Protein Expression and Purification 18, 182–192 (2000)
doi:10.1006/prep.1999.1179, available online at http://www.idealibrary.com on
Optimization of Inclusion Body Solubilization and
Renaturation of Recombinant Human Growth
Hormone from Escherichia coli
Ashok K. Patra, R. Mukhopadhyay, R. Mukhija,* Anuja Krishnan,*
L. C. Garg,* and Amulya K. Panda 1
Product Development Cell, *Gene Regulation Laboratory, National Institute of Immunology,
Aruna Asaf Ali Marg, New Delhi 110067, India
Received September 24, 1999, and in revised form November 10, 1999
Recombinant human growth hormone (r-hGH) was
expressed in Escherichia coli as inclusion bodies. In
10 h of fed-batch fermentation, 1.6 g/L of r-hGH was
produced at a cell concentration of 25 g dry cell
weight/L. Inclusion bodies from the cells were isolated
and purified to homogeneity. Various buffers with and
without reducing agents were used to solubilize r-hGH
from the inclusion bodies and the extent of solubility
was compared with that of 8 M urea as well as 6 M
Gdn-HCl. Hydrophobic interactions as well as ionic
interactions were found to be the dominant forces re-
sponsible for the formation of r-hGH inclusion bodies
during its high-level expression in E. coli. Complete
solubilization of r-hGH inclusion bodies was observed
in 100 mM Tris buffer at pH 12.5 containing 2 M urea.
Solubilization of r-hGH inclusion bodies in the pres-
ence of low concentrations of urea helped in retaining
the existing native-like secondary structures of r-hGH,
thus improving the yield of bioactive protein during
refolding. Solubilized r-hGH in Tris buffer containing
2 M urea was found to be less susceptible to aggrega-
tion during buffer exchange and thus was refolded by
simple dilution. The r-hGH was purified by use of
DEAE-Sepharose ion-exchange chromatography and
the pure monomeric r-hGH was finally obtained by
using size-exclusion chromatography. The overall
yield of the purified monomeric r-hGH was ⬃50% of the
initial inclusion body proteins and was found to be
biologically active in promoting growth of rat Nb2
lymphoma cell lines. © 2000 Academic Press
Key Words: recombinant human growth hormone;
Escherichia coli; inclusion bodies; purification;
bioactivity.
1
To whom correspondence should be addressed at Product Devel-
opment Cell, National Institute of Immunology, Aruna Asaf Ali
Road, New Delhi 110067, India. E-mail: amulya@nii.res.in.
182
High-level expression of recombinant proteins in
Escherichia coli often accumulates as insoluble aggre-
gates in vivo as inclusion bodies (1). The formation of
inclusion bodies is mainly attributed to the overexpres-
sion of proteins in the cell lacking the required acces-
sories for its folding to the native form (2). Endogenous
proteins when overexpressed in E. coli also accumulate
as inclusion bodies (3). There is no direct correlation
between the propensity of the inclusion body formation
of a certain protein and its intrinsic properties, such as
molecular weight, hydrophobicity, and folding path-
ways (4). In the case of proteins having disulfide bonds,
formation of protein aggregation as inclusion bodies is
anticipated since the reducing environment of bacte-
rial cytosol inhibits the formation of disulfide bonds.
Significant features of protein aggregates in inclusion
bodies are the existence of native-like secondary struc-
tures of the expressed protein (5) and their resistance
to proteolytic degradation (6). The aggregation leading
to inclusion body formation has also been reported to
be due to specific intermolecular interactions among a
single type of protein molecule (7). The formation of
inclusion bodies thus facilitates the easy isolation and
recovery of the expressed proteins in the denatured
form.
In general, proteins expressed as inclusion bodies
are solubilized by the use of high concentrations of
chaotropic solvents. Chaotropic agents such as urea,
guanidine hydrochloride (Gdn-HCl) 2, and thiocyanate
salts (8,9), detergents such as sodium dodecyl sulfate
2
Abbreviations used: r-hGH, recombinant human growth hor-
mone; Gdn-HCl, guanidine hydrochloride; CTAB, n-cetyltrimethyl-
ammonium bromide; NLS, sodium N-lauroyl sarcosine; BSA, bovine
serum albumin; HS, horse serum; FBS, fetal bovine serum; BCA,
bicinchoninic acid; IPTG, isopropyl-␤-D-thiogalactopyranoside; CD,
circular dichroism; SDS-PAGE, sodium dodecyl sulfate-polyacrylam-
ide gel electrophoresis; DMSO, dimethyl sulfoxide; PEG, polyethyl-
ene gycol; PMSF, phenylmethylsulfonyl fluoride.
1046-5928/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
 RECOMBINANT HUMAN GROWTH HORMONE
(SDS) (10), N-cetyltrimethylammonium chloride (11)
and sarkosyl (sodium N-lauroyl sarcosine; NLS) (12)
along with reducing agents like ␤-mercaptoethanol,
dithiothreitol, or cysteine have been extensively used
for solubilizing the inclusion body proteins. The soluble
proteins are then refolded to their native state after the
chaotropic agents or other salts are removed by dialyz-
ing the proteins in buffers containing reducing and
oxidizing agents (8,13). Often, additives such as ace-
tone, acetoamide, urea, DMSO, and PEG (14) are used
to enhance the yield of folded bioactive protein. Rena-
turation of proteins into the native conformation has
