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Process BioHu'mi~t O' V ol. 33, No. 6, pp. ~83-686, 1998
,~) 1998 Elsevier Science Lid. All rights reserved
P rinted in Grcal Britain
0032-9592/98 $19.1)(I +(1.(1(1

E LSEVIER
PII:

S0032-9592(98)00033-8

Human growth hormone production by high
cell density fermentation of recombinant
E scherichia coli
X ue-Wu Zhang,* Tao Sun, Xin Liu, De-Xiang Gu and Xiao-Ni Huang
Food EngineeringResearch Center of State Education Commission, Zhongshan University, 510275 Guangzhou,
People's Republic of China
( Received 1 December 1997; revised version received 16 Fcbruarv 1998: accepted I March 19c~8)

Abstract

ttigh cell density fermentation production of recombinant human growth hormone was studied in ILs'cherichia co/i.The results showed that by using glycerol instead of glucoseas carbon source and feeding in a
specific fed-batch mode,by controlling the stirrer speed and keeping the dissolved oxygen level at 20-25¢~:
saturation,cell density increases from 38.6 to 118.8 g/l,the production of r-hGH remained high and the
f ermentation time was reduced from 16 to 10h. © 1998 Published by Elsevier Science Ltd. All rights
reserved
Kcywords: H uman growth hormone, highcell density fermentation, Escherichia coil, glycerol.

b y-products, such as acetate. Acetate fl)rmation can be
r educed or prevented by altering the formulation of
t he medium, for example, acetate is not produced
w hen glycerol is used as a carbon source [1]. The
o bjective of this study was to investigate the effects of
f ermentation conditions including the types of carbon
s ource(glucose and glycerol), dissolved oxygen and
c ulture system (normal and high cell density fermentat ion) on the production of recombinant human growth
h ormone in E scherichia coli.

I ntroduction

R ecombinant human growth hormone (r-hGH) is used
t o treat dwarf patients, and to enhance the physiol ogical functions of middle-aged or elderly persons so
t hat senility can be delayed. As withother proteins
s uch as intcrferons, colony-stimulating factors, insulinl ike growth factors and human serum albumin, r-hGH
h as been successfully produced using recombinant
E scherichia coli [1]. Because most proteins are intrac ellularly accumulated in recombinant Escherichia coli, productivity is proportional to the final cell-density
a nd the specific productivity (i.e. the amount of
product formed per unit cell mass per unit time). High
c ell-density fermentation (HCDF) techniques for cult uring E scherichia coli h ave been developed to improve
p roductivity,and to provide advantages such as reduced
v olume,lower production costs and reduced investment
in equipment.
F ed-batch processes have most often been used to
o btain high cell-density [2-7]. However, this technique
has drawbacks, the formation of growth-inhibitory

M aterials and methods

Otig,anism and culture media
E scherichia coil k802 was used as the host organism, plasmid pSSM was provided by the Microbial Institute
o f Guangdong Province, Guangzhou, China. LB
m edium was used for seed culture. The composition is
p olypeptone (10g/l), yeast extract (5 g/I), and NaCI
( 5g/I) and pH was adjustedat 7.0. Fermentation
m edium has the following compositions: KHzPO4, 45 g;
N aeHPO4-12H20, 256g; NH4CI. 15g; MgSO4-7H20,

*Corresponding author.
(~83

684

X.-W. Zhang et al.

7.5 g; CaCI2, 0.5 g; NaCk 7.5 g tryptone, 750 g; yeast
e xtract 375 g; 12 1 of reverse osmosis water was added
t o the medium. Feed solutions consisted of tryptone,
2 00 g/l; yeast extract, 100 g/l; glycerol, 2I; 2 1 of reverse
o smosis water was added to the solution.

{.'~experimental for glucose
experimental for glycerol
O calculated by eqn(1)
I a-calculated by eq.n(2)

1 40

~"

1 20
1 00

B atch fermentations
S eed cultures for fermentations were started by picking
a n isolated colony freeze-preserved in glycerol solut ions and transferring by loop to a 1 1 flask containing
200...