scholarly journals PURIFICATION OF RAVE COMPLEX FROM Saccharomyces cerevisiae USING FLAG TAG-AFFINITY PURIFICATION METHOD

2017 ◽  
Vol 51 (5) ◽  
pp. 543
Author(s):  
Nguyen Thanh Trung ◽  
Pham Thi Tam ◽  
Ta Thi Thu Thuy ◽  
Chunyin Gu ◽  
Zhenyu Zhang
Keyword(s):  
Affinity Purification ◽  
Yeast Cells ◽  
Essential Factor ◽  
Purification Method ◽  
Small Column ◽  
C Terminus ◽  
Endosomal Membranes ◽  
Experimental Process ◽  
Cell Disruptor ◽  
Better Than

RAVE (Regulator of the H+-ATPase of the Vacuolar and Endosomal membranes) is an            essential factor of assembly and reversible disassembly of V-ATPase. RAVE complex has three subunits, which are Rav1p, Rav2p and Skp1p. There are few studies on RAVE so it is very             important to study structure of RAVE complex to understand more about the regulation of the assembly and reassembly at V-ATPase. In this study the RAVE complex was purified by affinity purification by fussing FLAG tag to subunit Rav1p or Rav2p. Experimental process: yeast cells were incubated in 8 L YEPD medium at 30 oC, 200 rpm (OD600nm around of 3). Furthermore                   harvested cells were broken by a French pressure cell disruptor at 25,000 p.s.i in TBSE (50 mM Tris/Cl, 150 mM NaCl, 1 mM EDTA, pH 7.4) with 1 mm PMSF. The cell lysate was centrifuged at 20,000 xg for 20 minutes at 4 oC. Then, the supernatant, was achieved by centrifugation, and loaded onto a small column contained 1 ml of anti-FLAG M2 gel. After washing anti-FLAG column with TBSE, RAVE complex was eluted with TBSE containing 100 µg/ml FLAG               peptides. The results showed RAVE complex purification from strain with FLAG tag fused in                 C-terminus of Rav2 is better than RAVE complex purification from the yeast strain S. cerevisiae with FLAG tag fused in N-terminus of Rav1 or C-terminus of Rav1.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Affinity Tags for Protein Purification

2020 ◽  
Vol 21 (8) ◽  
pp. 821-830
Author(s):  
Vibhor Mishra
Keyword(s):  
Protein Purification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Domain ◽  
Affinity Tag ◽  
Small Peptides ◽  
Affinity Tags ◽  
C Terminus ◽  
Solubility Enhancers ◽  
As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

scholarly journals Improved chromatic and sensory characteristics of Plavac Mali wines – efficiency of maceration enzymes

2017 ◽  
Vol 35 (No. 3) ◽  
pp. 236-245 ◽  
Author(s):  
IVANA ALPEZA ◽  
KARIN KOVAČEVIĆ GANIĆ ◽  
ANDREJA VANZO ◽  
STANKA HERJAVEC
Keyword(s):  
Sensory Quality ◽  
Yeast Cells ◽  
Enzyme Preparations ◽  
Pectolytic Enzymes ◽  
Colour Parameters ◽  
The Moment ◽  
Red Grape ◽  
Different Characteristics ◽  
Physical And Chemical ◽  
Better Than

Two commercial enzyme preparations were used in the production of wine from the Croatian autochthonous red grape variety Plavac Mali in order to improve the extraction of polyphenolic components from grapes, chromatic parameters, and sensory quality. During two vintages, the conventional maceration without enzymes was compared with the maceration using products with different characteristics: pectinase with additional cellulase and hemicellulase activity and pectinase with inactive yeast cells. Both products affected polyphenolic extraction and colour parameters: intensity and hue, and ratio between the yellow, red, and blue colour in young wines (2 months after fermentation) and at the moment of bottling (9 months after fermentation). The correlation between anthocyanins and colour intensity was very strong. The expected reduction of quantitative chromatic parameters during aging was confirmed. Significantly better results were observed in wines produced with pectinase, in relation to all analysed physical and chemical parameters. The sensory analysis showed that wines produced with pure pectolytic enzymes were significantly better than those produced without the enzymes. A product of the combination of pectolytic enzymes and inactive yeast cells had a partial influence on the improvement of the phenolic and sensory quality. The overall quality was significantly more expressed in wines produced with pectolytic enzymes, especially in young wines.

scholarly journals Identification of a functionally conserved surface region of rat cytochromes P450IA

1991 ◽  
Vol 278 (3) ◽  
pp. 749-757 ◽  
Author(s):  
R J Edwards ◽  
A M Singleton ◽  
B P Murray ◽  
S Murray ◽  
A R Boobis ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Cytochromes P450 ◽  
Surface Region ◽  
Carrier Protein ◽  
Peptide Antigen ◽  
Aryl Hydrocarbon ◽  
C Terminus ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
The Difference ◽  
Peptide Antibodies

A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.

scholarly journals Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

2007 ◽  
Vol 73 (18) ◽  
pp. 5711-5715 ◽  
Author(s):  
Sung Kuk Lee ◽  
Howard H. Chou ◽  
Brian F. Pfleger ◽  
Jack D. Newman ◽  
Yasuo Yoshikuni ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cross Talk ◽  
Biological Systems ◽  
Wild Type ◽  
Dimerization Domain ◽  
Expression Levels ◽  
Inducible Promoters ◽  
C Terminus ◽  
Increased Sensitivity ◽  
Better Than