also been reported by using immobilized minichaper-
ones (15) and size-exclusion chromatography (16). De-
spite several protocols available for protein solubiliza-
tion and refolding, the overall recovery of bioactive
proteins from inclusion body is often very low. It is
expected that the overall yield of purified bioactive
proteins from the inclusion bodies can be improved if
the existing secondary structure of the proteins is pro-
tected during solubilization without the use of high
concentrations of chaotropic agents (17).
Human growth hormone (hGH), a single chain
polypeptide containing 191 amino acid residues, apart
from stimulating cell growth, plays an important role
in a variety of metabolic, physiologic, and anatomic
processes (18). The protein folds into a four-helix bun-
dle structure with two disulfide bridges, one connecting
distant parts of the molecule involving amino acid res-
idues 53 and 165 (large loop) and another between
residues 182 and 189 (small loop) (19). The large-scale
requirement of r-hGH necessitates its high-level ex-
pression in E. coli as inclusion bodies (20). However,
expression of the protein along with fusion tag and
subsequent use of high concentrations of chaotropic
reagents for solubilization and purification makes the
overall process more complex and expensive as the
yield of bioactive r-hGH is lowered (21). In this report,
we have described a simple and efficient process for the
production of bioactive r-hGH from the inclusion bodies
of E. coli. The solubilization behavior of r-hGH inclu-
sion bodies in different buffers was analyzed for an
understanding of the nature of protein aggregation in
inclusion bodies. Solubilization of r-hGH from purified
inclusion bodies was carried out without disturbing the
existing native-like secondary structure and the solu-
bilized r-hGH was subsequently purified and refolded
into the bioactive form.
MATERIALS AND METHODS
Chemicals
Urea, acrylamide and bis-acrylamide, sodium dode-
cyl sulfate, deoxycholate, and CTAB were of analytical
grade, obtained from Amresco (U.S.A.). Sephacryl
S-200 and DEAE-Sepharose were from Pharmacia Bio-
tech (Sweden). NLS, BSA, prolactin, and RPMI 1640
183
were from Sigma Chemicals (U.S.A.). Fetal bovine se-
rum (FBS) and horse serum (HS) were from Gibco-BRL
(U.S.A.). Commercially available recombinant human
growth hormone was from Boehringer Mannheim
(Germany). All other chemicals were of analytical
grade. The spectral measurements and analytical
HPLC were performed in degassed and filtered buffers
prepared in Milli-Q water.
Cloning and Expression of r-hGH
To clone hGH without any tag and its signal se-
quence, the cDNA fragment coding for hGH was ex-
cised with HinfI-HindIII from pRMhGH (20). This ex-
cised cDNA fragment was lacking the first 18 bp. The
18 bp were chemically synthesized with HinfI over-
hang at the 3⬘-end and NcoI overhang at the 5⬘-end
[5⬘-CATG TTC CCA ACT ATT CCA CTG-3⬘; 3⬘-AAG
GGT TGA TAA GGT GAC TCA-5⬘].
This synthetic oligonucleotide and HinfI-HindIII-di-
gested cDNA fragments were inserted into NcoI-Hin-
dIII-digested pQE-60 expression vector (Qiagen,
U.S.A.). The construct thus obtained has hGH with
just one extra methionine at the N-terminus under the
control of the phage T5 promoter. The NcoI site origi-
nally present in pQE-60 was lost during construction of
this plasmid. E. coli M15 cells containing the recombi-
nant expression plasmid (pQE 60-hGH) were grown in
LB or complex medium in the presence of kanamycin
(25 ␮g/ml) and ampicillin (50 ␮g/ml). The cultures were
induced with 1 mM IPTG and were further grown for
4 h. Expression of r-hGH in the total cell extracts from
both uninduced and induced cultures was checked by
SDS-PAGE.
Fermentation
For large-scale production of r-hGH, recombinant E.
coli cells were grown in a 3.5-L fermenter (2-L working
volume) in complex medium. The composition of the
complex medium was as described (22), except that the
initial glucose and yeast extract concentrations were
10 g/L. Fermentation was carried out at 37°C with
vigorous aeration and agitation and the pH of the me-
dium was maintained at 7 by use of 5 N NaOH. After
3 h of batch growth, the cells were grown in a fed-batch
mode with a continuous supply of glucose and yeast
extract. Details of the fed-batch fermentation strategy
are described elsewhere (23). The culture at OD 600 (op-
tical density) of 40 was induced with 1 mM IPTG,
cultivated for another 4 h, and then harvested. The
harvested cells were checked for expression and pro-
cessed for purification of inclusion bodies. Samples
were collected at regular intervals during the fermen-
tation to check cell growth, glucose consumption, and
r-hGH expression. Cell density was determined by
measuring the OD of the culture at 600 nm with a
Kontron (Kontron AG, Switzerland) UV-visible spec-
184 PATRA ET AL.
trophotometer. Higher OD samples were diluted suit-
ably to have an absorbance in the range of 0.2– 0.6. Dry
cell weight was determined by centrifuging the sample
broth at 4000g for 20 min and drying the washed cell to
constant weight at 105°C. One absorbance unit was
equivalent to 0.35 g L ⫺1 of dry cell weight for uninduced
culture and 0.4 g L ⫺1 dry cell weight for induced cul-
