Biomedical subjects

Song-Ping Liang

Publications and source records attributed to Song-Ping Liang.

24 records · Page 2Linked to original sources

PubMed2002

[Expression and purification of recombinant huwentoxin-I in Pichia pastoris].

HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX-I expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity. The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.

Amino Acid Sequence↗

PubMed2001

Preliminary Function Study of NAG7 Using Two-dimensional Electrophoresis and Mass Spectrometry.

In search of mechanisms of function of NAG7 gene, a powerful new tool for the unambiguous characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. NAG7, a novel putative tumor suppressor gene, located on 3p25.3, was introduced into HNE1 cells by liposome transfection. We used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to identify proteins that were overexpressed in NAG7 transfected cells. After staining and image analysis, spots of interest were isolated and subjected to mass spectrometry (MS). We found nine proteins, which up-regulated in NAG7 transfected cells and be identified by MS. These proteins included growth arrest specific protein, DNA binding protein, c-myc promoter-binding protein and caspase 6 etc., which involved in cell cycling, transcription regulation, and apoptosis. NAG7 may exert the function by up-regulating the expression of these proteins.

Journal Article↗

PubMed2001

Sequence-specific Assignment of (1)H-NMR Resonance and Determination of the Secondary Structure of HWTX-II.

Huwentoxin-II (HWTX-II)is an insecticidal peptide purified from the venom of spider Selenocosmia huwena. The structure of this toxin in solution was investigated using 2D-NMR. The complete sequence-specific assignments of proton resonance in the (1)H-NMR spectra of HWTX-II were obtained by analyzing a series of 2D spectrum, including COSY, DQF-COSY, TOCSY and NOESY. All the backbone protons and side chain protons, except epsilonNH(2) protons of Lys residues, were identified by d(alphaN), d(alpha&dgr);, d(NN) and d(betaN) connectivities. The results provide a basis for further determination of the solution conformation of HWTX-II. Furthermore the secondary structure of HWTX-II was determined from NMR data. It contained mainly extended conformation, especially a double-stranded anti-parallel beta-sheet with Trp27--Cys29 and Cys34--Lys36 at the C terminal, and it lacked helix. These characters of the secondary structure of HWTX-II were similar to those spider toxins which structure in solution had been reported.

Journal Article↗

PubMed2000

Identification of Protein Spots in Silver-stained Two-dimensional Gels by MALDI-TOF Mass Peptide Map Analysis.

Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including i) In-gel reduction, alkylation and enzymatic digestion ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)) iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrate that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this the procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S.Huwena were analyzed and identified.

Journal Article↗

PubMed2000

A Chimera Polypeptide with Active Sites of HWTX-I and AAI.

The functional amino acid sequence and its neighbouring fragments in the molecule of Amaranth alpha-amylase inhibitor isolated from seeds of the Mexican crop plant Amaranthus hypochondriacus were transferred, by solid phase chemical synthesis, into N-terminal region of the huwentoxin-I(HWTX-I). The synthetic chimera polypeptide was confirmed by Edman degradation and MALDI-TOF mass spectroscopy. The formation of three disulfide bonds and special conformation of the synthetic chimera was induced by the addition of glutathione. Renatured chimera polypeptide was purified by ion-exchange and reversed phase HPLC. The results showed that the engineered chimera polypeptide exerted obvious inhibitory activity to alpha-amylase from digestive tube of the roach (Periplaneta Americana) at pH 5.5 with the concentration of 9.5x10(-5) mol/L, and also exerted 36% of the neurotoxic activity of the natural huwentoxin-I as shown by the experiments of blockage of the neuromuscular transmission of isolated mouse phrenic nerve-diaphragm preparations. The experiments demonstrated that the structural motif of HWTX-I is well promising for protein engineering, and the solid-phase peptide synthesis is adequate rapid for the engineering of artificially designed small proteins.

Journal Article↗

PubMed1997

Preliminary Study on the Primary Structure of Dragline Fibroin from the Spider Araneus ventrocosus.

The amino acid composition of the dragline fibroin from the spider fibroin of the spider Araneus ventrocosus was analysed and compared with that of the fibroins from different species of spiders. By means of partial acid hydrolysis and high performance liquid chromatography, several peptide fragments of the dragline fibroin were purified. The amino acid sequence analysis showed the sequences of these peptides to be different from that of the fibroin from the spider Nephila clavipes except for one common fragment with the sequence of GYGPG.

Journal Article↗