I keep them
murine scFv library specific for hLCN6 was produced with a simple and rapid method using phage display technology.
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Functional anti-hLCN6 scFv with good specificity and affinity was solubly expressed and purified from E.coli in high purity.
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This scFv is a potencial tool for sperm separation from forensic mixed stains by immunomagnetic beads method at low cost.
Protein Expression and Purification
Volume 171, July 2020, 105627
Selection, preparation and characterization of scFv against human lipocalin 6 by phage display technology
Author links open overlay panelJiongChenaWeiFenga
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https://doi.org/10.1016/j.pep.2020.105627Get rights and content
Highlights
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A murine scFv library specific for hLCN6 was produced with a simple and rapid method using phage display technology.
•
Functional anti-hLCN6 scFv with good specificity and affinity was solubly expressed and purified from E.coli in high purity.
•
This scFv is a potencial tool for sperm separation from forensic mixed stains by immunomagnetic beads method at low cost.
Abstract
Human lipocalin 6 (hLCN6) is a newly discovered epididymal-specific secreted protein, capable of binding to the head and tail of spermatozoa and involved in sperm maturation. Anti-hLCN6 monoclonal antibody coupled immunomagnetic beads (IMBs) can be effectively used for the separation and forensic identification of sperm cells from mixed stains. But the source of monoclonal antibody is limited. In this study, an immunized mouse phage display antibody library was constructed and the single-chain variable fragments (scFvs) against hLCN6 were screened. The selection was performed using four rounds of biopanning and positive clones were validated by phage ELISA. Two anti-hLCN6 scFv clones with highest affinity were selected and sequencing result showed that the two sequences were identical. After prokaryotic expression and purification, the purified scFv could specifically recognize the hLCN6 in the lysate of human sperm cells and epididymis by western blot analysis, without any cross-reactivity with cellular antigens in female epithelial cells. The dissociation constant (Kd) of anti-hLCN6 scFv was 6.69 × 10−7 mol/L measured by indirect ELISA. Therefore, our work not only provides a useful tool for further exploration of the biological functions of hLCN6, but also opens up new research avenues for the separation of sperm cells from mixed stains based on immuno-binding reaction.
HNPCC mutations in hMSH2 result in reduced hMSH2-hMSH6 molecular switch functions
Mutations in the human mismatch repair (MMR) gene hMSH2 have been linked to approximately 40% of hereditary nonpolyposis colorectal cancers (HNPCC). While the consequences of deletion or truncating mutations of hMSH2 would appear clear, the detailed functional defects associated with missense alterations are unknown. We have examined the effect of seven single amino acid substitutions associated with HNPCC that cover the structural subdomains of the hMSH2 protein. We show that alterations which produced a known cancer-causing phenotype affected the mismatch-dependent molecular switch function of the biologically relevant hMSH2-hMSH6 heterodimer. Our observations demonstrate that amino acid substitutions within hMSH2 that are distant from known functional regions significantly alter biochemical activity and the ability of hMSH2-hMSH6 to form a sliding clamp.
Gene
Volume 137, Issue 2, 31 December 1993, Pages 195-202
Mouse cDNAs encoding a trifunctional protein of de novo purine synthesis and a related single-domain glycinamide ribonucleotide synthetase
Author links open overlay panelJulie L.C.Kana∗∗Richard G.Moranacd
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https://doi.org/10.1016/0378-1119(93)90006-OGet rights and content
Abstract
Three of the enzymatic activities of de novo purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART), can be catalyzed by a single 110-kDa protein in mouse cells. Western blots using a polyclonal antibody (Ab) to this protein identified two species, the trifunctional 110-kDa protein and a 50-kDa cytosolic protein with GARS, but not GART activity. We used Ab and, subsequently, oligodeoxyribonucleotide screens to isolate cDNAs corresponding to these two proteins from mouse T-cell cDNA expression libraries. The sequence of one class of these cDNAs and the partial sequence of a corresponding genomic clone defined an open reading frame (ORF) encoding a 1010-amino-acid (aa) protein, individual domains of which showed high homology to each of the monofunctional bacterial GARS, AIRS and GART proteins, and to each domain of chicken and human trifunctional GARS-AIRS-GARTs. cDNAs corresponding to the smaller protein contained a 1.3-kb ORF with complete identity to the GARS domain of, but with a 3' untranslated region different from, the trifunctional cDNAs. Hence, both cDNAs appear to derive from the same gene due to either differential splicing or use of an intronic polyadenylation signal. The functional requirement for the expression of both trifunctional protein with GARS activity and monofunctional, catalytically active GARS is unknown.