ture. Residual glucose in the fermentation broth was
measured by a glucose kit (Sigma), and the acetic acid
concentration was monitored by an acetic acid kit
(Boehringer Mannheim).
Isolation, Purification, and Estimation of r-hGH from
Inclusion Bodies
Induced E. coli cells sampled at different time points
during fed-batch fermentation were centrifuged at
4000g for 30 min and the cell pellet was dissolved in 50
mM Tris-HCl buffer (pH 8.0) containing 5 mM EDTA
and 1 mM PMSF. Cells were lysed by sonication and
centrifuged at 8000g for 30 min to isolate r-hGH inclu-
sion bodies. For large-scale isolation and purification of
r-hGH, induced E. coli cells were lysed by a French
press at 18000 psi and the inclusion bodies were recov-
ered by centrifugation at 8000g. The inclusion body
pellets thus obtained were washed with 50 mM Tris-
HCl buffer (pH 8.0) containing 5 mM EDTA and 2%
deoxycholate (17). Finally, the inclusion bodies were
washed with distilled water to remove contaminating
salt and detergent and centrifuged at 8000g for 30 min
and the pellet was used for estimation of r-hGH. At this
stage, inclusion bodies contained mostly r-hGH
(⬎90%), the majority in the form of monomer around
21 kDa in SDS-PAGE along with some high molecular
aggregates. The inclusion bodies were completely sol-
uble in 50 mM Tris-HCl buffer (pH 8.5) containing 1%
SDS and the solubilized r-hGH samples were diluted
appropriately and estimated using BCA protein assay.
SDS-PAGE was carried out using the method de-
scribed by Laemmli (24).
Solubilization of r-hGH from Inclusion Bodies
Buffers of different pH, denaturants, combination of
denaturant and salt, and ionic detergent as well as
oxidizing and reducing agents were used to solubilize
the r-hGH inclusion bodies. Purified r-hGH inclusion
bodies were solubilized in 100 mM Tris buffer at dif-
ferent pH (3–13) both in the presence and in the ab-
sence of urea. Other solubilizing buffers such as 2 M
Tris buffer at pH 12, 2 M Tris buffer at pH 12 contain-
ing 2 M Urea, 2 M Tris buffer at pH 12 with reduced:
oxidized glutathione (5:1 and 10:1 mM), 1% N-lauroyl
sarcosine in Tris buffer at pH 8.5, 1% N-cetyltrimeth-
ylammonium bromide in 50 mM Tris at pH 8.5, 8 M
urea, 6 M guanidine hydrochloride, 1% SDS in 50 mM
Tris at pH 8.5, and 100 mM Tris buffer at pH 12.5 with
2 M urea along with reduced:oxidized glutathione
(10:1) were used to soubilize r-hGH from the inclusion
bodies. One hundred microliters of purified inclusion
bodies (8 mg/ml) in 50 mM Tris buffer pH 8.5 was
taken in different microcentrifuge tubes and centri-
fuged. The supernatant was discarded and 1 ml of each
of the above solubilizing buffers was added to the pel-
lets. The suspension was vortexed and left for 30 min
at room temperature and the turbidity of the solution
was measured at 450 nm. The samples were again
centrifuged at 12000 rpm for 10 min. The supernatant
after filtering through a 0.45-␮m Millipore filter was
measured at 280 nm for an estimation of protein con-
tent. The same protocol was followed to measure the
effect of pH and urea on inclusion body solubilization.
To dissociate the r-hGH oligomers present in inclu-
sion bodies into monomers, different concentrations of
␤-mercaptoethanol (2 to 20 mM) in 100 mM Tris buffer
at pH 12.5 containing 2 M urea were used. Solubiliza-
tion of inclusion bodies in 8 M urea with different
concentrations of ␤-mercaptoethanol (100 –200 mM)
was also tried to dissociate the oligomers into the
monomeric form during solubilization. The extent of
solubilization in these buffers was determined as de-
scribed above.
Purification of r-hGH
For large-scale purification of r-hGH, pure inclusion
bodies were isolated (⬃104 mg protein isolated from 65
ml of high cell density fermentation broth) and solubi-
lized in 16 ml of 100 mM Tris buffer, pH 12.5, contain-
ing 2 M urea. The solubilized r-hGH was diluted five
times with Milli-Q water and the pH was brought down
to 8.5 by adding 1 N HCl. A DEAE-Sepharose ion-
exchange column (5 ⫻ 5 cm) equilibrated with 20 mM
Tris buffer containing 5 mM EDTA, 0.4 M urea, and
0.02% sodium azide, pH 8.5, was used for the purifica-
tion of r-hGH. The solution was loaded to an ion-ex-
change column at a flow rate of 0.5 ml/min. The column
was washed with 5 column volumes of equilibration
buffer followed by 3 column volumes of equilibration
buffer containing 0.1 M NaCl. Recombinant hGH was
eluted with a gradient of 0.1– 0.25 M NaCl in the equil-
ibration buffer at a flow rate of 1.5 ml/min. The absor-
bance was measured on line with a Pharmacia UV
monitor. Peak fractions showing r-hGH were pooled,
checked in SDS-PAGE, and lyophilized. The lyophi-
lized r-hGH solubilized in 5 ml of 20 mM Tris buffer
containing 5 mM EDTA, 0.02% (w/v) sodium azide, and
1 mM PMSF was loaded onto a Sephacryl S-200 col-
umn (bed volume ⫽ 90 ⫻ 1.6 cm, flow rate of 20 ml/h)
for further purification. The fractions which showed a
single r-hGH protein band on SDS-PAGE were pooled
together, dialyzed against 20 mM Tris buffer at pH 8.5,
and lyophilized. Lyophilized r-hGH was used for phys-
icochemical and bioactivity assays and were stored at
⫺20°C for further use. HPLC analysis of the pure
 RECOMBINANT HUMAN GROWTH HORMONE 185

