Protein purification

Pwo DNA polymerase is an enzyme that is widely used in PCR due to its thermal stability and proofreading activity (3′-5′ exonuclease activity) with high accuracy. Optimizing the expression and purification of the protein is crucial to reducing the cost of production for this important enzyme. In this study, the higher expression level of the gene encoding Pwo DNA polymerase in bacteria strain E. coli BL21(DE3) was achieved by optimizing codon usage.

11p xuanphongdacy06 18-09-2024 2 1   Download

In this study, the DNA fragment encoding for the C-terminus domain of the AopB from Aeromonas hydrophila AH-1 was cloned into pET-M expression vector and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain existed as a mixture of tetramer, trimer, dimer, and monomer forms.

6p guitaracoustic02 08-12-2021 18 3   Download

RNA polymerase II (Pol II) is a 12 subunit protein complex from yeast to human that is required for gene expression. Gdown1 containing Pol II [Pol II (G)] is a special form of Pol II that is catalytically inactive and heavily depends on the 30-subunit Mediator complex for its activator and basal dependent function in vitro. Here we report for the first time, the identification and the generation of a 15-subunit human Mediator complex via the novel multibac baculovirus expression system that is fully responsive to Pol II (G).

9p thiencuuchu 27-11-2021 21 3   Download

The objectives of the thesis: Research in the toxin/protein having bioactive isolated from Heterometrus laoticus (H. laoticus) scorpion and krait Bungarus fasciatus (B. fasciatus) snake venom.

32p extraenglish 24-05-2021 13 3   Download

The objectives of the thesis: Research in the toxin/protein having bioactive isolated from Heterometrus laoticus (H. laoticus) scorpion and krait Bungarus fasciatus (B. fasciatus) snake venom.

32p extraenglish 24-05-2021 19 3   Download

In this study, the recombinant endoglucanase was purified by his - tag affinity chromatography with differrent processes, such as using phosphate buffer with or without sodium cloride, pretreatment of samples with ammonium sulphate before supplying into affinity column, using various concentration of imidazole for washing.

9p tamynhan10 24-10-2020 17 1   Download

Purified viral protein was obtained from Potato virus Y (PVY) pure culture maintained on potato (Solanum tuberosum L.) plants. The average concentration of the purified materials was found to be 153.1 ng/µl. PVY purified virus preparation was used for immunizing rabbit for production of polyclonal antisera against the virus. Good quality antisera were collected one week post boosters (AS4b, AS5b, AS6b and AS7b) and the IgG fractions were separated through ammonium sulphate precipitation.

6p cothumenhmong5 17-05-2020 19 4   Download

Pisum sativump16 is a protein present in the chromatin of ungerminated embryonic axes. The purification of p16 and the isolationof a cDNAclone ofpsp54, the gene encoding its precursor have been recently reported [Castillo, J., Rodrigo, M. I.,Ma´rquez, J. A., Zu´n˜iga, AandFranco, L. (2000)Eur. J.Biochem.267, 2156–2165]. In thepresentpaper,wepresent data showing that p16 is a nuclear protein.

8p research12 23-04-2013 34 2   Download

An extracellular lipase, LipA, extracted fromAcinetobacter sp. RAG-1 grown on hexadecane was purified and proper-ties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to sta-tionary phase, and purified with 22% yield and 10-fold purification. The protein demonstrates little affinity for anionexchange resins,withcontaminatingproteins removed by passing crude supernatants over a Mono Q column....

9p tumor12 22-04-2013 47 2   Download

Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of thegenes encoding individualmrps is still not complete and quite a fewof the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have con-structed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. ...

12p tumor12 22-04-2013 52 2   Download

Thea-phycoerythrocyanin subunits of the different phy-coerythrocyanin complexes of the phycobilisomes from the cyanobacterium Mastigocladus laminosusperform a remarkable photochemistry. Similar to phytochromes – the photoreceptors of higher plants – the spectral pro-perties of the molecule reversibly change according to the irradiation wavelength. To enable extensive analyses,the protein has been produced at high yield by improving purification protocols.

10p tumor12 22-04-2013 45 3   Download

aIIbb3, a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif.However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage.

8p tumor12 20-04-2013 48 2   Download

The peptide hormone endothelin transmits various signals throughGprotein-coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin-1, expressed in Sf9, COS-1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR.

12p fptmusic 16-04-2013 27 2   Download

GlutathioneS-transferases (GSTs) play critical roles in detoxification, response to oxidative stress, regeneration of S-thiolated proteins, and cata-lysis of reactions in nondetoxification metabolic pathways. Liver GSTs were purified from the anoxia-tolerant turtle,Trachemys scripta elegans. Purification separated a homodimeric (subunit relative molecular mass¼ 34 kDa) and a heterodimeric (subunit relative molecular mass¼32.6 and 36.8 kDa) form of GST.

13p fptmusic 11-04-2013 45 3   Download

We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cor-tex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distri-buted iron storage protein, ferritin....

0p awards 05-04-2013 50 2   Download

Human kynurenine aminotransferase I/glutamine trans-aminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids anda-keto acids on the activity of the enzyme. The experi-ments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme.

11p awards 05-04-2013 40 4   Download

Cell-free protein synthesis offers rapidaccess toproteins that are selectively labelledwith [ 15 N]aminoacids andsuitable for analysis by NMR spectroscopy without chromatographic purification. A system based on anEscherichia colicell ex-tractwas optimizedwith regard toproteinyieldandminimal usage of 15 N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reactionmedium were obtained by expression of a synthetic gene. ...

0p awards 05-04-2013 41 2   Download

The LmbB1 protein, participating in the biosynthesis of lincomycin,was heterologously expressed inEscherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agree-ment with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid.

6p awards 05-04-2013 36 5   Download

We describe the expression, purification, and biochemical characterization of two homologous enzymes, with amido-hydrolase activities, of plant (Lupinus luteuspotassium-independent asparaginase, LlA) and bacterial (Escherichia coli, ybiK/spt/iaaA gene product, EcAIII) origin. Both enzymes were expressed inE. colicells, with (LlA) or with-out (EcAIII) aHis-tag sequence. The proteins were purified, yielding6 or 30 mgÆL )1 of culture, respectively.

0p awards 05-04-2013 22 2   Download

The genesRv1318c, Rv1319c, Rv1320cand Rv3645 1 of Mycobacterium tuberculosisare predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded aden-ylyl cyclase activity for all four recombinant proteins.

6p dell39 03-04-2013 35 3   Download