Animal Biotechnology, 16: 117–126, 2005

Copyright # Taylor & Francis Inc.

ISSN: 1049-5398 print=1532-2378 online
DOI: 10.1080/10495390500263229

MOLECULAR CLONING OF GIANT PANDA PITUITARY

PROLACTIN CDNA AND ITS EXPRESSION IN
Escherichia coli

Zhang Zhi-He, Zheng Xu, Hu Xi-lian, and Zhu Mu-Yuan

Institute of Genetics, College of Life Sciences, Zhejiang University,
Hangzhou, China

Hou Rong, Shen Fu-Jun, and Zhang Liang

Key Laboratory for Reproduction and Conservation Genetics of Endangered
Wildlife of Sichuan Province, Chengdu Research Base of Giant Panda Breeding,
Chengdu, China

Liao Ming-Juan
Department of Biology, Hangzhou Teachers College, Hangzhou, China

Lv Xiao-Ping
CITES China Office

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed
in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229
amino acids including a putative signal peptide of 30 amino acids and a mature protein of
199 residues with one potential N-glycosylation site. Sequence comparison indicated that
panda PRL shares a high degree of identity to other known PRL sequences ranging from
98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved
residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed
through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved
in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein
was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 trans-
formed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature
panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could
be recognized by antibody against human PRL. Our results would contribute to further eluci-
dating the structural and functional characteristics of pituitary PRL and provide a basis for
the production of recombinant panda prolactin for future use in the breeding of giant panda.

Keywords: Giant panda (Aliuropoda melanoleuca); Cloning; Fusion expression; Pituitary PRL

INTRODUCTION
The giant panda (Aliuropoda melanoleuca) is one of the most endangered ani-
mals in the world and indigenous to China. Currently ex situ conservation by captive

Address correspondence to Zhu Mu-Yuan, Institute of Genetics, College of Life Sciences, Zhejiang
University, Hangzhou, 310012, China. E-mail: myzhu@zju.eu.cn

117
118 ZHI-HE ET AL.

breeding has become a necessary and important strategy for the giant panda popu-
lation. However, some reproductive problems such as low reproductive rates (1,2)
and high infant mortality (3) adversely affect ex situ breeding programs. Poor quality
of male semen (4) and ovulation failure probably resulting from insufficient release
of pituitary reproductive hormone in female (5,6) was believed to be associated with
the reproductive disorders in the breeding of giant panda. However, little is known
concerning the mechanism underlying these reproductive disorders. Therefore, there
is a need for an increase in the understanding of reproductive biology, especially the
structural, functional, and regulatory characteristics of reproductive hormones, to
improve reproductive in the ex situ giant panda population.
Prolactin (PRL) is a polypeptide hormone of approximately 23 kD, which is
primarily synthesized and secreted by the anterior pituitary gland. It was confirmed
that PRL is a multifunctional hormone with over 3000 separate biological activities
(7). Apart from its major action on milk production during pregnancy and lactation,
it also plays a significant role in reproduction, including regulation of luteal function,
mammary development, female receptivity, and parental behavior (8). Based on its
structural and functional properties, the PRL gene belongs to a gene family that also
includes homologous genes of growth hormone (GH) and placental lactogen (PL), all
evolving from a common ancestor that occurred by gene duplication about 400 mil-
lion years ago (9). To date, the genes, or cDNA, encoding PRL have been identified in
a wide variety of species including many mammalian, avian, amphibian, and teleost
species. A varying degree of homology almost reflecting the phylogenetic relationship
was demonstrated among the PRL sequences from different species (10). Despite
many studies that examine reproductive hormones of the giant panda at different
physiological stages (e.g., 11) and related gene cloning (2,3), to date no study has
examined its pituitary PRL at a molecular level. The aim of this study was isolate
the cDNA that encodes panda pituitary PRL and express it in E. coli. Our results
would contribute to elucidating the structural, functional, and regulatory character-
istics of panda PRL that is necessary for a successful artificial breeding of giant
panda. They would also provide a basis for producing recombinant PRL hormone,
which would be used in combination with other reproductive hormones to improve
assisted reproduction technology for the ex situ giant panda population.

MATERIALS AND METHODS

Extraction of RNA
Following the natural death of a female giant panda at Chengdu Zoo (June, 20,
2000), total RNA was extracted from fresh pituitary tissue ground in liquid nitrogen
using TRIzol reagent (Invitrogen) according to the manufacturers protocol. Total
RNA was dissolved in 0.1% DEPC-treated water and stored at
70C until use.

RT-PCR
Two PCR primers (sense primer: 50 -AAG CAT CAC CAC CAT GGA C-30 ;
anti-sense primer: 50 -TGG GCT TAG CAG TTG CTG T-30 ) were designed by
comparing PRL sequences from reported mammals including cat (GenBank
 MOLECULAR CLONING OF GIANT PANDA CDNA 119