Abbreviations
aa
amino acid(s)
Ab
antibody(ies)
AIRS
aminoimidazole ribonucleotide synthetase
bp
base pair(s)
DDATHF
5,10-dideaza-5,6,7,8-tetrahydrofolate
GAR
glycinamide ribonucleotide
GARS
GAR synthetase
GART
GAR formyltransferase
kb
kilobase(s) or 1000 bp
nt
nucleotide(s)
ORF
open reading frame
RBS
ribosome-binding site
SDS
sodium dodecyl sulfate
SSC
149 mM NaCl/15mM Na3 β citrate pH 7.0
TBS
50 mM Tris pH 7.5/150 mM NaCl
UTR
untranslated region(s)
Abstract
The electronic structure of mono, bis and trihydroxylated derivatives in the B-ring of the flavylium cation was studied. The AM1 semiempirical method of molecular orbital calculations was used. Data showed that substitution in the B-ring causes resonance increase between this ring and the C-ring, except for substitution on C(3′). Bond orders were interpreted through the variation in the contribution of resonance structures and they helped to explain the relative stability of the several dissubstituted isomers. Another factor that contributes to this stability is the formation of a hydrogen bond in the B-ring and between the B-and C-rings. The rotation enthalpies in the B-ring were calculated and they confirm that the rotation on the C(2)-C(1′) bond is free in the compounds with substitution on position 3′. The molecule planarity can be attributed to the interaction between the oxygen atom in the C-ring and the hydrogen atoms in the B-ring, or between the hydrogen atoms in these rings. The charge variations are in agreement with the proposed resonance structures. Substitution in the B-ring does not affect the sites of basic and nucleophilic attack.
Tetrahedron
Volume 48, Issue 33, 1992, Pages 6863-6874
A novel cyclocondensation of xanthates containing active methylene groups with isothiocyanates. Spectral data and X-ray structures of the products
Author links open overlay panelG.V.TormosaS.V.Belyakovb
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https://doi.org/10.1016/S0040-4020(01)89877-XGet rights and content
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A cyclocondensation reaction of S-(alkoxycarbonylmethyl)-and S-(cyanomethyl)dithiocarbonate O-esters with isothiocyanates in the presence of sodium tert-butoxide is described. Spectral data and X-ray structures of the main products are given.
A cyclocondensation reaction of S-(alkoxycarbonylmethyl)- and S-(cyanomethyl)dithiocarbonate O-esters with isothiocyanates in the presence of sodium tert-butoxide is described.
Journal of Molecular Structure
Volume 327, Issue 1, 20 October 1994, Pages 1-21
A vibrational molecular force field of model compounds with biological interest. VII. Harmonic dynamics of N-acetyl-α-d-muramic acid and N-acetyl-β-d-neuraminic acid in the crystalline state
Author links open overlay panelI.Kouach-AlixaG.Vergotena
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https://doi.org/10.1016/0022-2860(94)08351-7Get rights and content
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Combining a modified Urey—Bradley—Shimanouchi intramolecular potential energy function with an appropriate intermolecular energy function, normal coordinate calculations have been performed for N-acetyl-α-d-muramic acid and for N-acetyl-β-d-neuraminic acid in the crystalline state. IR and Raman spectra were recorded. Overall agreement between the observed and the calculated frequencies leads to an average error of 4 cm−1. The computed potential energy distribution was found to be compatible with previous assignments for N-acetyl-α-d-glucoasamine for the pyranose ring and the acetamido group, and for pivalic acid for the carboxylic group. The sets of force constants used for the two monosaccharides were taken from previous works on monosaccharides.
The acetylene complex ()-(N,N-bis(diphenylphosphino)-1-phenethylamine) (hexafluorobut-2-yne)platinum(O) has been prepared, and its structure determined by X-ray analysis. Crystals are monoclinic, space group P21, with a cell of dimensions a = 11.177(2), b = 15.690(3), c = 19.655(4) Å and β = 94.46(1)°. Intensity data collected on an automated four-circle diffractometer was used for full-matrix least-squares refinement on F, which converged at R = 0.059, 5851 observations. There are two molecules in the asymmetric unit. The coordination at the Pt atoms is essentially planar, and the CC bond distances are 1.26(2) and 1.25(2) Å. The deviations from linearity of the alkyne ligand upon coordination are 38(2)° and 46(2)° in one molecule, while values of 40(2)° and 39(2)° are found in the other. IR, 19F NMR and 31P NMR spectra are reported and discussed for this species and for the series of hexafluorobut-2-yne complexes of general formula ML2-(acetylene) which we have studied by X-ray techniques.