Abstract：
:The gene encoding S. cerevisiae Kex2 protease derivative Kex2-667 (encoding the N-terminal 20th to 667th amino acid residues of Kex2 protease, containing the propeptide, catalytic domain, P domain and Ser/Thr enrichment region) and its 225th amino acid residue mutant K225L were overexpressed in Pichia pastoris. Proteases were purified by dialysis and anion exchange chromatography (Q-FF). Their properties were further investigated. For catalysis efficiency, the value of Kcat/Km of Kex2-667-K225L was 3 folds higher than that of Kex2-667. Both were quite stable at 25 °C and 37 °C after 8 h of incubation at pH5.6, while Kex2-667 remained nearly 90% of the total activity while Kex2-667-K225L remained only 80%. The stability of Kex2-667-K225L was lower than that of Kex2-667 from pH4.0 to pH9.0. Due to the mutation site K225 was located at one of the calcium ion binding sites, it resulted in a tighter calcium ion binding region, which may be the reason why the catalytic efficiency of Kex2-667-K225L was improved while the stability was a little decreased.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Yang F,Liu L,Liu Y,Li Sdoi
10.1016/j.pep.2020.105725subject
Has Abstractpub_date
2020-12-01 00:00:00pages
105725eissn
1046-5928issn
1096-0279pii
S1046-5928(20)30316-8journal_volume
176pub_type
杂志文章abstract：:Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1232
更新日期：2000-06-01 00:00:00
abstract：:The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified recombinant cyclin H is similar to the endogenous one according to the following criteria...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0693
更新日期：1997-03-01 00:00:00
abstract：:Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification pr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.03.025
更新日期：2008-08-01 00:00:00
abstract：:Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein,...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.01.015
更新日期：2005-06-01 00:00:00
abstract：:Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective express...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.01.022
更新日期：2006-08-01 00:00:00
abstract：:Post-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specif...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.10.018
更新日期：2008-02-01 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：: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：:BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-e...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00675-7
更新日期：2003-03-01 00:00:00
abstract：:DNA gyrase is a type IIA topoisomerase found in bacteria but not in humans. The enzyme is required for bacterial DNA replication and transcription, and is an important antibacterial target that is sensitive to the widely-used fluoroquinolone drugs. Due to the emergence of fluoroquinolone resistance, the discovery of n...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.11.009
更新日期：2015-03-01 00:00:00
abstract：:Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in h...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.10.004
更新日期：2012-02-01 00:00:00
abstract：:Accurate diagnosis is essential for the treatment, prevention, and control of tuberculosis. Poor specificity of the tuberculin skin test in BCG-vaccinated populations and constraints to implementation of PCR and CMI-based diagnostic assays in developing countries warrant development of easy-to perform robust serologic...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.04.014
更新日期：2005-11-01 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：: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：:Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secre...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.09.002
更新日期：2019-01-01 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：:Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.12.006
更新日期：2006-07-01 00:00:00
abstract：:Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, fu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.09.014
更新日期：2006-04-01 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：:Streptavidin is a versatile molecule for many in vitro and in vivo applications. To optimize the production of the full-length streptavidin in a soluble and functional form via secretion using Bacillus subtilis as the expression host, three different strategies were used. These strategies include the construction of a...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1582
更新日期：2002-04-01 00:00:00
abstract：:An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open re...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1456
更新日期：2001-08-01 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 numerous proteins occluded within the molluscan shell play a key role in the control of the mineralization process. Although extensively studied, these proteins are still poorly known, mainly because they are difficult to fractionate. In the present paper, we present, for the first time, a simple combined strategy...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1487
更新日期：2001-10-01 00:00:00
abstract：:Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-la...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.08.002
更新日期：2004-11-01 00:00:00
abstract：:The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CR...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.08.011
更新日期：2016-12-01 00:00:00
abstract：:Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications....
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.05.011
更新日期：2011-09-01 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：:Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be opti...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.09.012
更新日期：2005-01-01 00:00:00
abstract：:Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.10.005
更新日期：2008-02-01 00:00:00
abstract：:Plasmids pQE-60 and pQE-30 containing 6 x His-tag sequence were used for expression of fragments of HCV structural and non-structural proteins in Escherichia coli (E. coli). The following fragments were used: core (1-98 aa), NS3 (202-482 aa), and tetramer of hypervariable region 1 (HVR1) of E2 protein. The constructed...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.06.011
更新日期：2006-11-01 00:00:00
