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 methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.03.010
更新日期：2009-08-01 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：: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：:Der p 2, a major allergen derived from the house dust mite Dermatophagoides pteronyssinus, is one of the most clinically relevant allergens worldwide. Recombinant Der p 2 (rDer p 2) is useful in clinical diagnosis and disease-specific immunotherapy. However, previous studies showed that Der p 2 can only be expressed i...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.01.012
更新日期：2016-05-01 00:00:00
abstract：:Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.05.007
更新日期：2012-08-01 00:00:00
abstract：:2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia col...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2016.05.009
更新日期：2016-10-01 00:00:00
abstract：:The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1070
更新日期：1999-07-01 00:00:00
abstract：:A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)(n)-repeated linker inserted between albumin and EPO. Albumin-EPO fusion genes were co-transfected with the dhfr gene. Albumin-EPO fusion protein has three kinds of sub-types (IALE,...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2009.07.003
更新日期：2009-12-01 00:00:00
abstract：:Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with speci...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2014.12.003
更新日期：2015-06-01 00:00:00
abstract：:PLD's (Phospholipases D) are ubiquitously expressed proteins involved in many transphosphatidylation reactions. They have a bi-lobed structure composed by two similar domains which at their interface reconstitute the catalytic site through the association of the two conserved HxKx(4)Dx(6)GSxN motifs. PLD1 interacts wi...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.02.012
更新日期：2008-06-01 00:00:00
abstract：:Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.10.023
更新日期：2009-04-01 00:00:00
abstract：:Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glu...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2012.09.016
更新日期：2012-12-01 00:00:00
abstract：:Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl dipho...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.12.008
更新日期：2016-04-01 00:00:00
abstract：:The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2006.06.008
更新日期：2007-02-01 00:00:00
abstract：:Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-la...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2004.08.002
更新日期：2004-11-01 00:00:00
abstract：:Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltr...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2018.05.012
更新日期：2018-10-01 00:00:00
abstract：:Amyloid-beta (Aβ) peptide mediates several neurodegenerative diseases. The 42 amino acid (Aβ1-42) is the predominant form of peptide found in the neuritic plaques and has been demonstrated to be neurotoxic in vivo and in vitro. The availability of large quantities of Aβ peptide will help in several biochemical and bio...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.05.015
更新日期：2015-10-01 00:00:00
abstract：:Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2020.105636
更新日期：2020-08-01 00:00:00
abstract：:The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.2002.1621
更新日期：2002-06-01 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：:Rat granulocyte macrophage colony-stimulating factor (rGM-CSF) cDNA was amplified and cloned, and recombinant-rGM-CSF (R-rGM-CSF) was expressed and isolated from Escherichia coli. The synthesis of R-rGM-CSF was directed by a modified, inducible maltose binding protein (MBP) gene fusion expression vector, pMTR-23, and ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1995.1077
更新日期：1995-10-01 00:00:00
abstract：:The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1999.1040
更新日期：1999-06-01 00:00:00
abstract：:An active form of a single-chain antibody fragment (scFv) from the murine monoclonal antibody ABL-1, which is specific for B-cell-activating factor of the TNF family, was produced in Escherichia coli. The complementary DNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were connected by a ...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2005.04.022
更新日期：2005-10-01 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：:The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-lengt...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1006/prep.1998.0896
更新日期：1998-07-01 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：:Chan Su is a traditional medicine prepared from toxic secretions from the auricular and skin glands of Chinese toads. Previous studies show that active components in Chan Su can inhibit the proliferation of tumor cells. To study the effect of Chan Su peptides on angiogenesis, fresh Chan Su was collected and its compon...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2019.105445
更新日期：2019-11-01 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
更新日期：1996-05-01 00:00:00
abstract：:The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2008.05.008
更新日期：2008-10-01 00:00:00
abstract：:The market of therapeutic glycoproteins (including coagulation factors, antibodies, cytokines and hormones) is one of the profitable, fast-growing and challenging sectors of the biopharmaceutical industry. Although mammalian cell culture is still expensive and technically complex, the ability to produce desired post-t...
journal_title：Protein expression and purification
pub_type： 杂志文章
doi：10.1016/j.pep.2015.08.008
更新日期：2015-11-01 00:00:00
