Enzymatic Synthesis of Heparin - Sustainable Biotechnology: Sources of Renewable Energy - page 265

3 Technical Details-Materials and Methods

3.1 Purification of Heparosan from E. coli

E. coli K5 strain was used to produce heparosan, a bacterial polysaccharide with
repeating disaccharide N -acetylated glucosamine and glucuronic acid residues.
Heparosan is structurally similar to unepimerized and unsulfated HS backbone. In
this study, heparosan can be used to mimic HS backbone and can be modified by
HS biosynthetic enzymes. Heparosan is harvested from E. coli cells in large quan-
tities. Separation of heparosan from most proteins and other impurities was done
using a diethylaminoethyl (DEAE) Sephacel column. To further remove any protein
impurities, the eluted material was precipitated with ethanol followed by ammonium
sulfate precipitation and phenol/chloroform treatment. Each liter of E. coli culture
can yield up to 300 mg of heparosan.

Ta b l e 1 List of HS biosynthetic enzymes expressed in E. coli

Bacterial
expression cells

Yield
(mg/L) Enzyme Type of fusion protein References

NDST-2 Rosetta-gami-B

with GroEL

MBP-fusion protein 2 [46]

NDase-1 a Rosetta-gami-B

with GroEL

Thioredoxin-fusion
protein

5 Unpublished

NDase-2 a Rosetta-gami-B

with GroEL

MBP-fusion protein 10 [46]

NDase-3 a Rosetta-gami-B

with GroEL

Thioredoxin-fusion
protein

5 Unpublished

NST b

BL21 GST-fusion protein 20 [47]

Epi Rosetta-gami-B

with GroEL

MBP-fusion protein 15 [52]

2-OST Rosetta-gami-B MBP-fusion protein 12 [40]

6-OST-1 Rosetta-gami-B

with GroEL

MBP-fusion protein 14 [40]

6-OST-2 Rosetta-gami-B

with GroEL

(His) 6 -fusion protein 4 unpublished

6-OST-3 Rosetta-gami-B

with GroEL

(His) 6 -fusion protein 6 [41]

3-OST-1 BL21(DE3)RIL (His) 6 -fusion protein 8 [43]

3-OST-3 BL21(DE3)RIL (His) 6 -fusion protein 3 [44]

3-OST-5 Rosetta-gami-B

with GroEL

(His) 6 -fusion protein 8 [45]

KfiA BL21 Star with

GroEL

(His) 6 -fusion protein 10 [49]

pmHS2 BL21 Star (His) 6 -fusion protein 20 unpublished

a NDase-1, -2, and -3 represents the N -deacetylase domain of NDST-1, NDST-2, and NDST-3,
respectively.
b NST represents the N -sulfotransferase domain of NDST-1.