Abstract：
:We have used Ni(2+)-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed in Halobacterium salinarium by integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling function in vivo. Immobilized Ni(2+)-affinity chromatography of membranes solubilized in 1% layryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (> or = 90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pKa as the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer protein in vitro.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Krebs MP,Spudich EN,Spudich JLdoi
10.1006/prep.1995.0009subject
Has Abstractpub_date
1995-12-01 00:00:00pages
780-8issue
6eissn
1046-5928issn
1096-0279pii
S1046-5928(85)70009-5journal_volume
6pub_type
杂志文章abstract：:The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E wer...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.10.003
更新日期：2016-02-01 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：:The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0705
更新日期：1997-06-01 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：: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：: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：:Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant protei...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(03)00075-5
更新日期：2003-07-01 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：:Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.03.013
更新日期：2010-08-01 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：: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：:Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthes...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2013.01.004
更新日期：2013-05-01 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：:Pigment epithelium-derived factor (PEDF) is a neurotrophic protein and a member of the serine protease inhibitor superfamily. Here we describe the identification of PEDF in bovine eyes and optimization of its purification from this natural source. We have developed a polyclonal antibody to recombinant human PEDF, Ab-r...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1995.1060
更新日期：1995-08-01 00:00:00
abstract：:The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease defi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.05.019
更新日期：2004-09-01 00:00:00
abstract：:Pullulanases are well-known starch-debranching enzymes that are widely used for hydrolysis of a-1,6-glycosidic linkages in starch, pullulan, amylopectin, and other oligosaccharides. Escherichia coli is a popular heterologous expression host for generating target enzymes. However, cells have to be disrupted to obtain t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.09.011
更新日期：2019-03-01 00:00:00
abstract：:In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.09.004
更新日期：2009-01-01 00:00:00
abstract：:The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. Th...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1504
更新日期：2001-11-01 00:00:00
abstract：:A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.11.003
更新日期：2006-05-01 00:00:00
abstract：:This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of im...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2011.08.021
更新日期：2011-09-03 00:00:00
abstract：:Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) tra...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.12.017
更新日期：2007-06-01 00:00:00
abstract：:To expand the application of the streptavidin-biotin technology for reversible affinity purification of biotinylated proteins, a novel form of monomeric streptavidin was engineered and produced using Bacillus subtilis as the expression host. By changing as little as two amino acid residues (T90 and D128) to alanine, t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00021-9
更新日期：2002-08-01 00:00:00
abstract：:G-protein coupled receptors (GPCRs) are seven transmembrane helical proteins involved in cell signaling and response. They are targets for many existing therapeutic agents, and numerous drug discovery efforts. Production of large quantities of these receptors for drug screening and structural biology remains challengi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.10.017
更新日期：2007-04-01 00:00:00
abstract：:Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.08.004
更新日期：2014-10-01 00:00:00
abstract：:Transcription regulation in the cell occurs in the context of chromatin. It follows that a thorough investigation of the mechanism of transcription regulation must take into account the role of chromatin structure. Through classical and molecular genetic experiments in yeast, great strides have been made in understand...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0716
更新日期：1997-06-01 00:00:00
abstract：:To produce a monoclonal antibody specific to a mouse major histocompatibility complex (MHC) class II protein, we synthesized the complementary DNAs for the heavy and light chains of a monoclonal antibody by PCR amplification. These cDNAs were then introduced separately into tobacco plant cells. After performing Northe...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2007.02.015
更新日期：2007-07-01 00:00:00
abstract：:This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.04.011
更新日期：2005-08-01 00:00:00
abstract：:NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression sy...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1098
更新日期：1999-10-01 00:00:00
abstract：:Enzymatic hydrolysis of the N-iminylamide was investigated in this study. An enzyme possessing N-iminylamidase activity from pig liver was purified to electrophoretic homogeneity. This enzyme was also active, however, with imides and appears to be identical to pig liver imidase. The identification was confirmed by cop...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.12.008
更新日期：2005-03-01 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
