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, CXCL8((3-72))K11R/G31P (hG31P) has been developed. It has been demonstrated that hG31P is a high affinity antagonist for both CXCR1 and CXCR2. To obtain large quantities of hG31P, we have successfully constructed and expressed hG31P in Escherichia coli. Moreover, we have developed a new protocol for high-yield purification of hG31P and for the removal of lipopolysaccharide (LPS, endotoxin) associated with hG31P due to the expression in E. coli. The purity of hG31P is more than 95% and the final yield is 9.7mg hG31P per gram of cell paste. The purified hG31P was tested by various biological assays. In addition, the structural properties of hG31P were studied by circular dichroism (CD), ultracentrifuge, isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that this purification protocol is very simple and easy to amplify at a large scale. The results of this study will provide an effective route to produce enough hG31P for future clinical studies.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Cheng HT,Huang KC,Yu HY,Gao KJ,Zhao X,Li F,Town J,Gordon JR,Cheng JWdoi
10.1016/j.pep.2008.04.007subject
Has Abstractpub_date
2008-09-01 00:00:00pages
65-72issue
1eissn
1046-5928issn
1096-0279pii
S1046-5928(08)00117-4journal_volume
61pub_type
杂志文章abstract::Insoluble expression of intrinsically soluble proteins with native activity is potentially a promising alternative to soluble expression of folded protein or insoluble expression of unfolded protein requiring refolding. Here, we attempted to express highly soluble halophilic His-rich metal binding protein (HP) as inso...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2019.01.001
更新日期:2019-04-01 00:00:00
abstract::Human pancreatic ribonuclease (HP-RNase) has considerable promise as a therapeutic agent. Structure-function analyses of HP-RNase have been impeded by the difficulty of obtaining the enzyme from its host. Here, a gene encoding HP-RNase was designed, synthesized, and inserted into two expression vectors that then direc...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1996.0036
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abstract::Hereditary enzymopenic methemoglobinemia is a rare disease that predominantly results from defects in either the erythrocytic (type I) or microsomal (type II) forms of the enzyme NADH:cytochrome b5 reductase (EC 1.6.2.2). All 25 currently identified type I and type II methemoglobinemia mutants have been expressed in E...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/s1046-5928(03)00046-9
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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
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abstract::ABCA4 is a member of the A subfamily of ATP-binding cassette transporters that consists of large integral membrane proteins implicated in inherited human diseases. ABCA4 assists in the clearance of N-retinylidene-phosphatidylethanolamine, a potentially toxic by-product of the visual cycle formed in photoreceptor cells...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.02.010
更新日期:2014-05-01 00:00:00
abstract::A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purifi...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.11.018
更新日期:2008-04-01 00:00:00
abstract::The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in t...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2006.12.019
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abstract::Mammalian target of rapamycin (TOR) controls cell growth and metabolism in response to the availability of nutrients, growth factors, and the cellular energy status. Misregulation of TOR can result in a pathogenic increase or decrease in organ size and in cancer. TOR can be inhibited by binding of a complex of rapamyc...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.01.014
更新日期:2008-05-01 00:00:00
abstract::We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of criti...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.10.012
更新日期:2005-02-01 00:00:00
abstract::Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chrom...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2008.10.012
更新日期:2009-04-01 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::A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0890
更新日期:1998-07-01 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::Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.02.007
更新日期:2014-05-01 00:00:00
abstract::Splicing factor proline- and glutamine-rich (SFPQ) is an RNA-binding protein, playing significant roles in gene regulation and subnuclear body formation. Our recent serendipitous discovery showed that SFPQ binds zinc directly and forms an infinite polymer that is induced by zinc binding to the protein. The zinc-induce...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2020.105626
更新日期:2020-07-01 00:00:00
abstract::A growing number of studies require the purification of multiple proteins simultaneously and the development of simple economical high-throughput purification methods is essential. We have tested the purification of two related proteins in a variety of conditions to benchmark the semi-automated affinity chromatography...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2014.01.014
更新日期:2014-04-01 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 purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4 FF. Further purification on Superdex 200 resulted in another 29-fold pu...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.04.011
更新日期:2015-09-01 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::One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purifie...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.2001.1586
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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
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abstract::A cDNA clone, lambda GTHP1del, encoding glutathione transferase (GST) P1-1, was isolated from a human K562 erythroleukemia cell line cDNA library. The coding sequence was lacking the codons for the N-terminal 34 amino acids. A DNA segment was designed in order to obtain the missing portion and a structure representing...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1995.1034
更新日期:1995-06-01 00:00:00
abstract::Recombinant human interleukin-5 (rhIL-5) was expressed in baculovirus-infected insect cells and purified to homogeneity from the culture medium in a single chromatographic step. Beginning with a cDNA encoding the full-length precursor form of human IL-5, including the authentic secretory leader sequence, recombinant b...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1995.1009
更新日期:1995-02-01 00:00:00
abstract::Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning ...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2015.01.012
更新日期:2015-07-01 00:00:00
abstract::Heterologous protein expression in Escherichia coli is commonly used to obtain recombinant proteins for a variety of downstream applications. However, many proteins are not, or are only poorly, expressed in soluble form. High level expression often leads to the formation of inclusion bodies and an inactive product tha...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2011.08.030
更新日期:2012-01-01 00:00:00
abstract::The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chrom...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1998.0995
更新日期:1999-02-01 00:00:00
abstract::Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market....
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2013.01.011
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abstract::Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fus...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2004.01.003
更新日期:2004-05-01 00:00:00
abstract::Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved i...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1016/j.pep.2007.04.010
更新日期:2007-12-01 00:00:00
abstract::Human GRO alpha, GRO beta, and GRO gamma are neutrophil chemoattractants structurally related to IL-8 and compete with IL-8 for binding to IL-8 receptors on neutrophils. These proteins are part of a large superfamily of chemotactic cytokines, the "chemokines," members of which share striking structural similarities. W...
journal_title:Protein expression and purification
pub_type: 杂志文章
doi:10.1006/prep.1994.1050
更新日期:1994-08-01 00:00:00