scholarly journals The rapid “teabag” method for high-end purification of membrane proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jenny Hering ◽  
Julie Winkel Missel ◽  
Liying Zhang ◽  
Anders Gunnarsson ◽  
Marie Castaldo ◽  
...  
Keyword(s):  
Applied Sciences ◽  
Membrane Proteins ◽  
Size Exclusion ◽  
Purification Method ◽  
New Approach ◽  
Purification Time ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Processing Steps

Abstract Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast “teabag” purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2455-2462 ◽  
Author(s):  
Masaru Nagai ◽  
Maki Kawata ◽  
Hisayuki Watanabe ◽  
Machiko Ogawa ◽  
Kumiko Saito ◽  
...  
Keyword(s):  
Lentinula Edodes ◽  
Veratryl Alcohol ◽  
Size Exclusion ◽  
Melanin Synthesis ◽  
Sds Page ◽  
Diammonium Salt ◽  
Exclusion Chromatography ◽  
Homogeneous Preparation

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2020 ◽  
Author(s):  
Fei Jin ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
Motoyuki Hattori
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
Size Exclusion ◽  
Expression Screening ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Exclusion Chromatography

AbstractMembrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

scholarly journals Cost-Effective Paramagnetic Bead Technique for Purification of Cycle Sequencing Products

Sequencing ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Slavica Mijatovic-Rustempasic ◽  
Michael A. Frace ◽  
Michael D. Bowen
Keyword(s):  
Size Exclusion Chromatography ◽  
Cost Effective ◽  
Size Exclusion ◽  
Procedure Time ◽  
Cycle Sequencing ◽  
Reagent Cost ◽  
Exclusion Chromatography ◽  
Paramagnetic Bead

The quality of sequencing results depends greatly upon the quality and purity of the template as well as the purity of the fluorescently labeled products generated by cycle sequencing. Numerous approaches have been used for purification of cycle sequencing products, including alcohol precipitation, affinity-based chromatography, size exclusion chromatography, commercially-available proprietary methods, and paramagnetic bead technology. In this paper, we describe an affordable paramagnetic technology method using BioMag Carboxyl beads. Compared to other well-established, proprietary methods for purification of cycle sequencing products, this method produced consistently good results, with a very low reagent cost and short procedure time.

Effect of Some Hydrolytic Parameters in the Action of Subtilisin and Pancreatin on Whey Protein Concentrate

2013 ◽  
Vol 9 (1) ◽  
pp. 55-66 ◽  
Author(s):  
Marialice P.C. Silvestre ◽  
Wendel O. Afonso ◽  
Carlos O. Lopes Junior ◽  
Viviane D.M. Silva ◽  
Mariana W.S. Souza ◽  
...  
Keyword(s):  
Whey Protein ◽  
Reaction Times ◽  
Point Of View ◽  
Size Exclusion ◽  
Whey Protein Concentrate ◽  
Protein Concentrate ◽  
Exclusion Chromatography ◽  
Peptide Profiles ◽  
Hydrolysis Of

AbstractIn this work, the influence of some reactional parameters in the hydrolysis of whey protein concentrate (WPC) was evaluated, in terms of the nutritional quality of peptide profiles of the hydrolysates as well as the reduction of costs for scaling-up the process. Two enzymes (subtilisin and pancreatin) were used for preparing 18 hydrolysates, using different E:S ratios and reaction times, and the distribution of peptides according to chain length was analyzed by size-exclusion chromatography. The studied parameters affected the peptide profiles of WPC hydrolysates and the best result was similar for subtilisin and pancreatin, both using an E:S ratio of 4:100, after 5 h and 10 h, respectively. In these conditions, these enzymes gave rise to the smallest large peptide contents (12.28% and 12.34%, respectively) and one of the highest amount of di- and tripeptides (13.34% and 13.00%, respectively) as well as of free amino acids (45.56% and 47.26%, respectively). However, in terms of number of samples the action of pancreatin was more advantageous than subtilisin, since among the nine hydrolysates, four showed appropriate peptide profiles (P1, P2, P5, and P6), from the nutritional point of view, while the same happened only with one hydrolysate prepared by using subtilisin (S3). Also, the economical advantage of using smaller E:S ratio and reaction time was observed in several cases for both enzymes.

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