Abstract:
:l-glutamate oxidase (GLOD), encoded by the gox gene, catalyses the transformation of l-glutamic acid into α-ketoglutaric acid (α-KG). In the present study, Pichia pastoris was used for heterologous production of GLOD following optimization of the gox coding sequence for expression in the yeast host. A series of constructs based on the pHBM905BDM plasmid were engineered and transformed into P. pastoris to increase the gox copy number. The results indicated that GLOD protein levels and enzyme activity increased with increasing gox copy number. Strain PGLOD4, which contained four copies of the target gene, was chosen for subsequent fermentation experiments, and a fermentation strategy involving two exponential feeding phases was developed. During the preinduction phase, glycerol was fed exponentially at μG = 0.15/h. When the cell density reached 300 g/l, methanol was fed exponentially at μM = 0.03/h to induce GLOD production. After 84 h of cultivation, the final cell density and total enzyme activity reached 420 g/L and 247.8 U/mL, respectively. The recombinant enzyme displayed an optimum temperature of 40 °C, which was higher than recombinant enzyme expressed in E. coli. This is important because increasing the temperature could accelerate enzymatic transformation of l-glutamic acid to α-KG. Experiments also demonstrated superior thermo-stability for the enzyme produced in yeast, which further enhances its potential for industrial applications.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
YaPing W,Ben R,Hong Y,Rui H,Li L,Ping'an L,Lixin Mdoi
10.1016/j.pep.2016.09.014subject
Has Abstractpub_date
2017-01-01 00:00:00pages
108-114eissn
1046-5928issn
1096-0279pii
S1046-5928(16)30266-2journal_volume
129pub_type
杂志文章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::As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pa...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2018.07.013
更新日期:2018-12-01 00:00:00
abstract::Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00697-6
更新日期:2003-04-01 00:00:00
abstract::Acyl coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-t...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.010
更新日期:2008-04-01 00:00:00
abstract::Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:
更新日期:1992-04-01 00:00:00
abstract::Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the G...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.09.008
更新日期:2004-02-01 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::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::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::A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-s...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/1046-5928(90)90041-v
更新日期:1990-09-01 00:00:00
abstract::Post-translational processing of host defense cathelicidin peptide LL-37 in human sweat and skin generates new antimicrobial peptides. To understand structure and mechanism of action of these LL-37 derivatives, this article presents the cloning and expression of SK-29, KR-20, LL-29, and LL-23. We also provide a two-st...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.04.023
更新日期:2007-10-01 00:00:00
abstract::Autophagy is the process of degradation of intracellular proteins through the lysosome. Members of the tripartite motif (TRIM) proteins have shown to directly recognize autophagic cargo and also to act as a hub for the phagophore nucleation complex. The TRIM proteins are classically characterized by the presence of an...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2017.10.014
更新日期:2018-03-01 00:00:00
abstract::The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guaran...
journal_title:Protein expression and purification
pub_type: 杂志文章,评审
doi:10.1016/j.pep.2013.10.016
更新日期:2014-02-01 00:00:00
abstract::Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion...
journal_title:Protein expression and purification
pub_type: 杂志文章,评审
doi:10.1016/j.pep.2007.05.014
更新日期:2007-10-01 00:00:00
abstract::The ELR-CXC chemokines are important to neutrophil inflammation in many acute and chronic diseases. Among them, CXCL8 (interleukin-8, IL-8), binds to both the CXCR1 and CXCR2 receptors with high affinity and the expression levels of CXCL8 are elevated in many inflammatory diseases. Recently, an analogue of human CXCL8...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.04.007
更新日期:2008-09-01 00:00:00
abstract::Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00573-9
更新日期:2003-02-01 00:00:00
abstract::The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2003.11.023
更新日期:2004-04-01 00:00:00
abstract::Large amounts of soluble fibronectin were easily purified from cryoprecipitated or fresh citrated human blood plasma by a three-step combination of gelatin and heparin-cellufine affinity chromatography. The elution conditions were optimized to obtain a homogeneous fraction on SDS-PAGE and Western blot under reducing c...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1103
更新日期:1999-10-01 00:00:00
abstract::Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion p...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.07.004
更新日期:2013-09-01 00:00:00
abstract::Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.03.009
更新日期:2014-07-01 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
abstract::The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The simil...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2005.05.002
更新日期:2005-09-01 00:00:00
abstract::The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1416
更新日期:2001-06-01 00:00:00
abstract::Recombinant protein expression in insect cells varies greatly from protein to protein. A fusion tag that is not only a tool for detection and purification, but also enhances expression and/or solubility would greatly facilitate both structure/function studies and therapeutic protein production. We have shown that fusi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.07.010
更新日期:2008-11-01 00:00:00
abstract::In this work, we present the production of an active 43 aa recombinant human beta-defensin-1 (rhBD-1(43)) in Escherichia coli AD202 cells using specific pLMM1-rhBD-1 expression system. Unique solubility properties of the C-terminal fragment of light meromyosin (LMM) allowed us to overcome foreseeable problems with iso...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.04.024
更新日期:2004-09-01 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::Heparinase I (HepA) was originally isolated from Flavobacterium heparinum (F. heparinum) and specifically cleaves heparin/heparan sulfate in a site-dependent manner, showing great promise for producing low molecular weight heparin (LMWH). However, expressing recombinant HepA is extremely difficult in Escherichia coli ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.03.016
更新日期:2012-06-01 00:00:00
abstract::Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is cr...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.02.004
更新日期:2019-06-01 00:00:00
abstract::The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stres...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.01.016
更新日期:2008-05-01 00:00:00
abstract::PagP, a beta-barrel membrane protein found in Gram-negative bacteria, expresses robustly in inclusion bodies when its signal sequence is removed. We have developed a new fusion protein expression system based on PagP and demonstrated its utility in the expression of the unstructured N-terminal region of human cardiac ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.07.007
更新日期:2012-09-01 00:00:00