Abstract：
:Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Jayalakshmi R,Sumathy K,Balaram Hdoi
10.1006/prep.2001.1610keywords:
subject
Has Abstractpub_date
2002-06-01 00:00:00pages
65-72issue
1eissn
1046-5928issn
1096-0279pii
S1046592801916109journal_volume
25pub_type
杂志文章abstract：:Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.11.012
更新日期：2010-12-04 00:00:00
abstract：:Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5'-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In or...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0935
更新日期：1998-10-01 00:00:00
abstract：:Vascular endothelial growth factor (VEGF) is one of the most significant mediators of angiogenesis, which interacts with a specific membrane receptor: VEGF receptor 2 (VEGFR2). Studies elsewhere have shown that, a VEGF-blocker can regulate several vital processes of tumor promotion. However, there is no literature evi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.04.010
更新日期：2013-08-01 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：:The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-pro...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.01.021
更新日期：2012-04-01 00:00:00
abstract：:Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.11.023
更新日期：2016-05-01 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 method for the simplified, reproducible production of both normal and altered versions of human proinsulin has been developed. A polyhistidine/proinsulin fusion protein was expressed using a prokaryotic expression system and partially purified by affinity chromatography. Disulfide bonds within the polypeptide were f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.1024
更新日期：1999-04-01 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：:The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.05.004
更新日期：2004-10-01 00:00:00
abstract：:The major active form of human thrombin, alpha-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2-0M sodium chloride (5-20 min). Total analysis time was 30 min per ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00596-x
更新日期：2003-01-01 00:00:00
abstract：:Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escher...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.02.019
更新日期：2006-09-01 00:00:00
abstract：:The hepatitis E virus (HEV) capsid antigen has been proposed as a candidate subunit vaccine for the prevention of hepatitis E. The full-length HEV ORF2 protein product is predicted to contain 660 amino acids and to weigh 72,000 daltons. Expression of the HEV ORF2 capsid gene from recombinant baculoviruses in insect ce...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1997.0817
更新日期：1998-02-01 00:00:00
abstract：:Wild-type and deglycosylated forms of human prostate-specific antigen were expressed in Chinese hamster ovary (CHO) cells as zymogens. ProPSA was collected from conditioned medium and purified using a single cation-exchange chromatographic step for the deglycosylated form and cation-exchange followed by gel filtration...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1342
更新日期：2000-12-01 00:00:00
abstract：:The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B(max) values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.06.018
更新日期：2004-10-01 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：: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：:The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-prote...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.01.012
更新日期：2014-04-01 00:00:00
abstract：:This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of im...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.08.021
更新日期：2011-09-03 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：: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：:The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containin...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.08.020
更新日期：2012-11-01 00:00:00
abstract：:Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.07.008
更新日期：2015-12-01 00:00:00
abstract：:Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.01.001
更新日期：2009-06-01 00:00:00
abstract：:Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.03.006
更新日期：2015-07-01 00:00:00
abstract：:Antibodies specific to β-Glucocerebrosidase were selected from phage displayed naïve scFv libraries. Biopannings were performed against recombinant human protein β-Glucocerebrosidase immobilized on polystyrene surface, specific phages were eluted with 50% ethylene glycol in citrate buffer (pH 6.0). Several specific bi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105573
更新日期：2020-06-01 00:00:00
abstract：:The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1070
更新日期：1999-07-01 00:00:00
abstract：:Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved i...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.04.010
更新日期：2007-12-01 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：:The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the wid...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.105546
更新日期：2020-03-01 00:00:00