refolded r-hGH was carried out through a gel-filtration

Shodex (Protein KW-804, Waters) column. The flow
rate of the solvent was 1 ml/min. The absorbance was
monitored at 280 nm, using a Waters HPLC-UV Model
490 detector.

Spectroscopic Analysis
A circular dichroism spectrum was obtained at 25°C
in the wavelength range of 190 –250 nm using a
JASCO-Spectropolarimeter in 20 mM Tris buffer. The
sample was scanned 10 times for data accumulation
and the average spectrum was plotted. Similarly, the
UV spectrum of r-hGH was scanned within the wave-
length range 240 to 350 nm (Contron UV-Vis spectro-
photometer). Fluorescence emission spectrum was
taken by exciting the protein molecules at 280 nm and
measuring the emission in the wavelength region from
320 to 350 nm using a Schimadzu spectrofluoropho-
tometer (Model 1501).

Estimation of Extinction Coefficient

The purified and lyophilized r-hGH was used for
measurement of extinction coefficient. Prior to the
measurement, r-hGH was dialyzed extensively against
Milli-Q water. Absorbance of water-dialyzed r-hGH
was recorded and 1 ml of the protein was dried at
105°C. The dried fraction was weighed and the molar
extinction coefficient of r-hGH was derived from the
formula
FIG. 1. Fed-batch fermentation kinetics of E. coli for the produc-
tion of r-hGH. Cells were grown in batch mode until the initial
glucose was consumed. After 3 h, glucose feeding was started at a
predetermined rate for fed-batch growth of cells. At OD 600 of 40, the
cells were induced with 1 mM IPTG. Samples were taken at intervals
to monitor glucose (E), cell OD at 600 nm (‚), acetic acid (䊐), and
r-hGH concentration (F).

carried out in triplicate under atmospheric conditions

A ⫽ ⑀ bC,
with 5% CO 2 at 37°C.

where A is the absorbance at 280 nm, b is the path RESULTS

length of incident light in cm, C is the protein concen-
tration in moles per liter, and ⑀ is the molar extinction
coefficient.
Bioactivity Assay
The biological activity of r-hGH was determined by
its growth-promoting action on rat Nb2 lymphoma cell
lines. Commercially available recombinant human
growth hormone form Boehringer Mannheim was used
as standard. The Nb2 cell lines were maintained in
RPMI medium supplemented with 10% FBS and 10%
HS. Quiescent Nb2 cells arrested at the G 0/G 1 phases
were prepared by incubating cells in RPMI supple-
mented with 1% FBS and 10% HS. To initiate cellular
proliferation, different concentrations (1–25 ng/ml)
each of BSA, commercial hGH, or r-hGH were added to
the culture medium. The assay was set in a 96-well flat
bottom culture plates using RPMI as control. Growth-
promoting activity was evaluated by counting the num-
ber of cells every 24 h for 5 days. Experiments were
Fed-Batch Fermentation and Isolation of r-hGH
Inclusion Bodies
E. coli cells expressing r-hGH were grown in a fed-
batch fermentation process to produce large quantities
of r-hGH. The cultures at cell OD of 40 (20 g/L dry cell
weight) were induced with 1 mM IPTG (optimum IPTG
for induction was 0.01 mM/L/ OD culture) and grown
for another 4 h, and the batch was terminated at a cell
OD of 60 (Fig. 1). A maximum of 1.6 g/L of r-hGH was
expressed as inclusion bodies in 10 h of fed-batch fer-
mentation. The expression of r-hGH plateaued after
3 h of IPTG induction (Fig. 2) and the level of r-hGH
expression was around 13% of the total cellular pro-
tein. The specific cellular r-hGH yield was 26.6 mg/L/
OD. The residual glucose concentration was main-
tained around 0.5 to 1 g/L throughout the fed-batch
operation and the acetic acid accumulation was also
very low (⬍2.5 g/L). Inclusion bodies of r-hGH from E.
coli cells were isolated and purified as described earlier
for ovine growth hormone (17). Extensive washing with
186 PATRA ET AL.

