Abstract:
:General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enzyme is obtained in a nearly pure state by stepwise elution with KCl. The eluate is directly applied to hydroxylapatite, and the homogeneous enzyme is eluted with a gradient of potassium phosphate. The method has been successful for the purification of recombinant wild-type enzyme and all mutants thus far examined. The entire purification procedure has been automated using a commonly available FPLC system and can be performed in several hours with minimal operator time.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Kealey JT,Santi DVdoi
10.1016/s1046-5928(05)80039-7keywords:
subject
Has Abstractpub_date
1992-10-01 00:00:00pages
380-5issue
5eissn
1046-5928issn
1096-0279pii
S1046-5928(05)80039-7journal_volume
3pub_type
杂志文章abstract::The SaeRS two-component system in Staphylococcus aureus controls the expression of a series of virulence factors, such as hemolysins, proteases, and coagulase. The response regulator, SaeR, belongs to the OmpR family with an N-terminal regulatory domain and a C-terminal DNA binding domain. To improve the production an...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105765
更新日期:2021-01-01 00:00:00
abstract::Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic an...
journal_title:Protein expression and purification
pub_type: 杂志文章,评审
doi:10.1006/prep.2001.1465
更新日期:2001-07-01 00:00:00
abstract::Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrod...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.02.001
更新日期:2017-04-01 00:00:00
abstract::Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.06.002
更新日期:2006-01-01 00:00:00
abstract::Thymosin α1-thymopentin (Tα1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1-TP5 more effectively and economically, Tα1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.04.013
更新日期:2012-07-01 00:00:00
abstract::Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously do...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.03.010
更新日期:2012-06-01 00:00:00
abstract::Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2018.06.015
更新日期:2018-12-01 00:00:00
abstract::Streptomyces coelicolor is a soil-dwelling bacterium that undergoes an intricate, saprophytic lifecycle. The bacterium takes up exogenous nucleosides for nucleic acid synthesis or use as carbon and energy sources. However, nucleosides must pass through the membrane with the help of transporters. In the present work, t...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.02.004
更新日期:2015-05-01 00:00:00
abstract::The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 an...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105698
更新日期:2020-11-01 00:00:00
abstract::Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.10.023
更新日期:2009-04-01 00:00:00
abstract::Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (r...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.01.008
更新日期:2014-04-01 00:00:00
abstract::The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 g (wet weight) of cells (0.5 L of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molec...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.03.002
更新日期:2008-06-01 00:00:00
abstract::A TRAIL-CM4 fusion protein in soluble form with tumor selective apoptosis and antibacterial functions was expressed in the Escherichia coli expression system and isolated through dialysis refolding and histidine-tag Nickel-affinity purification. Fresh Jurkat cells were treated with the TRAIL-CM4 fusion protein. Trypan...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2016.02.015
更新日期:2016-06-01 00:00:00
abstract::The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic r...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0806
更新日期:1998-02-01 00:00:00
abstract::Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is cl...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.04.010
更新日期:2014-07-01 00:00:00
abstract::Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.10.004
更新日期:2018-02-01 00:00:00
abstract::The purpose of this work was to characterize the functions of two sesquiterpene synthase genes from moso bamboo (Phyllostachys edulis). Two novel sesquiterpene synthase genes, belonging to the Tpsa subfamily, were isolated from moso bamboo. MoTPS2 was 1641 bp in length and encoded a protein of 63 kDa, whereas MoTPS6 w...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.11.019
更新日期:2016-04-01 00:00:00
abstract::Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direc...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0036
更新日期:1996-05-01 00:00:00
abstract::A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purifi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.018
更新日期:2008-04-01 00:00:00
abstract::A maize dehydrin with an apparent molecular weight of 20 kDa was purified from whole kernels of maize inbred line G50. Kernels were ground in a seed mill, stirred overnight in extraction buffer, and centrifuged to extract soluble proteins. The sample was heated to 89 degrees C and centrifuged to remove heat-insoluble ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1040
更新日期:1994-06-01 00:00:00
abstract::Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1014
更新日期:1994-02-01 00:00:00
abstract::An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the peripl...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0741
更新日期:1997-08-01 00:00:00
abstract::Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants. Here we report the heterologous expression of glycolate oxidase in Escherichia coli and an isolation procedure which results in 4 mg pure protein per gram cell paste in only 1.5 days. This corresponds to a more than 50-fold impro...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0103
更新日期:1996-11-01 00:00:00
abstract::The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.03.010
更新日期:2009-08-01 00:00:00
abstract::In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhance...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.06.001
更新日期:2017-08-01 00:00:00
abstract::One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purifie...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1586
更新日期:2002-04-01 00:00:00
abstract::We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.05.007
更新日期:2013-08-01 00:00:00
abstract::Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.03.013
更新日期:2004-07-01 00:00:00
abstract::Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.08.004
更新日期:2008-12-01 00:00:00
abstract::Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.07.016
更新日期:2013-10-01 00:00:00