Abstract:
:Escherichia coli glutamic acid decarboxylase is a pyridoxal phosphate-dependent enzyme that catalyzes the alpha-decarboxylation of glutamate to yield 4-amino-butyrate and CO2. The E. coli chromosome contains two genes encoding for this enzyme, gadA and gadB, which map at distinct loci. Their protein products differ in only five amino acid residues, four of which are located in the N-terminal region (Smith et al., 1992, J. Bacteriol. 174, 5820-5826). Herein, we report the sequences of the two gad genes, including their regulatory regions. Both genes were separately cloned into the vector pQE60, for overexpression under the control of the lac promoter. In this way, we have succeded in separately expression large quantities of each pure isoform. The two isoforms were characterized biochemically and all evidence, including that from analysis of the complex pre-steady-state kinetic behavior of the enzymes, indicates that the functional properties of the two isoenzymes are identical.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
De Biase D,Tramonti A,John RA,Bossa Fdoi
10.1006/prep.1996.0121subject
Has Abstractpub_date
1996-12-01 00:00:00pages
430-8issue
4eissn
1046-5928issn
1096-0279pii
S1046592896901217journal_volume
8pub_type
杂志文章abstract::The sequence and structure of the target protein exert a marked effect on its soluble expression in Escherichia coli. The effects of the mutation of an amylase isolated from Bacillus licheniformis (BLA) on its soluble expression in E. coli were investigated. A random mutation library of BLA was constructed to screen f...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.12.010
更新日期:2016-04-01 00:00:00
abstract::A full-length cDNA sequence of plant type CRY (designated Hae-P-CRY) was cloned from the green alga Haematococcus pluvialis. The cDNA sequence was 3608 base pairs (bp) in length, which contained a 2988-bp open reading frame encoding 995 amino acids with molecular mass of 107.7 kDa and isoelectric point of 6.19. Multip...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105633
更新日期:2020-08-01 00:00:00
abstract::The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.08.006
更新日期:2004-12-01 00:00:00
abstract::The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0087
更新日期:1996-09-01 00:00:00
abstract::A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.11.003
更新日期:2006-05-01 00:00:00
abstract::We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system. The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase. In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.02.004
更新日期:2004-06-01 00:00:00
abstract::Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding pr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1158
更新日期:1999-12-01 00:00:00
abstract::The introduction of an affinity tag offers an attractive approach to isolation of membrane proteins. The type of affinity tag and its positioning in the protein is determined by the desired subsequent experimental uses of the isolated protein. To minimize the risk of interference, membrane proteins may preferentially ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.06.005
更新日期:2009-11-01 00:00:00
abstract::A novel and rapid procedure for the isolation of catalase from mouse liver, after prior treatment with the peroxisome proliferator perfluorooctanoic acid was developed using immobilized metal ion affinity chromatography involving chelation with zinc ions. The purification developed is simple, rapid (requiring 3 hours ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0827
更新日期:1998-03-01 00:00:00
abstract::Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.02.018
更新日期:2007-07-01 00:00:00
abstract::Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the hos...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0955
更新日期:1998-11-01 00:00:00
abstract::Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypept...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90095-z
更新日期:1991-10-01 00:00:00
abstract::Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant protei...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00075-5
更新日期:2003-07-01 00:00:00
abstract::Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0870
更新日期:1998-06-01 00:00:00
abstract::Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0988
更新日期:1999-02-01 00:00:00
abstract::alpha 1-Acid glycoprotein could be isolated by a one-step extraction method from human sera and plasma. Protein recovered in the water phase after extraction with phenol at 70 degrees C for 20 min was verified as human alpha 1-acid glycoprotein when it was compared with the reference standard human alpha 1-acid glycop...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(91)90006-5
更新日期:1991-02-01 00:00:00
abstract::PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts wi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.02.012
更新日期:2008-06-01 00:00:00
abstract::We describe a single method to purify milligram amounts of authentic wild-type and mutant HIV-1 and HIV-2 Tat proteins overexpressed in Escherichia coli. This method takes advantage of the highly basic, positively charged RNA binding domain present in both HIV-1 and HIV-2 Tat, which also facilitated purification of HI...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0096
更新日期:1996-09-01 00:00:00
abstract::Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0709
更新日期:1997-04-01 00:00:00
abstract::The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-t...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.08.008
更新日期:2015-11-01 00:00:00
abstract::Human Fas ligand is a medically important membrane glycoprotein that induces the apoptosis of harmful cells. A new secretory expression and purification method was devised for the production of a large amount of recombinant human Fas ligand extracellular domain (hFasLECD) by Pichia pastoris. The expression plasmid con...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.08.006
更新日期:2006-12-01 00:00:00
abstract::The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 12...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.02.005
更新日期:2005-06-01 00:00:00
abstract::Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large solub...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00135-9
更新日期:2003-09-01 00:00:00
abstract::Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficul...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1181
更新日期:2000-03-01 00:00:00
abstract::The superoxide dismutases (EC 1.15.1.1) are a family of enzymes that catalyze the dismutation of superoxide radical anion to dioxygen and hydrogen peroxide. The active site contains a critical metal ion such as manganese, iron, or copper. The copper-containing protein also has one zinc ion bound per subunit. The stand...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1241
更新日期:2000-06-01 00:00:00
abstract::A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin bindin...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.07.013
更新日期:2007-02-01 00:00:00
abstract::Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly ex...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.01.019
更新日期:2004-06-01 00:00:00
abstract::An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified SacI and PstI restricted fragment was cloned in-fra...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0869
更新日期:1998-04-01 00:00:00
abstract::Plantaricin JK (PlnJK) is a Class IIb LAB bacteriocin that includes two peptides; i.e., PlnJ and PlnK, which can synergistically halt many types of gram-positive bacteria, including food spoilage organisms. Purification of these peptides from natural lactic acid bacteria is difficult therefore, their application remai...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.02.013
更新日期:2019-07-01 00:00:00
abstract::Separation of copurifying protease activity from recombinant human Müllerian inhibiting substance (rhMIS) bound to a monoclonal antibody immunoaffinity column by a high-salt wash results in cleaner preparations of rhMIS resistant to cleavage upon storage. In addition, an inhibitor of rhMIS antiproliferative activity i...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(92)90020-w
更新日期:1992-06-01 00:00:00