carried out to enhance the solubility of r-hGH from the

inclusion bodies. Higher solubilization of r-hGH from
inclusion bodies was observed by incorporating 2 M
urea in 100 mM Tris buffer at pH 12.5 (Fig. 3B). Fur-
ther addition of urea in 100 mM Tris buffer at pH 12.5
did not help in solubilizing higher amounts of r-hGH

FIG. 2. SDS-PAGE analysis of r-hGH expression during fed-batch

fermentation. Lanes 1 and 2, uninduced cells. Lanes 3– 6, induced
cells after 0.5, 1, 2, and 3 h of IPTG induction, respectively. Lane 7,
MW markers in kDa. Lane 8, pure r-hGH inclusion bodies isolated
from cells after extensive washing.

detergents resulted in pure inclusion bodies containing

⬎90% monomeric r-hGH (Fig. 2). Washing of the inclu-
sion body preparation in deoxycholate-containing
buffer helped in removing the majority of the contam-
inants. Dimers as well as multimers of r-hGH were
also present in pure inclusion body preparations. For-
mation of dimers was also observed at the early stages
of recombinant protein synthesis. Most of the purified
inclusion body proteins visualized in SDS-PAGE re-
acted with polyclonal hGH antibody (data not shown),
indicating the purity of the preparation. As this low
level of contaminants has little effect on the refolding
of proteins, the purified inclusion bodies containing
mostly r-hGH were used for subsequent solubilization
and refolding.

Solubilization of r-hGH from Inclusion Bodies

Solubilization of inclusion bodies at different pH. It
has been widely reported that growth hormone inclu-
sion bodies of different species expressed in E. coli can
be solubilized by alkaline pH (17,21,25). Thus, the pu-
rified r-hGH inclusion bodies were solubilized at differ-
ent pH in 100 mM Tris buffer (pH 3–13) and percent-
age solubilization of r-hGH was monitored (Fig. 3A).
Solubilization of r-hGH from inclusion bodies was ob-
served by increasing the pH from 8 to 12.5. High alka-
line pH (⬎12.5), even though it helped in solubilizing
r-hGH from inclusion bodies, resulted in extensive deg-
FIG. 3. (A) Effect of pH on the solubility of r-hGH inclusion bodies.
radation of r-hGH (SDS-PAGE data not shown). A A constant amount of r-hGH inclusion bodies was solubilized at
maximum of 2 mg/ml of r-hGH was solubilized in 100 different pH in 100 mM Tris buffer and the turbidity was measured
mM Tris buffer at pH 12.5 without the addition of urea at 450 nm. The sample solution after centrifugation and filtration
or guanidine hydrochloride. was used for estimation of protein by taking the absorbance at 280
nm. (B) Effect of urea concentration on the solubility of r-hGH
Effect of urea and ␤-mercaptoethanol. Solubiliza- inclusion bodies. Different concentrations of urea were added to 100
tion experiments in 100 mM Tris buffer at pH 12.5 mM Tris buffer, pH 12.5, and solubilization behavior was monitored
containing different molar concentrations of urea were by estimating the amount of protein at 280 nm.
 RECOMBINANT HUMAN GROWTH HORMONE 187

