonal antibody intended for either tumor diagnosis, prognosis and/or therapy.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

抗人卵巢癌单域抗体V_H 基因的克隆、表达及放射免疫显像

 为研究小分子抗体作为导向载体的作用 ,制备抗人卵巢癌单克隆抗体COC1 83B2 单域抗体(重链可变区 ,VH) ,并进行放射免疫显像 (RII) .利用PCR技术从COC1 83B2 杂交瘤细胞中扩增COC1 83B2 的VH 基因片段 ,并将其克隆入高效表达载体pTHA90中 .重组子转化大肠杆菌TOP10 ,IPTG诱导表达VH 融合蛋白 ,经改进氯化亚锡法进行锝 ( 99mTc)的标记 ,在卵巢癌裸鼠皮下瘤模型上进行RII.结果表明 :( 1 )VH 融合蛋白以包涵体形式获高效表达 ;( 2 )改进氯化亚锡法成功进行VH 融合蛋白99mTc的标记 ,标记率达 96%以上 ;( 3)用99mTc标记VH 融合蛋白进行RII,肿瘤显像早 ,图像清晰 .说明99mTc可用于小分子抗体标记 ,应用单域抗体VH 可改善RII     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Development of annexin V mutants suitable for labeling with Tc(i)-carbonyl complex

(99m)Tc-annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V. Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues. These molecules were expressed cytoplasmically in E. coli and purified with a final yield of 33 mg of protein/L of culture. Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations. Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine. Using the Tc-carbonyl reagent, both proteins could be labeled with (99m)Tc to specific activities of at least 10-20 microCi/microg with full retention of bioactivity. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc-carbonyl without altering its high affinity for cell membranes.     

Comparative binding of 125I-and 99mTc-d-labeled native and glycated low-density lipoprotein to human microvascular endothelial cells-potential for atherosclerosis imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with 125I or 99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-gamma receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the KDs using either 125I-or 99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that 99mTc-g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for 99mTc-nLDL and all the 125I-labeled LDL forms. Binding studies using both 125I-and 99mTc-nLDL indicated a weak binding affinity (KD 10- 7mol/L) to human microvascular endothelial cells. The binding affinity of 125I-g/goLDL to these cells was significantly higher (KD 10- 9mol/L) and could be increased further by preactivation of the endothelial cells using TNFalpha. Incubation with 99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with 99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with 125I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only 125I-labeled goLDL should be used, whereas nLDL may be applied as 125I-or 99mTc-labeled ligand.     

Ca2+-dependent effect of ATP on spin-labeled sarcoplasmic reticulum.

Vesicular fragments of sarcoplasmic reticulum (SR) were labeled with the --SH-directed spin label 2,2,6,6-tetra-methyl,4-amino(N-iodoacetamide). Colorimetric titrations of the remaining --SH residues and determinations of unbound spin label indicated that primarily 3 residues/enzyme molecule were labeled under saturating conditions. This labeling was accompanied by minimal losses in activity, providing precautions were taken to prevent sulfhydryl oxidation during the labeling process. Additions of ATP produced a new "highly constrained" component in the ESR spectrum of the labeled SR, an effect not noted in previous studies. It is demonstrated that the changes produced by ATP are reversible, and require both substrate binding and Ca2+ binding. However, hydrolysis of the substrate is not required. It is further demonstrated that the labeled residue(s) responsible for the spectral change is not in the immediate vicinity of the ATP binding site. It is apparent that the observed spectral change is related to a conformational effect of ATP and Ca2+ on the ATPase protein, which is associated with a large free energy change occurring on binding. It is also suggested that the conformational effect extends to a significant distance from the nucleotide binding site and may be a precursory step to Ca2+ translocation.     

Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR

Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.     

Split Inteins介导的多肽链两端标记

多肽链多位点特异性标记有助于了解蛋白质的结构与功能,特别是在蛋白质的动态构象研究方面. 但是,现有的多肽链多位点特异性标记方法各有局限性,并且种类有限,所以有必要开发新的多肽链多位点特异性标记方法以满足研究需求. 本文以Diub (ubiquitin dimer) 蛋白为研究对象,借助S1和S11型2种不同的断裂蛋白质内含子 (split inteins) 的蛋白质反式剪接,将含有不同荧光基团的两种小肽成功剪接至靶蛋白的两端,最终达到对靶蛋白的末端标记目的.     

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labeling preformed liposomes with technetium-99m (^99mTc) has been developed which is simple to perform and stable in vivo. Previous ^99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (> 90%) with excellent stability. A commercially available radiopharmaceutical kit—hexamethylpropyleneamine oxime (HM-PAO)—is reconstituted with ^99mTcO^−₄ and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity.This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with ^99mTc may also have applications in diagnostic imaging.