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Analysis of E. coli K5 capsular polysaccharide heparosan

Ly, Mellisa, Wang, Zhenyu, Laremore, Tatiana N., Zhang, Fuming, Zhong, Weihong, Pu, Dennis, Zagorevski, Dmitri V., Dordick, Jonathan S., Linhardt, Robert J.
Analytical and bioanalytical chemistry 2011 v.399 no.2 pp. 737-745
Escherichia coli, anticoagulants, fermentation, fractionation, heparin, ionization, mass spectrometry, molecular weight, polyacrylamide gel electrophoresis, swine
Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (MN), weight-averaged MWs (MW), and MW ranges of 3,000 to 150,000 Da.