from the inclusion bodies. In 100 mM Tris buffer at pH

12.5 containing 2 M urea, a maximum of 6 mg/ml of
r-hGH was solubilized from the inclusion bodies. Sol-
ubility of r-hGH was comparable to that of 8 M urea in
Tris buffer at pH 8. Solubilized r-hGH in 100 mM Tris
buffer at pH 12.5 containing 2 M urea was analyzed in
CD and fluorescence spectra and were found to have
native-like secondary structures (data not shown).
Isolation of r-hGH and their subsequent solubiliza-
tion at alkaline pH were always associated with the
presence of dimer (44 kDa, in SDS-PAGE), which con-
stituted around 5– 8% of the total inclusion body pro-
tein. These r-hGH dimers were also observed in the
SDS-PAGE despite the high reducing and denaturing
environment. Addition of increasing amounts of ␤-mer-
captoethanol (2–20 mM ) in 100 mM Tris buffer at pH
12.5 containing 2 M urea had very little effect on dis-
sociating oligomers into monomeric r-hGH. Dissocia-
tion of oligomers and dimers to monomeric r-hGH was
also not observed in 8 M urea solution containing 100
to 200 mM ␤-mercaptoethanol (SDS-PAGE data not
shown).
Effect of different solvents. The solubilizing effect of FIG. 4. Effect of different solvents on the solubility of r-hGH inclu-
sion bodies. A fixed amount of r-hGH inclusion bodies was used and
100 mM Tris, pH 12.5, buffer containing 2 M urea on the turbidity and solubility were measured at 450 and 280 nm,
r-hGH inclusion bodies was compared with different respectively. (A) 2 M Tris buffer at pH 12. (B) 2 M Tris buffer at pH
solubilizing buffers as described under Materials and 12 with 2 M urea. (C) 2 M Tris buffer at pH 12 with 2 M urea and R:O
Methods. It was observed that the solubility achieved glutathione (5:1). (D) 2 M Tris buffer at pH 12 with 2 M urea and R:O
glutathione (10:1). (E) 1% SDS in 50 mM Tris buffer at pH 8.5. (F) 8
in 8 M urea, 6 M Gdn-HCl, 1% SDS, and 1% CTAB in M urea at pH 8.5. (G) 100 mM Tris buffer at pH 12.5. (H) 100 mM
50 mM Tris buffer at pH 8.5 was comparable to that of Tris buffer at pH 12.5 with 2 M urea. (I) 100 mM Tris buffer at pH
100 mM Tris buffer containing 2 M urea at pH 12.5 12.5 with 2 M urea and R:O glutathione (10:1). (J) 50 mM Tris buffer
(Fig. 4). Other buffers used for solubilization were not at pH 8.5. (K) 1% CTAB in Tris buffer at pH 8.5. (L) 1% NLS in Tris
as effective as 100 mM Tris buffer at pH 12.5 contain- buffer at pH 8.5.
ing 2 M urea. The solubility of the r-hGH inclusion
bodies in 1% NLS in 50 mM Tris at pH 8.5 was also r-hGH were passed through a DEAE-Sepharose col-
very low. The solubility of r-hGH from inclusion bodies umn for purification. Recombinant human growth hor-
in 2 M Tris containing 2 M urea was lower than 100 mone which eluted between the conductivity range of
mM Tris buffer containing 2 M urea at pH 12.5. The 14 to 16 mS/cm (Fractions 20 to 28) was found to be
presence of reduced/oxidized glutathione helped to in- homogeneous and was 40% of the total protein. How-
crease the solubility of r-hGH from inclusion bodies in ever, some amount of r-hGH was coeluted along with
2 M Tris buffer containing 2 M urea at pH 12 but had r-hGH dimer between conductivities of 22 to 25 mS/cm
little effect when used in 100 mM Tris buffer at pH 12.5 (Fractions 35 to 45) which constituted about 25–30% of
with 2 M urea. In 100 mM Tris buffer at pH 12.5 the total protein. The overall recovery of r-hGH from
containing 2 M urea, 6.5 mg/ml of r-hGH was solubi- ion-exchange matrix was around 65%. Pure r-hGH con-
lized from the inclusion bodies. As observed earlier, taining dimers/oligomers was passed through the size-
none of these buffers could dissociate r-hGH dimers/ exclusion chromatography column for further purifica-
oligomers present in the inclusion body preparation tion. The dimeric or higher forms of the proteins were
into monomers. removed through gel filtration (Fig. 5A). The overall
yield of the purified refolded r-hGH from the inclusion
Purification of r-hGH bodies of E. coli was ⬃50% (Table 1). HPLC analysis of
the purified r-hGH showed a single peak at 11.38 min
Pure r-hGH inclusion bodies solubilized in 100 mM
(Fig. 5B) and SDS-PAGE showed a single band (Fig.
Tris buffer at pH 12.5 containing 2 M urea were fur-
5C), thus indicating the high purity of the r-hGH.
ther purified using ion-exchange and gel-filtration
chromatography. The solubilized r-hGH was diluted
five times and the pH of the buffer was adjusted to 8.5. Characterization and Bioactivity
No aggregation of the solubilized r-hGH was observed Authenticity of the purified r-hGH was further con-
during dilution and buffer exchange. Solubilized firmed from the N-terminal analysis of r-hGH and from
188 PATRA ET AL.

