Abstract：
:Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the pathogenesis of this disease; elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia. Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein with the glucagon peptide at its C-terminus (GCGR-ECD-Gc). We utilized the maltose binding protein (MBP) fusion method and disulfide bond isomerase DsbC co-expression approach for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in Escherichia coli. We also obtained a high yield production of secreted GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. We first utilized isothermal titration calorimetry approach to determine the in vitro binding affinities of glucagon to the GCGR-ECD. No significant differences were found between the prokaryotic expressed GCGR-ECD (7.6μM) and the eukaryotic glycosylated one (6.6μM). The observation of the intra ligand-receptor binding within the fusion protein GCGR-ECD-Gc suggests it as a good candidate for further structural study.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Wu L,Zhai Y,Lu J,Wang Q,Sun Fdoi
10.1016/j.pep.2013.04.004subject
Has Abstractpub_date
2013-06-01 00:00:00pages
232-40issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(13)00071-5journal_volume
89pub_type
杂志文章abstract：:Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overprodu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.09.001
更新日期：2012-01-01 00:00:00
abstract：:NAD(+)-dependent malic enzyme (NAD-ME) gene from Escherichia coli K12 was inserted into an expression vector pET24b(+) and transformed into E. coli BL21 (DE3). Recombinant NAD-ME was expressed upon IPTG induction, purified with affinity chromatography, and biochemically characterized. The results showed that recombina...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.11.017
更新日期：2007-05-01 00:00:00
abstract：:A baculovirus vector system that expresses cloned DNA sequences as glutathione S-transferase fusion proteins was developed. This system was used to express and purify the lymphocyte-specific tyrosine kinase p56lck. This recombinant p56lck was purified to homogeneity in a single chromatography step using glutathione re...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1993.1051
更新日期：1993-10-01 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：:The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0087
更新日期：1996-09-01 00:00:00
abstract：:A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00614-9
更新日期：2003-02-01 00:00:00
abstract：:Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2000.1206
更新日期：2000-04-01 00:00:00
abstract：:Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a clone...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.1015
更新日期：1999-03-01 00:00:00
abstract：:The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression. The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically impo...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/S1046-5928(03)00193-1
更新日期：2003-11-01 00:00:00
abstract：:Endostatin, a 20kDa C-terminal fragment of collagen XVIII, is a potent anti-angiogenic protein and inhibitor of tumor growth. Recombinant endostatin was prepared from Escherichia coli deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant human ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.09.021
更新日期：2005-06-01 00:00:00
abstract：:A genomic DNA of 1416 bp containing an open reading frame encoding a manganese superoxide dismutase (Mn-SOD) from Tatumella ptyseos ct was cloned. Sequence analysis of this new gene revealed that it translates 205 amino acid residues. The deduced amino acid sequence showed variable identities (41-91%) with sequences o...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.10.003
更新日期：2005-03-01 00:00:00
abstract：:The expression of recombinant proteins in bacterial hosts may alter the biophysical properties of the protein of interest as a result of differences in post-translational processing from that observed when produced in the native cell. For example, recombinant human interleukin-1beta (IL-1beta) is produced as three iso...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.01.010
更新日期：2005-05-01 00:00:00
abstract：:Proteus mirabilis (P. mirabilis) is a zoonotic pathogen that has recently presented a rising infection rate in the poultry industry. To develop an effective vaccine to protect chickens against P. mirabilis infection, OmpA, one of the major outer membrane proteins of P. mirabilis, was expressed in Pichia pastoris. The ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.10.001
更新日期：2015-01-01 00:00:00
abstract：:Recombinant protein expression from lac derived promoters by the autoinduction regime is based on diauxic growth of Escherichia coli on glucose and lactose. Glycerol is used as a supporting carbon source during the lactose-induced expression. While this glycerol-based formulation usually provides high cell densities, ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.07.016
更新日期：2013-10-01 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：:We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "porta...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.09.018
更新日期：2009-02-01 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：:One-step chromatography on a Mono S column allows the purification of high mobility group (HMG) proteins 1 and 2 under nondenaturing conditions. Chromatography of HMG1 and -2 on Mono S can be achieved with three of the most widely employed extraction techniques for chromosomal proteins, 0.35 M sodium chloride, 0.74 M ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(90)90051-y
更新日期：1990-09-01 00:00:00
abstract：:Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanoma...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.09.010
更新日期：2011-02-01 00:00:00
abstract：:A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactos...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(91)90073-r
更新日期：1991-04-01 00:00:00
abstract：:Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.06.018
更新日期：2011-10-01 00:00:00
abstract：:The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.08.006
更新日期：2004-12-01 00:00:00
abstract：:Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.09.015
更新日期：2015-01-01 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：:Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V(H)) and light chain (V(L)) cloned from the ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2003.12.008
更新日期：2004-05-01 00:00:00
abstract：:Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and exp...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.07.021
更新日期：2006-03-01 00:00:00
abstract：:Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escher...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.02.019
更新日期：2006-09-01 00:00:00
abstract：:A maize dehydrin with an apparent molecular weight of 20 kDa was purified from whole kernels of maize inbred line G50. Kernels were ground in a seed mill, stirred overnight in extraction buffer, and centrifuged to extract soluble proteins. The sample was heated to 89 degrees C and centrifuged to remove heat-insoluble ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1994.1040
更新日期：1994-06-01 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 second enzyme of the naphthalene degradation pathway in Pseudomonas putida G7 is NahB, a dehydrogenase that converts cis-1,2-dihydroxy-1,2-dihydronaphthalene to 1,2-dihydroxynaphthalene. We report the cloning, optimization of expression, purification, kinetic studies and preliminary structural characterization of ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2017.01.005
更新日期：2017-04-01 00:00:00
