Amperometric immunosensor for carcinoembryonic antigen detection with carbon nanotube-based film decorated with gold nanoclusters

In a recent article, we described the application of phasor analysis to fluorescence intensity decay data on in vitro samples. As detailed in that article, this method provides researchers with a simple graphical method for viewing lifetime data that can be used to quantify individual components of a mixture as well as to identify excited state reactions. In the current article, we extend the use of in vitro phasor analysis to intrinsic protein fluorescence. We show how alterations in the excited state properties of tryptophan residues are easily visualized using the phasor method. Specifically, we demonstrate that protein–ligand and protein–protein interactions can result in unique shifts in the location of phasor points, indicative of protein conformational changes. Application of the method to a rapid kinetic experiment is also shown. Finally, we show that the unfolding of lysozyme with either urea or guanidine hydrochloride results in different phasor trajectories, indicative of unique denaturation pathways.     

Fabrication of protein gradients for cell culture using a miniature squeegee

We present a straightforward method to create spatial gradients of substrate bound protein for live cell studies using only mechanical parts. Protein concentration gradients on a micron scale can be fabricated in several minutes for a relatively low cost using a method that is generally applicable to any protein and substrate combination. We describe the details of the device construction, and provide examples of mammalian cells grown on substrates patterned with protein concentration gradients using this technique.     

An immunoassay method for quantitative detection of proteins using single antibodies

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose-response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01 ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose-response was 3.3 to 0.01 ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.     

鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定

目的建立一种高效提取、分离、纯化鼠尾胶原蛋白的方法。方法通过对鼠尾进行剥离获得鼠尾腱,用Tris-HCl缓冲液、胃蛋白酶处理获得鼠尾胶原蛋白原液、反复使用氯化钠溶液进行分级盐析、醋酸溶液复溶进行鼠尾胶原蛋白的纯化。超纯水透析除去无机盐类获得纯化的鼠尾胶原蛋白。通过SDS-PAGE蛋白质电泳、氨基酸含量分析等技术手段鉴定。结果本研究建立的方法可以获得高纯度的鼠尾胶原蛋白,纯度达到电泳纯。与国外进口的商业化鼠尾胶原蛋白产品相比无差异。研究了提取、分离、纯化参数对得率、纯度的影响,建立了最优的鼠尾胶原蛋白提取条件,胃蛋白酶用量：1∶500,酶解时间：72 h,盐析浓度：2 mol/L,提取所用酸溶液：0.05mol/L醋酸溶液。结论为鼠尾胶原蛋白的扩大化生产提供了合适的工艺参数,为大量获得鼠尾胶原蛋白并进行更深层次的功效方面研究提供了理论支持和实践基础。     

Ratiometric Raman spectroscopy for quantification of protein oxidative damage

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement because it requires only one conjugation reaction, uses a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is approximately 0.33 pmol carbonyl per measurement. Fluorescence and/or immunoassay-based techniques only detect a signal from the labeling molecule and, thus, yield no structural or quantitative information for the modified protein, whereas the RMRS technique allows protein identification and protein carbonyl quantification in a single experiment.     

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).     

Methods for mapping of interaction networks involving membrane proteins

Nearly one-third of all genes in various organisms encode membrane-associated proteins that participate in numerous protein-protein interactions important to the processes of life. However, membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is limited with respect to the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.     

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.     

雄激素含量测定用蛋白微阵列的制备

利用分子生物学技术对鼠雄激素受体结合域(rARLBD)重组表达,结合新兴的蛋白质微阵列技术建立了一种快速无污染的检测方法,用于临床雄激素检测及新药发现。实验将编码ARLBD的cDNA片段(888bp)插入到含有多聚组氨酸标签的表达载体pET32a中构建了表达质粒pET32a/AR,并将其转化到大肠杆菌BL21中。经IPTG低温诱导,得到高效表达的可溶性ARLBD融合蛋白产物,并通过NiNTA凝胶亲和吸附纯化。将纯化得到的ARLBD使用芯片点样仪固定到硅烷化-多糖表面片基,制备得到ARLBD蛋白质微阵列。实验得到的特异性结合曲线表明在微阵列上的受体蛋白能够保持功能构像。通过Scatchard方程计算得到微阵列上ARLBD与荧光标记的睾酮结合的Kd值为5.32nmol/L。浓度依赖性标准曲线表明这一方法有较高的灵敏度,最低检测限达1pmol/L。应用这一方法对224例健康中国老年人血清雄激素水平进行了测定,研究证明了该方法的可靠性,并首次提供了一定样本量的我国老年人血清活性雄激素水平的参考值。这一技术的建立将为生物工程技术产品与临床检测和分子水平化合物筛选有效地结合提供一条新的途径。     

Protein aggregation and degradation during iodine labeling and its consequences for protein adsorption to biomaterials

Protein adsorption on modified and unmodified polymer surfaces investigated through radiolabeling experiments showed a tendency for higher than expected albumin and immunoglobulin G (IgG) adsorption. Possible enhanced protein aggregation and degradation caused by the iodine labeling method used were analyzed through chromatography and spectroscopy techniques. Results show that the iodine labeling method using chloramine-T (CAT) as an oxidizing agent can cause both enhanced aggregation and fragmentation of proteins. Albumin shows an enhanced tendency to aggregate after iodine labeling using the CAT method, and higher amounts of fragmentation are observed for CAT-labeled IgG molecules relative to unlabeled IgG molecules as well as to IgG molecules labeled using the Iodo-Gen method. These results show that the widely applied method of radioisotope labeling for quantitative assessment of protein adsorption should be used with caution and preferably should be validated by a label-free methodology for each combination of radiolabel and protein. The results obtained in this study can be used to optimize investigation of protein adsorption on surfaces of materials for biomedical devices.     

