Document Detail


Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.
MedLine Citation:
PMID:  22326251     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.
Authors:
Jong Youn Baik; Leyla Gasimli; Bo Yang; Payel Datta; Fuming Zhang; Charles A Glass; Jeffrey D Esko; Robert J Linhardt; Susan T Sharfstein
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-02-06
Journal Detail:
Title:  Metabolic engineering     Volume:  14     ISSN:  1096-7184     ISO Abbreviation:  Metab. Eng.     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-03-09     Completed Date:  2012-07-03     Revised Date:  2014-09-17    
Medline Journal Info:
Nlm Unique ID:  9815657     Medline TA:  Metab Eng     Country:  Belgium    
Other Details:
Languages:  eng     Pagination:  81-90     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
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MeSH Terms
Descriptor/Qualifier:
Amidohydrolases / biosynthesis*,  genetics
Animals
CHO Cells
Cricetinae
Cricetulus
Gene Expression*
Heparin / biosynthesis*,  genetics
Humans
Metabolic Engineering*
Mice
Sulfotransferases / biosynthesis*,  genetics
Transfection / methods
Transgenes
Grant Support
ID/Acronym/Agency:
R01 GM038060/GM/NIGMS NIH HHS; R01 GM090127/GM/NIGMS NIH HHS; R01 GM090127-01/GM/NIGMS NIH HHS; R01 GM093131/GM/NIGMS NIH HHS; R01 GM093131-01/GM/NIGMS NIH HHS; R01GM090127/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
9005-49-6/Heparin; EC 2.8.2.-/NDST2 protein, human; EC 2.8.2.-/Sulfotransferases; EC 2.8.2.8/heparitin sulfotransferase; EC 3.5.-/Amidohydrolases
Comments/Corrections
Comment In:
Bioengineered. 2012 Jul-Aug;3(4):227-31   [PMID:  22714556 ]

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


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