scholarly journals Generation of Mature Nα-Terminal Acetylated Thymosinα1 by Cleavage of Recombinant Prothymosinα

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Bo Liu ◽  
Xin Gong ◽  
Shaohong Chang ◽  
Peng Sun ◽  
Jun Wu
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Expression System ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Reverse Phase ◽  
E Coli ◽  
Amino Acid Peptide

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. Nα-terminal acetylated thymosinα1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosinα(ProTα) inE. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTαwas cleaved by human legumain expressed inPichia pastoristo release Tα1in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of anE. coliexpression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides inE. coli.

scholarly journals A Polysaccharide Deacetylase Homologue, PdaA, in Bacillus subtilis Acts as an N-Acetylmuramic Acid Deacetylase In Vitro

2005 ◽  
Vol 187 (4) ◽  
pp. 1287-1292 ◽  
Author(s):  
Tatsuya Fukushima ◽  
Toshihiko Kitajima ◽  
Junichi Sekiguchi
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
High Performance Liquid Chromatography ◽  
Bacillus Subtilis ◽  
Liquid Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Reverse Phase

ABSTRACT A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-l-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without dl-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.

scholarly journals Characterization of the In Vivo and In Vitro Metabolites of Linarin in Rat Biosamples and Intestinal Flora Using Ultra-High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2140 ◽  
Author(s):  
Xinchi Feng ◽  
Yang Li ◽  
Chenxi Guang ◽  
Miao Qiao ◽  
Tong Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Time Of Flight ◽  
Intestinal Flora ◽  
Ring Cleavage ◽  
Drug Candidate

Linarin, a flavone glycoside, is considered to be a promising natural product due to its diverse pharmacological activities, including analgesic, antipyretic, anti-inflammatory and hepatoprotective activities. In this research, the metabolites of linarin in rat intestinal flora and biosamples were characterized using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS). Three ring cleavage metabolites (4-hydroxybenzoic acid, 4-hydroxy benzaldehyde and phloroglucinol) were detected after linarin was incubated with rat intestinal flora. A total of 17 metabolites, including one ring cleavage metabolite (phloroglucinol), were identified in rat biosamples after oral administration of linarin. These results indicate that linarin was able to undergo ring fission metabolism in intestinal flora and that hydrolysis, demethylation, glucuronidation, sulfation, glycosylation, methylation and ring cleavage were the major metabolic pathways. This study provides scientific support for the understanding of the metabolism of linarin and contributes to the further development of linarin as a drug candidate.

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