Spectroscopic determination of protein concentrations from proteinase K digests

A method to determine protein concentrations and absorptivities based on absorbance measurements of proteinase K digests has been developed. Molar absorptivities of proteinase K digests at 56 degrees C can be predicted by using the following equation: epsilon (M)(280)=5318 x (No.of Trp) + 1227 x (No.of Tyr) + 133 x (No.of Cys-Cys). Protein concentration in the digest can be determined by dividing the corrected digest solution absorbance by the calculated epsilon(M)(280). The absorptivity of a native protein can then be calculated by dividing the absorbance of the intact protein solution by the concentration value obtained for the digest solution. Precision of the experimental data is within +/-3%, and the error of the method does not exceed 4.5%. The accuracy of determination does not depend on the size of the protein, Trp/Tyr ratio, presence or absence of certain chromophores, or other structural factors. The method requires amounts of protein routinely used for absorbance measurements.     

A new method for protein domain recognition

A fuzzy cluster method is presented to recognize protein domains. This algorithm can identify domains globally. A protein structure set was used to test the algorithm. Among 219 proteins, 66.7% yielded results that agreed with the reference definitions, 30.6% showed minor differences, and only 2.7% (six proteins) showed major differences with the reference. The new method is more than 20 times fast than previous algorithms. Received: 9 November 1998 / Revised version: 20 December 1999 / Accepted: 20 December 1999     

Strategies for folding of affinity tagged proteins using GroEL and osmolytes

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.     

An automated high-throughput screening method for the identification of high-yield, soluble protein variants using cell-free expression and systematic truncation

A highly automated method for rapidly identifying soluble protein variants with good expression yields has been developed. This method is based on a commercially available in vitro protein expression system. It consists of two polymerase chain reactions (PCR) followed by in vitro protein expression and protein quantification by dot blot. The PCR protocols have been improved and optimized to allow automation using commercial fluid handling devices. A PCR primer design program has also been implemented to streamline protein variant design. This automated protocol is highly reliable and has tremendously improved the throughput of expression screening as compared to conventional cell-based methods and manual in vitro methods. We have applied this method to 32 problematic targets from the TB Structural Genomics Consortium. Experimental results of these studies are reported.     

Analysis of complex protein-polypeptide systems for proteomic studies

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.     

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.     

Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies

Here we present a method to purify large amounts of highly pure and stably arrested ribosome-nascent chain complexes (RNCs) from Escherichia coli cells. It relies on the combined use of translation-arrest sequences to generate nascent polypeptides of specified length and subsequent tag purification of the RNCs. Moreover, we adapted this method for the in vivo production of RNCs with specific isotope labeling of the nascent chains for nuclear magnetic resonance (NMR) studies. This method opens therefore possibilities for a wide range of biochemical and structural studies exploring conformations of nascent chains during the early steps of protein folding and targeting.     

Two-color selection for amplified co-production of proteins in mammalian cells

Structural and functional analyses for many mammalian systems depend on having abundant supplies of recombinant multi-protein complexes that can be produced best, or only, in mammalian cells. We present an efficient fluorescence marking procedure for establishing stable cell lines that overexpress two proteins in co-ordination, and we validate the method in the production of monoclonal antibody Fab fragments. The procedure has worked without fail on all seven of seven trials on Fabs, which are being used in the crystallization of G-protein coupled receptors. This manner of efficient selection may readily be adapted for the co-production of other complexes of two or more proteins.     

Rapid estimation of potato tuber total protein content with coomassie brilliant blue G-250

Summary A new method for measuring protein with Coo-massie Brilliant Blue G-250 has been adapted for use as a screening method in a potato tuber protein improvement breeding program. The method is simple, fast and inexpensive, and has successfully estimated the total protein content of a broad range of tuber genotypes having dissimilar amino acid profiles and tuber maturities. Correlation between the Coomassie method and a modified micro-Kjeldahl method, the standard method used in the potato breeding program, was 0.93. Free amino acids and other compounds which interfere with other methods for measuring protein do not interfere with the Coomassie Brilliant Blue procedure.This research was supported in part by the Rockefeller Foundation. Scientific Journal Series Article # 10,157 of the Minnesota Agricultural Experiment Station     

Phylogeny reconstruction for protein sequences based on amino acid properties

This paper presents an essentially new method used to construct phylogenetic trees from related amino acid sequences. The method is based on a new distance measure which describes sequence relationships by means of typical steric and physicochemical properties of the amino acids and is advantageous in some essential points. The method was applied to different sets of protein sequences and the results were compared with other well-established methods.