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

Identification and Partial Characterization of Fanconi Anemia Associated Polypeptides (FAAPs) Using a Versatile Multiprotein-Complex Purification Method.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 989-989 ◽  
Author(s):  
Abdullah M. Ali ◽  
Thiyam R. Singh ◽  
Ruhikanta A. Meetei
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Affinity Purification ◽  
Gene Products ◽  
Core Complex ◽  
Purification Method

Abstract Fanconi Anemia (FA) is an autosomal recessive and X-linked disorder characterized by congenital abnormalities, progressive bone marrow failure, and a high incidence of hematological (acute leukemia) and non-hematological malignancies (squamous cell carcinomas of the head and neck or gynecologic system). FA is genetically heterogeneous disease and to date 12 complementation groups are known of which 11 gene products have been identified (FANC- A, B, C, D1, D2, E, F, G, J, L, M). Eight of the FA gene products, FANCA, FANCB, FANC, FANCE, FANCF, FANCG, FANCL and FANCM form a multiprotein FA core complex. This complex is required for the monoubiquitination of FANCD2 upon DNA damage by various genotoxic agents. The other two FA proteins; FANCD1/BRCA2 and FANCJ are believed to act “downstream” of FANCD2. In order to understand the role of FA proteins in DNA repair pathway it is necessary to find all the FA genes and their interacting partners. We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCM and FANCL proteins; we have established two different HeLa cell lines; HeLa-HF-FANCM and HeLa-HF-FANCL, stably expressing HF-FANCM and HF-FANCL recombinant proteins respectively. Two step affinity purification was carried out to isolate the complexes from the extracts prepared from stable cell lines. Two polypeptides, namely, FAAP16 and FAAP100 were identified by mass-spectrometry as major interacting partners of FANCM and FANCL respectively. The interaction of FAAP16 and FAAP100 with other FA core complex proteins was confirmed by reciprocal affinity purification coupled mass-spectrometry using HeLa cells stably expressing HF-FAAP16 and HF-FAAP100 proteins. Furthermore, suppression of FAAP16 and FAAP100 in HeLa cells using siRNA resulted in a reduced MMC-induced FANCD2 monoubiquitination. Studies are being carried out to understand the precise role of these proteins in the FA core complex. These data suggest additional proteins interact with FA core complex members and demonstrate the utility of the purification method in delineating interacting proteins involved in FA.

Reversing the oncogenic roles of misdirected chromatin remodeling: Mechanistic insights into the SS18-SSX fusion protein in synovial sarcoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10515-10515
Author(s):  
Cigall Kadoch ◽  
Gerald R. Crabtree
Keyword(s):  
Amino Acids ◽  
Synovial Sarcoma ◽  
Chromatin Remodeling ◽  
Affinity Purification ◽  
Cell Types ◽  
Molecular Feature ◽  
C Terminus ◽  
Therapeutic Development ◽  
Chromatin Remodeling Complexes ◽  
Rhabdoid Tumors

10515 Background: Synovial sarcoma (SS) accounts for ~10% of soft-tissue malignancies and is generally resistant to chemotherapy-based approaches, underscoring the need for a mechanistic understanding of its pathogenesis and the development of disease-specific biologic agents. The hallmark molecular feature is a precise and uniform translocation, t(X;18), which results in the fusion of exactly 78 amino acids of SSX to the SS18 C-terminus. Because the SS18-SSX genetic lesion is observed in 100% of cases, it is likely the driving oncogenic event in these tumors; however, the molecular basis for its role in oncogenesis is undefined. Methods: We performed an affinity purification-/mass spectrometry-based analysis of endogenous mSWI/SNF (BAF) chromatin remodeling complexes in several primary cell types. Using these data in combination with protein biochemical methods, we discovered that SS18 is a dedicated, non-exchangeable subunit of these complexes with a binding affinity comparable to that of ribosomal subunits. Subsequent biochemical and functional investigations were performed to assess the oncogenic consequences of addition of 78aa of SSX to the SS18 subunit in SS. Results: We demonstrate that the SS18-SSX fusion incorporates into BAF complexes, evicting both wild-type (WT) SS18 and the tumor suppressor subunit, hSNF5 (BAF47), known to be biallelically inactivated in pediatric malignant rhabdoid tumors (MRTs). The altered complex binds the Sox2 locus, reversing polycomb-mediated repression and activating Sox2. Sox2, a pro-pluripotency transcription factor, is uniformly expressed in SS tumors and is essential for proliferation. Remarkably, increasing the concentration of WT SS18 leads to reassembly of WT complexes, retargeting of BAF complexes, returned polycomb-mediated repression at the Sox2 locus and cessation of SS cell proliferation. This mechanism of transformation depends on a region of only two amino acids of SSX, and hence provides a strong foundation for therapeutic intervention. Conclusions: These studies provide a novel oncogenic mechanism for SS tumors and inform strategies for therapeutic development in this intractable cancer.

scholarly journals The contribution of the C-terminal sequence to the catalytic activity of GST2, a human Alpha-class glutathione transferase

1991 ◽  
Vol 275 (1) ◽  
pp. 171-174 ◽  
Author(s):  
P G Board ◽  
B Mannervik
Keyword(s):  
Catalytic Activity ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Affinity Purification ◽  
Cumene Hydroperoxide ◽  
Plasmid Vector ◽  
Competitive Inhibitor ◽  
Terminal Segment ◽  
Truncated Form ◽  
C Terminus

A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.

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