Abstract：
:Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a "funnel approach" to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Lewinson O,Lee AT,Rees DCdoi
10.1016/j.jmb.2007.12.059subject
Has Abstractpub_date
2008-03-14 00:00:00pages
62-73issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(07)01704-4journal_volume
377pub_type
杂志文章abstract：:Oncogenes may play a crucial role in the genetic program of cellular differentiation; even, probably, at a very early stage in this program, which can be described as pre-commitment. We have investigated the variation in, and the control level of, the accumulation of the transformation-associated cellular protein p53 ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(88)90333-6
更新日期：1988-03-05 00:00:00
abstract：:The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2005.09.011
更新日期：2005-11-11 00:00:00
abstract：:Phase transitions that alter the physical state of ribonucleoprotein particles contribute to the spacial and temporal organization of the densely packed intracellular environment. This allows cells to organize biologically coupled processes as well as respond to environmental stimuli. RNA plays a key role in phase sep...
journal_title：Journal of molecular biology
pub_type： 杂志文章,评审
doi：10.1016/j.jmb.2018.05.003
更新日期：2018-11-02 00:00:00
abstract：:Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate prec...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2009.11.045
更新日期：2010-02-19 00:00:00
abstract：:We have analysed the hydration of main-chain carbonyl and amide groups in 24 high-resolution well-refined protein structures as a function of the secondary structure in which these polar groups occur. We find that main-chain atoms in beta-sheets are as hydrated as those in alpha-helices, with most interactions involvi...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(91)80080-e
更新日期：1991-09-20 00:00:00
abstract：:beta-1,4-Galactosyltransferase-I (beta4Gal-T1) transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3-arm), and the other to the 6-...
journal_title：Journal of molecular biology
pub_type： 杂志文章
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1997.1316
更新日期：1997-10-24 00:00:00
abstract：:Using homopolymeric units of either phenylalanine or tryptophan to replace the natural core segment of the Escherichia coli alkaline phosphatase signal peptide, the hydrophobicity requirements for protein export and processing were further explored. The mutant signal peptide containing polyphenylalanine functioned at ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(92)90577-7
更新日期：1992-03-05 00:00:00
abstract：:The proteins coded by the five major capsid genes of herpes simplex virus 1, VP5 (gene UL19), VP19c (UL38), VP23 (UL18), pre-VP22a (UL26.5), and pre-VP21 (UL26), assemble into fragile roundish "procapsids", which mature into robust polyhedral capsids in a transition similar to that undergone by bacteriophage proheads....
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/s0022-2836(96)80018-0
更新日期：1996-11-01 00:00:00
abstract：:The pathological and physiological hallmarks of the protein alpha-synuclein (aS) are its misfolding into cytotoxic aggregates and its binding to synaptic vesicles, respectively. Both events are mediated by seven 11-residue amphiphilic pseudorepeats and, most generally, involve a transition from intrinsically unstructu...
journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：2009-07-17 00:00:00
abstract：:CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2010.09.067
更新日期：2010-12-10 00:00:00
abstract：:Transcription of the ribosomal RNA operons (rrn) in Escherichia coli is subject to an antitermination mechanism whereby RNA polymerase is modified to a termination-resistant form during transit through the rrn leader region. This antitermination mechanism is unable to overcome the T1-T2 terminator cluster located at t...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(91)90737-q
更新日期：1991-11-05 00:00:00
abstract：:Regulation of eukaryotic genes is largely governed by multiple cis-acting DNA sequences recognized by specific transcription factors. The transcription factor NF-kappa B has been implicated as an important regulator of cellular and viral genes, including those of immunoglobulin kappa light chain, interleukin-2, beta-i...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/0022-2836(90)90187-q
更新日期：1990-07-20 00:00:00
abstract：:Mycobacteriophage Brujita is an unusual temperate phage in which establishment of superinfection immunity is dependent on chromosomal integration. Integration is mediated by a non-canonical tyrosine integrase (Int) lacking an N-terminal domain typically associated with binding to arm-type sites within the phage attach...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2016.04.023
更新日期：2016-06-05 00:00:00
abstract：:Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2011.10.042
更新日期：2012-01-06 00:00:00
abstract：:The terminal deoxynucleotidyl transferase gene (TdT) is expressed in mice only in early B and T lymphoid precursors a few days after birth. Transactivating factors have been shown to contribute to the lymphoid specific expression of TdT, but they do not account entirely for the restriction of its expression to early p...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1999.3079
更新日期：1999-09-17 00:00:00
abstract：:Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefota...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2012.07.012
更新日期：2012-10-26 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.2000.4016
更新日期：2000-08-25 00:00:00
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pub_type： 杂志文章
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journal_title：Journal of molecular biology
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doi：10.1006/jmbi.1998.1727
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journal_title：Journal of molecular biology
pub_type： 杂志文章
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journal_title：Journal of molecular biology
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更新日期：2014-05-15 00:00:00
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journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.2001.4576
更新日期：2001-05-04 00:00:00
abstract：:Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated ...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1996.0110
更新日期：1996-03-01 00:00:00
abstract：:The refined structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 reveals a membrane protein with protein-protein interactions in the trans-membrane region exclusively of a van der Waals nature. The dominant factors in the formation of the complex appear to be extramembrano...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1997.0966
更新日期：1997-05-02 00:00:00
abstract：:The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes...
journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：1984-01-15 00:00:00
abstract：:Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values...
journal_title：Journal of molecular biology
pub_type： 杂志文章
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更新日期：1990-07-20 00:00:00
abstract：:The B-DNA decanucleotide C-G-A-T-C-G-6meA-T-C-G has been crystallized under the same conditions used earlier for C-G-A-T-C-G-A-T-C-G, but is found to adopt a new trigonal P3(2)21 packing mode instead of the expected orthorhombic P2(1)2(1)2(1) form. Unit cell dimensions a = b = 33.38 A, c = 98.30 A, gamma = 120 degrees...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1006/jmbi.1993.1325
更新日期：1993-06-05 00:00:00
abstract：:Aldo-keto reductases (AKRs) are a large superfamily of NADPH-dependent enzymes that catalyze the reduction of aldehydes, aldoses, dicarbonyls, steroids, and monosaccharides. While their precise physiological role is generally unknown, AKRs are nevertheless involved in the detoxification of a broad range of toxic metab...
journal_title：Journal of molecular biology
pub_type： 杂志文章
doi：10.1016/j.jmb.2010.02.021
更新日期：2010-04-23 00:00:00