FIG. 5. (A) Final purification of r-hGH through gel-filtration chromatography. Lyophilized powder of r-hGH was solubilized in 5 ml of buffer
and loaded onto a gel-filtration column. Fractions of 3.5 ml were collected and the peaks were pooled separately. The majority of the r-hGH
eluted as a monomeric form between Fractions 30 and 40. (B) Monomeric pure r-hGH analyzed on a Shodex Protein KW-804 HPLC column
at 280 nm showing a single peak. (C) SDS-PAGE analysis of purified r-hGH eluted from S-200 gel-filtration column. Lane 1, pure r-hGH.
Lane 2, molecular weight markers.

spectroscopic analysis. UV spectrum of the purified
r-hGH showed an absorbance maxima at 276.8 nm,
and a shoulder at 283 nm, which was comparable to
that of native human growth hormone reported by
Bewley and Li, in 1984 (29) (Fig. 6A). The fluorescence
spectrum of refolded r-hGH was found to be identical to
the native hGH which gave a peak at 340 nm (Fig.
6B).The molar extinction coefficient of pure r-hGH was
found to be 18,800 M ⫺1 cm ⫺1 which is very close to the
reported value of 18,890 M ⫺1 cm ⫺1 for native hGH (26).
Growth kinetics of the prolactin-dependent Nb2 lym-
phoma cell line was monitored to evaluate the bioac-
 RECOMBINANT HUMAN GROWTH HORMONE
Purification of r-hGH from High Cell Density Culture a
Total protein
Steps
Cell lysate
Pure inclusion body
Solubilization
Ion-exchange chromatography
Gel-filtration chromatography
a
Sixty-five milliliters of high density culture at OD 600 nm ⫽ 60 contained about 1.6 g dry cell weight. Step and overall yields were calculated
starting from the pure inclusion body preparation.
b
Purity is defined as the percentage of monomeric r-hGH in the purified protein preparation.
tivity of purified r-hGH. The addition of prolactin, com-
mercial hGH, and r-hGH promoted growth of Nb2 cells
arrested at G 0/G 1 phases by serum deprivation.
Growth of Nb2 cells in the presence of different con-
centrations of r-hGH was found to be comparable to
that observed for the commercial hGH (Fig. 7). No
growth stimulation was observed in the presence of
BSA which was used as a negative control.
DISCUSSION
Fed-batch fermentation of E. coli resulted in high
volumetric yield of r-hGH. In 10 h of fed-batch fermen-
tation, 1.6 g/L of r-hGH was produced in comparison to
30 – 40 mg/L expressed in shaker flask culture. The
r-hGH accumulated as inclusion bodies in E. coli and
was isolated to more than 90% purity by extensive
washing in 2% deoxycholate in 50 mM Tris buffer, pH
8.5. However, among the last 5–10%, the dimers and
oligomers of r-hGH were the major contaminants. The
purity and homogeneity of the r-hGH inclusion body
preparation were in agreement with the proposed com-
position of the inclusion bodies that they are formed
due to specific aggregation of single protein intermedi-
ates (4,7). As the inclusion bodies consisted of mostly
r-hGH, solubilization and refolding were carried out
before further purification.
Inclusion body proteins have been reported to have
extensive native-like secondary structure (5,27) and
thus when solubilized while retaining the native-like
secondary structure results in high recovery of the
bioactive protein (17). In order to protect the native-
like secondary structure, r-hGH inclusion bodies were
solubilized at alkaline pH, containing mild concentra-
tions of chaotropic agents (2 M urea). Charge distribu-
tion provided by high alkaline pH along the protein
chain was responsible for higher solubilization of
r-hGH from inclusion bodies. This suggested that pH
has a crucial role in destabilizing the inclusion body
aggregation. Changing the charge distribution along
the protein molecule by changing the pH generally
affects the protein stability and may lead to an unfold-
189
TABLE 1
Step yield Overall yield Purity b
(mg) (%) (%) (%)
800 — — 13
104 100 100 90
98 94 94 92
67 71 64 95
52 77 50 99
ing of the native protein (28). However, for human
growth hormone, there did not seem to be any indica-
tions of general unfolding of the secondary structure
(29,30) and the pH-induced unfolding has been re-
ported to be reversible for a similar protein [bovine
growth hormone (31)]. Use of 2 M urea at alkaline pH
improved r-hGH solubilization from inclusion bodies
without disturbing the existing native-like secondary
structure of the proteins. Although diminishing hydro-
phobic interaction between water and protein molecule
is a linear function of urea concentration (32), this
effect also existed substantially at as low as 2 M urea
concentration. Since proteins could not be denatured at
such a low concentration of urea, urea was probably
only serving the purpose of physical separation of the
molecules by disrupting the hydrophobic interactions.
Thus in conclusion, r-hGH was solubilized from the
inclusion bodies without its existing native-like sec-
ondary structure being disturbed. The extent of solu-
bilization in 100 mM Tris buffer, pH 12.5, containing 2
M urea was found to be comparable with that of 8 M
urea and a maximum amount of 6.5 mg/ml of r-hGH
could be solubilized from the inclusion bodies. In-
creased solubility of r-hGH in the above buffer could be
an effect of both urea and pH, indicating the existence
both ionic and hydrophobic interactions in the inclu-
sion bodies.
Expression of r-hGH in E. coli as inclusion bodies
was always associated with the formation of dimers
and oligomers which were also observed at the early
stage of protein synthesis. Bacterial cytosol being very
