Abstract:
:The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Jin HJ,Yang YDdoi
10.1006/prep.2002.1621keywords:
subject
Has Abstractpub_date
2002-06-01 00:00:00pages
149-59issue
1eissn
1046-5928issn
1096-0279pii
S1046592802916219journal_volume
25pub_type
杂志文章abstract::The gene that was inferred to encode the TyrR protein of Haemophilus influenzae Rd was synthesized by polymerase chain reaction and inserted into a T7-based expression vector. Methods were developed to overexpress the TyrR protein of H. influenzae in Escherichia coli and to purify the protein on a large scale. Both in...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1997.0757
更新日期:1997-07-01 00:00:00
abstract::Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1520
更新日期:2001-11-01 00:00:00
abstract::Conkunitzin-S1 from the cone snail Conus striatus is the first member of a new neurotoxin family with a canonical Kunitz domain fold. Conk-S1 is 60 amino acids long and lacks one of the three conserved disulfide bonds typically found in Kunitz domain modules. It binds specifically to voltage activated potassium channe...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.01.019
更新日期:2006-06-01 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::Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105636
更新日期:2020-08-01 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::Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 man...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2009.02.013
更新日期:2009-07-01 00:00:00
abstract::Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase) and this modification is essential for their biological activity. Rab Escort Protein (REP) is a molecular chaperone that assists in the prenylation reaction carried out by RabGGTase. Muta...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00506-5
更新日期:2002-10-01 00:00:00
abstract::Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble re...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105715
更新日期:2020-11-01 00:00:00
abstract::Telomerase is a specialized reverse transcriptase that catalyzes the addition of telomeric repeats, TTAGGG in all vertebrates, to the ends of chromosomes. The lack of recombinant purified human telomerase reverse transcriptase (hTERT) has hampered biochemical and structural studies. The primary problem in generating a...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.05.016
更新日期:2007-11-01 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::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::In order to produce sufficient quantities of fibroblast growth factor-saporin (rFGF-2-SAP) mitotoxin for preclinical evaluation in models of diseases such as cancer and restenosis, we have undertaken the large-scale expression, purification, and characterization of the recombinant molecule. The fusion gene encoding rF...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0079
更新日期:1996-08-01 00:00:00
abstract::Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.10.023
更新日期:2009-04-01 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::A family of restriction enzyme- and ligation-independent cloning vectors has been developed for producing recombinant His-tagged fusion proteins in Escherichia coli. These are based on pURI2 and pURI3 expression vectors which have been previously used for the successful production of recombinant proteins at the millig...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.10.013
更新日期:2011-03-01 00:00:00
abstract::A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2000.1331
更新日期:2000-12-01 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 plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.03.004
更新日期:2019-07-01 00:00:00
abstract::The assembly pathway of the insect cell Spodoptera frugiperda (Sf-9) was engineered to include expression of the murine chaperone immunoglobulin heavy chain binding protein (BiP) using the baculovirus vector. The impact of BiP coexpression on the production and secretion of functional and soluble recombinant immunoglo...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1082
更新日期:1994-12-01 00:00:00
abstract::Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.09.009
更新日期:2008-03-01 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::Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2012.05.007
更新日期:2012-08-01 00:00:00
abstract::The major capsid protein L1 of human papillomavirus (HPV) contains the immunodominant neutralization epitopes of the virus and can auto-assembles to form virus-like particles (VLPs). Therefore, HPV L1 capsid proteins have been well investigated as potential vaccine candidates. To express large quantities of human papi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.05.010
更新日期:2007-11-01 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::Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2010.07.013
更新日期:2010-12-01 00:00:00
abstract::Five variants of human β-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.02.007
更新日期:2013-05-01 00:00:00
abstract::The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1999.1186
更新日期:2000-04-01 00:00:00
abstract::A method for purifying acylation stimulating protein (ASP) from porcine serum is described. The mRNA encoding ASP was cloned by reverse transcriptase-polymerase chain reaction which predicted a 76 residue peptide. Based on this sequence, we generated antisera to a C-terminal peptide (ASP(1-20)) which aided ASP purific...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(02)00019-0
更新日期:2002-07-01 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