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Journal of Bacteriology, June 2002, p. 3242-3252, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3242-3252.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

Françoise Blain,,{dagger} A. Lydia Tkalec,,{dagger} Zhongqi Shao,,{ddagger} Catherine Poulin, Marc Pedneault,,{ddagger} Kangfu Gu,,§ Bernhard Eggimann,,{ddagger} Joe Zimmermann,,|| and Hongsheng Su,{dagger}*

IBEX Pharmaceuticals Inc., Montreal, Quebec H4P 1P7, Canada

Received 25 October 2001/ Accepted 19 March 2002

A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

* Corresponding author. Present address: Celmed Biosciences Inc., 2310 Alfred-Nobel Blvd., Saint Laurent, Quebec H4S 2A4, Canada. Phone: (514) 336-7800. Fax: (514) 336-7329. E-mail: hsu{at}celmedbio.com.

{dagger} Present address: Celmed Biosciences Inc., Saint Laurent, Quebec H4S 2A4, Canada.

{ddagger} Present address: BioMarin, Montreal, Quebec H4P 1P7, Canada.

§ Present address: Diversa Corporation, San Diego, CA 92121.

|| Present address: The Millennium Three Group, Rosemont, PA 19010.

Journal of Bacteriology, June 2002, p. 3242-3252, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3242-3252.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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