reducing in nature does not allow formation of disulfide
bonds, so the aggregates could not be due to intermo-
lecular disulfide bonds. Possibilities of mixed disulfide
bond formation leading to high molecular weight ag-
gregates may occur during cell lysis. However as the
r-hGH oligomers could not be dissociated in to mono-
mers in the presence of strong reducing agent such as
␤-mercaptoethanol or reduced:oxidized glutathione, it
can be concluded that non-disulfide covalent bonding is
responsible for oligomer formation in r-hGH inclusion
190 PATRA ET AL.
FIG. 6. Physicochemical analysis of pure r-hGH. (A) UV spectrum
of purified r-hGH (—) solubilized in 20 mM Tris buffer, pH 8.5
(scanned between 240 and 350 nm) and compared with the native
hGH. (– – –). (B) Fluorescence emission spectra of purified r-hGH
(—) and native hGH (– – –) solubilized in 20 mM Tris buffer, pH 8.5.
Emission intensity, measured at 280 nm excitation, showed the
maximum at 340 nm for hGH.
bodies. The existence of such non-disulfide covalent
bonds in r-bGH oligomers has been reported through
Raman spectroscopic studies by Thamann (33).
Even though Tris buffer at pH 12.5 with 2 M urea
solubilized the r-hGH from the inclusion bodies, buff-
ers containing 1% SDS, 1% CTAB, 8 M urea, and 6 M
guanidine hydrochloride also solubilized r-hGH to a
similar extent. Maximum solubility observed in Tris
buffer containing ionic detergent 1% SDS could be due
the ionic property of SDS which helps in the ionization
of the proteins, thus helping in its solubilization. Sol-
ubilization of inclusion bodies with the help of CTAB
restores the secondary structure of the expressed pro-
tein (34); however, its use was avoided as it requires
extensive washing and many chromatographic steps
for its removal from the protein mixture. Similarly,
SDS being a detergent binds to the protein tightly and
needs extensive washing for its complete removal from
the protein. High concentrations of urea and Gdn-HCl,
being strong denaturants, result in the loss of existing
native-like secondary structures of the inclusion body
proteins. This results in random coil structure forma-
tion of the solubilized proteins and makes them more
FIG. 7. Receptor-mediated growth-promoting activity of the puri-
fied r-hGH on Nb2 cell lines. The initial cell concentration was 0.5 ⫻
10 5 cells/ml. Cell growth was arrested by serum deprivation and
different concentrations of r-hGH (F) or commercial hGH (Œ) or BSA
(■) in RPMI medium was used for activation of cell growth. Cell
growth was monitored by counting cell numbers on different days.
Cell concentrations achieved at 96 h after activation are presented to
compare the bioactivity of r-hGH with that of commercial hGH
(c-hGH).
 RECOMBINANT HUMAN GROWTH HORMONE
prone to intermolecular aggregation during refolding.
Hence these buffers, despite their high solubilization
power, lower the overall recovery of the native folded
proteins and thus were avoided for the solubilization of
r-hGH.
Enhanced solubilization of the r-hGH from the inclu-
sion bodies in all these above-mentioned buffers indi-
cated that hydrophobic interaction is probably the
most dominant force for aggregation of proteins during
high-level expression. However, the presence of ionic
and/or covalent interactions in the formation and sta-
bilization of inclusion bodies cannot be ruled out. The
r-hGH inclusion bodies were devoid of disulfide bond
aggregates. This was supported by the ineffectiveness
of ␤-mercaptoethanol and reduced/oxidized glutathi-
one in dissociating the r-hGH multimers to the mono-
meric form.
As the extent of aggregation of partially denatured
solubilized r-hGH was low during buffer exchange, pu-
rification and refolding of the r-hGH were carried out
at high initial protein concentrations. Solubilized
r-hGH was purified and refolded using ion-exchange
and gel-filtration chromatography. Solubilization of
the r-hGH from inclusion bodies while retaining the
native-like secondary structures helped in lowering the
extent of protein aggregation during buffer exchange
and dilution. Despite the presence of two disulfide
bonds, extensive protein aggregation during refolding
due to incorrect disulfide bond formation was not ob-
served for r-hGH. This may be due to exposure to
sufficient air oxidation during the process of purifica-
tion, indicating that air oxidation has a profound effect
on the rearrangement of the disulfide bonds while pro-
tein was folding from a metastable state having exten-
sive secondary structures to the folded native state.
The overall yield of the r-hGH from the inclusion bod-
ies was ⬃50% in comparison to 20 to 25% achieved in
solubilizing the inclusion bodies in high concentrations
of chaotropic reagents. Recombinant human growth
hormone showed efficient growth-promoting activity in
Nb2 cell proliferation assays, indicating that the puri-
fied and refolded r-hGH has a biologically active con-
formation. Such solubilization and purification proto-
cols described in this communication could be applied
for maximizing the recovery of therapeutically impor-
tant proteins expressed as inclusion bodies in E.coli.
ACKNOWLEDGMENTS
This work is supported by the core grant from National Institute of
Immunology (NII), New Delhi, received from the Department of
Biotechnology, Government of India. The authors are grateful to Dr.
S. K. Basu, Director, NII, for his support and encouragement.
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