Document Detail

Two-step chromatographic purification of recombinant human thyrotrophin and its immunological, biological, physico-chemical and mass spectral characterization.
MedLine Citation:
PMID:  15679148     Owner:  NLM     Status:  MEDLINE    
A purification strategy for rapidly obtaining recombinant human thyrotropin (rhTSH) was designed based on size exclusion and reversed-phase high-performance liquid chromatographic (HPLC) analysis, carried out on hTSH-secreting CHO cell conditioned medium. These analyses permitted the identification of the main contaminants to be eliminated. Considering that hTSH is highly hydrophobic and elutes only with the addition of organic solvents, hydrophobic interaction chromatography was adopted as the first purification step; this resulted in the elimination of, among others, the major contaminant. A second purification step, based on size exclusion chromatography, was then utilized, being effective in the elimination of other previously identified contaminating proteins. Useful purity, as high as 99% at the chemical reagent level, and recoveries (37%) were obtained by adopting this two step strategy, which also provided adequate material for physico-chemical, immunological and biological characterization. This included matrix-assisted laser desorption ionization time-of-flight mass spectral analysis (MALDI-TOF-MS), Western blotting analysis, in vivo biological assay, size-exclusion HPLC (HPSEC) and reversed-phase HPLC (RP-HPLC) analysis, which confirmed the integrity and bioactivity of our rhTSH in comparison with the only two reference preparations available at the milligram level of native (hTSH-NIDDK) and recombinant (Thyrogen) hTSH. Thyrogen and rhTSH-IPEN, when compared to pit-hTSH-NIDDK, presented more than twice as much biological activity and about 7% increased molecular mass by MALDI-TOF-MS analysis, an accurate heterodimer mass determination providing the Mr values of 29,611, 29,839 and 27,829, respectively. The increased molecular mass of the two recombinant preparations was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPSEC analysis. Comparing the two recombinant preparations, minor though interesting physico-chemical and biological differences were also observed.
Fernanda de Mendonça; João Ezequiel de Oliveira; Paolo Bartolini; Maria Teresa C P Ribela
Related Documents :
17206598 - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabo...
19302738 - Effect of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry...
15476188 - Preliminary investigation of electrodynamic charged droplet processing to couple capill...
21372408 - The novel assay method for nicotine metabolism to cotinine using high performance liqui...
10910218 - Fast development of separation methods for the chiral analysis of amino acid derivative...
22885238 - On-line hplc-fluorescence detection-esi-ms profiling of human milk oligosaccharides der...
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of chromatography. A     Volume:  1062     ISSN:  0021-9673     ISO Abbreviation:  J Chromatogr A     Publication Date:  2005 Jan 
Date Detail:
Created Date:  2005-01-31     Completed Date:  2005-05-10     Revised Date:  2009-01-15    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  103-12     Citation Subset:  IM    
Biotechnology Department, IPEN-CNEN, Cidade Universitária, 05508-900 São Paulo, Brazil.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Blotting, Western
CHO Cells
Chromatography, High Pressure Liquid / methods*
Culture Media, Conditioned
Electrophoresis, Polyacrylamide Gel
Recombinant Proteins / isolation & purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Thyrotropin / isolation & purification*
Reg. No./Substance:
0/Culture Media, Conditioned; 0/Recombinant Proteins; 9002-71-5/Thyrotropin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Previous Document:  Comparison of voltammetric detection assisted by multivariate curve resolution with amperometric det...
Next Document:  Chromatographic preprocessing of GC-MS data for analysis of complex chemical mixtures.