Abstract：
:The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da. The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence. A likely ribosomal binding site was identified 9bp upstream of the translational start site. The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases. Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively. Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector. Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa. Thus, we conclude that the active form of native Nocardia GCH is a decamer. Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
He A,Simpson DR,Daniels L,Rosazza JPdoi
10.1016/j.pep.2004.02.008keywords:
subject
Has Abstractpub_date
2004-06-01 00:00:00pages
171-80issue
2eissn
1046-5928issn
1096-0279pii
S1046592804000828journal_volume
35pub_type
杂志文章abstract：:The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expressio...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.11.003
更新日期：2010-04-01 00:00:00
abstract：:A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to ac...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1256
更新日期：2000-08-01 00:00:00
abstract：:Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1412
更新日期：2001-06-01 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：:Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl dipho...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.12.008
更新日期：2016-04-01 00:00:00
abstract：:The bivalent anti-human anti-T cell immunotoxin A-dmDT390-bisFv(G(4)S) was developed for treatment of T cell leukemia, autoimmune diseases, and tolerance induction for transplantation. The multi-domain structure of the bivalent immunotoxin hinders efficient production in Escherichia coli and most eukaryotes are sensit...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00009-8
更新日期：2002-07-01 00:00:00
abstract：:Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.03.004
更新日期：2018-07-01 00:00:00
abstract：:We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of S...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00062-7
更新日期：2003-06-01 00:00:00
abstract：:Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.105463
更新日期：2019-12-01 00:00:00
abstract：:Amidase signature (AS) family amidases are known to exhibit broad substrate specificity. According to the available genome sequence data, a novel AS family amidase, Pl-Ami, was identified and cloned from the genome of Parvibaculum lavamentivorans ZJB14001. The recombinant amidase was overexpressed in Escherichia coli ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.09.005
更新日期：2017-01-01 00:00:00
abstract：:Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) prot...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.06.015
更新日期：2010-11-01 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：:Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.08.008
更新日期：2011-12-01 00:00:00
abstract：:A relatively inexpensive yet highly efficient and extremely rapid procedure has been developed for the isolation and purification of estrogen receptor from the goat uterine cytosol. Greater than 1 mg of purified receptor protein could be obtained from 75 g of uterine tissue within a period of < 24 h, following this pr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1993.1070
更新日期：1993-12-01 00:00:00
abstract：:To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector....
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1363
更新日期：2001-02-01 00:00:00
abstract：:Proteins are essential throughout the biological and biomedical sciences and the purification strategies of proteins of interest have advanced over centuries. Elastin-like polypeptides (ELPs) are compound polymers that have recently been highlighted for their sharp and reversible phase transition property when heated ...
journal_title：Protein expression and purification
pub_type： 杂志文章,评审
doi：10.1016/j.pep.2018.09.006
更新日期：2019-01-01 00:00:00
abstract：:Hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) was utilized for the separation of native human antithrombin (AT) and a partially denaturated form of AT, known as the latent form (L-AT). The AT used in this study is commercially available (Atenativ, Pharmacia & Upjohn, Sweden) and contains al...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1349
更新日期：2001-02-01 00:00:00
abstract：:Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74. The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1370
更新日期：2001-03-01 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：: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：:Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overprodu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.09.001
更新日期：2012-01-01 00:00:00
abstract：:PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.02.015
更新日期：2014-06-01 00:00:00
abstract：:A stable mammalian cell line expressing highly active bovine interferon-gamma (BoIFN-γ) was generated using Flp recombinase-mediated integration. This recombinant 293 cell line (B1) efficiently secreted FLAG-tagged BoIFN-γ protein into the culture supernatant, as determined by ELISA and Western blot. The recombinant B...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.04.012
更新日期：2014-07-01 00:00:00
abstract：:Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.07.007
更新日期：2014-10-01 00:00:00
abstract：:A highly efficient Escherichia coli expression system was established to obtain an appreciable quantity of antihypertensive peptide. The DNA-coding sequence for the Gly-Val-Tyr-Pro-His-Lys peptide was chemically synthesized and linked to form a ten-copy in tandem. It was cloned into the vector pET-15b and expressed in...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.05.001
更新日期：2015-09-01 00:00:00
abstract：:Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.10.005
更新日期：2007-04-01 00:00:00
abstract：:The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.11.005
更新日期：2013-07-01 00:00:00
abstract：:The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatogra...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.10.011
更新日期：2008-02-01 00:00:00
abstract：:Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1153
更新日期：2000-02-01 00:00:00
abstract：:Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.02.005
更新日期：2018-07-01 00:00:00
