Abstract：
:Antimicrobial peptide LL-37 plays an important role in human body's first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of (15)N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database (), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Li Y,Li X,Wang Gdoi
10.1016/j.pep.2005.10.022keywords:
subject
Has Abstractpub_date
2006-06-01 00:00:00pages
498-505issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(05)00380-3journal_volume
47pub_type
杂志文章abstract：:The major immunodominant integral outer membrane protein C (OmpC) from Salmonella typhi Ty21a was overexpressed, without the signal peptide, in Escherichia coli. The protein aggregates as inclusion bodies (IBs) in the cytoplasm. OmpC from IBs was solubilized with 4 M urea and refolded. This involved rapid dilution of ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.12.023
更新日期：2005-03-01 00:00:00
abstract：:Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.07.007
更新日期：2014-10-01 00:00:00
abstract：:The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the wid...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.105546
更新日期：2020-03-01 00:00:00
abstract：:For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0969
更新日期：1998-12-01 00:00:00
abstract：:Fusion proteins expressed in bacteria are often insoluble or inefficiently purified by standard procedures previously reported to work well for the non-fused proteins. We report here a simple but general procedure that can be used to quickly customize and optimize the purification of milligram quantities of most GST f...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00524-7
更新日期：2002-11-01 00:00:00
abstract：:Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.06.015
更新日期：2018-12-01 00:00:00
abstract：:Deformylation of the initiator N-formylmethionine does not always proceed to completion for proteins overexpressed in Escherichia coli. To overcome this limitation, the def gene encoding the Escherichia coli peptide deformylase was cloned into the plysS plasmid under the tetracycline (Tc) promoter control. The efficie...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.03.007
更新日期：2004-07-01 00:00:00
abstract：:The sequence of the gene from Nocardia sp. NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined. The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass o...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.02.008
更新日期：2004-06-01 00:00:00
abstract：:Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxid...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.10.008
更新日期：2006-03-01 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：:Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the hos...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0955
更新日期：1998-11-01 00:00:00
abstract：:Formation of biominerals often involves specific proteins that modulate the process of matrix assembly, nucleation, and crystal growth. AP7 is an aragonite-associated protein of 7 kDa and is intrinsically disordered. The structural disorder of AP7 makes it very difficult to express in Escherchiacoli. In this work, we ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.05.002
更新日期：2014-08-01 00:00:00
abstract：:A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpresse...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.05.016
更新日期：2006-01-01 00:00:00
abstract：:Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein co...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.10.005
更新日期：2013-02-01 00:00:00
abstract：:This paper reports the overproduction and the details of a rapid method to purify active sigmaS monomers from a T7 RNA polymerase-based protein expression system. This 2-day procedure involves solubilizing inclusion bodies in sarkosyl detergent, removal of sarkosyl by dialysis, and a single gel filtration column chrom...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1996.0069
更新日期：1996-08-01 00:00:00
abstract：:A crude cell extract from yeast Saccharomyces cerevisae was fractionated by affinity chromatography using the leucine tRNA gene as the recognition site. This approach enables the rapid purification of a protein, which retained its full DNA binding capacity during the enrichment procedure. The active fraction contains ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/1046-5928(90)90015-q
更新日期：1990-11-01 00:00:00
abstract：:A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding p...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.01.009
更新日期：2006-06-01 00:00:00
abstract：:Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor. We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence. Th...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2001.1402
更新日期：2001-04-01 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：: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：:Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.08.014
更新日期：2015-11-01 00:00:00
abstract：:Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. P...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.07.022
更新日期：2007-03-01 00:00:00
abstract：:Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which are involved in viral attachment and entry into target cells. We have obtained in insect cells infected by recombinant baculovirus a chimeric secreted recombinant protein, E1(341)E2(661,) containing the ectodomains of E1 and E2. The described proc...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2010.02.012
更新日期：2010-06-01 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：:Soluble oligomers and fibrillar deposits of amyloid beta (Abeta) are key agents of Alzheimer's disease pathogenesis. However, the mechanism of amyloid aggregation and its interaction with live cells still remain unclear requiring the preparation of large amounts of pure and different Abeta peptides. Here we describe a...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.02.009
更新日期：2009-07-01 00:00:00
abstract：:Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively hig...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/s1046-5928(02)00573-9
更新日期：2003-02-01 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：: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：:The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2017.04.002
更新日期：2017-06-01 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
