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 documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Marzahn MR,Bloom LBdoi
10.1016/j.pep.2012.03.010subject
Has Abstractpub_date
2012-06-01 00:00:00pages
135-44issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(12)00082-4journal_volume
83pub_type
杂志文章abstract：:We present a new streptavidin-binding peptide for both the purification and the detection of recombinant proteins. The peptide possesses nanomolar-affinity for streptavidin and therefore was termed Nano-tag. The Nano-tag(15) is 15 amino acids long and binds to streptavidin with a dissociation constant of 4 nM and the ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.08.014
更新日期：2004-01-01 00:00:00
abstract：:A novel and rapid procedure for the isolation of catalase from mouse liver, after prior treatment with the peroxisome proliferator perfluorooctanoic acid was developed using immobilized metal ion affinity chromatography involving chelation with zinc ions. The purification developed is simple, rapid (requiring 3 hours ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1997.0827
更新日期：1998-03-01 00:00:00
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 ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2002.1621
更新日期：2002-06-01 00:00:00
abstract：:The entire stx2 region from Escherichia coli O157:H7, containing two open reading frames (stx2a and stx2b), was cloned into pET-32a with a single promoter, and transformed into E. coli BL21 (DE3) pLysS. We used two methods of IPTG induction using LB medium and auto-induction using ZYM-5052 medium to express recombinan...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.05.005
更新日期：2009-10-01 00:00:00
abstract：:The gene coding for phosphoglucomutase (PGM) from Oryctolagus cuniculus (rabbit) has been expressed in Escherichia coli under a T7 expression system with a His-tag. About half of the expressed PGM protein was present in inclusion bodies, but this protein was inactive when solubilized. The protein in the soluble cell f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1288
更新日期：2000-10-01 00:00:00
abstract：:High-level extracellular production of Fusarium solani cutinase was achieved using a Pichia pastoris expression system. The cutinase-encoding gene was cloned into pPICZalphaA with the Saccharomyces cerevisiae alpha-factor signal sequence and methanol-inducible alcohol oxidase promoter by two different ways. The additi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.06.021
更新日期：2009-11-01 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：:Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.03.010
更新日期：2019-07-01 00:00:00
abstract：:Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00187-6
更新日期：2003-10-01 00:00:00
abstract：:We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We e...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.12.005
更新日期：2012-03-01 00:00:00
abstract：:Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of an...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.04.012
更新日期：2009-10-01 00:00:00
abstract：:The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00562-4
更新日期：2003-01-01 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：:Ndt80 is a Saccharomyces cerevisiae meiosis-specific transcription factor responsible for promoting the stage-specific expression of a family of genes referred to as middle sporulation genes. Many members of this gene family are essential for the completion of meiotic chromosome segregation. Thus, Ndt80 is essential f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.08.025
更新日期：2004-01-01 00:00:00
abstract：:Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.06.014
更新日期：2004-10-01 00:00:00
abstract：:Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, mo...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.10.007
更新日期：2013-02-01 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：:Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we repo...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.11.001
更新日期：2017-03-01 00:00:00
abstract：:Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.03.016
更新日期：2007-08-01 00:00:00
abstract：:Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Pla...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.01.003
更新日期：2015-04-01 00:00:00
abstract：:Hydroxypyruvate reductase (HPR), a plant leaf peroxisomal enzyme involved in the glycolate pathway, has been purified in two steps from a crude extract of parsley leaves during the purification of an unrelated ATP-dependent enzyme. HPR, a homogenous side-fraction arising from this purification procedure, was identifie...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0666
更新日期：1997-02-01 00:00:00
abstract：:The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastori...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.06.018
更新日期：2006-11-01 00:00:00
abstract：:Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recomb...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.03.021
更新日期：2005-07-01 00:00:00
abstract：:Flt3 ligand (FL) is a potent hematopoietic cytokine that affects the growth and differentiation of hematopoietic progenitor and stem cells both in vivo and in vitro. Pichia pastoris transformants secreting high-level rhFL were obtained using 'yeastern blotting' method and the expression level in liquid was about 30 mg...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.04.001
更新日期：2005-08-01 00:00:00
abstract：:The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.03.010
更新日期：2009-08-01 00:00:00
abstract：:Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells. AR has been shown to have an important role in the progression of prostate cancer...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.11.002
更新日期：2005-02-01 00:00:00
abstract：:We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protei...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.09.019
更新日期：2008-02-01 00:00:00
abstract：:Leishmaniasis is considered by the World Health Organization to be the second most important disease caused by a protozoan parasite. Biochemical and molecular biology studies can help in the understanding of the biology of the Leishmania parasite. All protozoan parasites, including Leishmania, are unable to synthesize...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.05.010
更新日期：2006-10-01 00:00:00
abstract：:Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.09.008
更新日期：2009-02-01 00:00:00
abstract：:Human small nuclear (sn) RNA genes are transcribed by either RNA polymerase II or III depending upon the arrangement of their core promoter elements. Regardless of polymerase specificity, these genes share a requirement for a general transcription factor called the snRNA activating protein complex or SNAP(C). This mul...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.02.015
更新日期：2006-08-01 00:00:00