accession no. U25974), cow (V00112), sheep (M27057), pig (X14068), human
(NM_000948), rat (NM_012629), and mouse (NM_011164).
The first chain of cDNA was synthesized using a reverse transcription system
(Promega). Briefly, reverse transcription was performed for 1 h at 42C in a 20 ml
reaction volume containing about 1 mg total RNA, 0.5 mg Oligo(dT)15 primer mix-
ture, 5 mM MgCl2, 1 mM each dNTP, 10 U=ml recombinant RNasin ribonuclease
inhibitor, and 15 U AMV reverse transcriptase. Target DNA fragment was amplified
by 30 cycles of PCR using 2 ml synthesized cDNA as a template in a 25 ml reaction
containing 1.5 mM MgCl2, 300 pM each primer, and 5 U Pfu DNA polymerase
(Sangon). Each PCR cycle consisted of 40 sec at 94C, 40 sec at 45C, and 50 sec at
72C. All PCR reactions were undertaken on a GeneAmp PCR System 9600.
Purified PCR products were ligated with SmaI-digested pUC18 (TaKaRa) at a
molar ratio of 3:1 at 22C overnight and then transformed into JM109 (SABC) com-
petent cells. Recombinant clones were selected using blue=white screening on
X-gal=IPTG=ampicillin LB plates. Following the identification by enzymatic diges-
tion, three positive clones were picked up for sequencing in both directions.

Sequence Analysis
Nucleotide sequence data were analyzed by DNASIS computer software. The
deduced amino acid sequences were aligned with available PRL of other species using
GENDOC (version 3.2) software. Based on multiple sequence alignments of mature
PRL amino from the giant panda and other mammalian species with CLUSTAL X
software, a gene phylogenetic tree was built with Mega 2.1 software according to
N-J method. Species used in comparison and accession numbers were as follows: mink
(AY249860), cat (U25974), pig (X14068), possum (AF054634), sheep (M27057),
cow (V00112), rat (NM_012629), mouse (BC061141), human (NM_000948), chicken
(J04614), duck (AB158610), frog (L07620), tilapia (M27010, M27011), catfish
(AF372653), salmon (D00249).

Generation of Expression Vector

DNA encoding mature panda PRL protein was amplified with the following
primers designed according to the PRL sequence we isolated. Forward primer
(50 -GGA ATT CCT GCC CAT CTG TCC CAC C 30 ) containing EcoR I site and
reverse primer (50 -CCT CGA GTT AGC AGT TGC TGT CGT AG-30 ) containing
Xho I site. The amplified fragment was digested by EcoR I and Xho I and subcloned
into the equivalent site of pGEX-4T-1 (Amersham) expression vector. The generated
expressed plasmid was designated as pGEX-PRL and transformed into competent
cells of E. coli BL21 (Amersham) for fusion expression.

Fusion Expression of Panda PRL

A single colony of BL21 containing pGEX-PRL was grown overnight at 37C
in LB medium containing 100 mg=ml ampicillin. Cultures were then diluted (1:20)
in fresh 50 ml LB medium and grown at 37C for approximately 2 h until the
OD reached approximately 0.6. Expression was initiated by the addition of
120 ZHI-HE ET AL.

isopropyl-b-D-thiogalactoside (IPTG) (Sigma) to a final concentration of 0.6 mM.

After 2 h induction, 1 ml culture was collected and centrifuged (12,000 g, 1 min).
The pellet was dissolved in 100 ml 1  SDS sample buffer (50 mM Tris–HCl, pH
6.8, 2% SDS, 0.1 M DTT, 10% glycerol, 0.1% bromophenol blue), heated at
100C for 3 min, and resolved on 12% SDS polyacrylamide gel electrophoresis
(SDS-PAGE). pGEX-PRL without IPTG induction and BL21 served as controls.
Gel was analyzed by Commassie blue staining or transferred to PVDF membrane
for Western blot analysis. For Western blot, the membrane was incubated with mouse
anti-GST antibody and polyclonal goat anti-hPRL antibody (Santa Cruz) at 1:1000
dilution. The secondary antibody was horseradish peroxidase-conjugated anti-mouse
or goat IgG (Santa Cruz) at 1:1000 dilution. Immunosignal was detected on X-ray
film (Pierce) using a Chemiluminescence detection system (Pierce).
In order to examine the solubility of expressed fusion protein, another 1 ml of
the culture by 2 h was harvested by centrifugation (4000 g, 20 min). A 100 ml cell lysis
buffer containing 50 mM Tris (pH 8.0), 0.2 mM PMSF, 1% Triton X-100, and
250 mg=ml bacteriolysin was added, followed by incubation for 30 min at 37C. Fol-
lowing freezing and melting several times, 10 mg=ml DNAase I was added to the sol-
ution to digest DNA for 30 min at 37C. After centrifugation for 15 min at 12,000 g,
the final pellet was dissolved in 1  SDS sample buffer and the supernatant was
mixed with 6  SDS sample buffer. Both the soluble part from supernatant and
insoluble part from pellet were subjected to SDS-PAGE analysis.

RESULTS
Amplification and Sequence Analysis
The PCR product of approximately 690 bp in length was amplified from the pitu-
itary total RNA of giant panda. To ensure that no wrong base might be introduced dur-
ing PCR cycles, three independent PCR amplifications were performed. Clones from
independent RT-PCR were sequenced with completely identical nucleotide sequence.
A BLAST search for this sequence in NCBI database indicated the most resemblance
to PRL sequences from other reported species, suggesting the obtained sequence repre-
sented pituitary PRL cDNA from giant panda. The new sequence has been submitted
to GenBank accession number AY161285. As depicted in Figure 1, pituitary PRL
cDNA of panda contains a 687-bp ORF encoding a PRL prehormone of 229 amino
acids, which shares the identical frame structure with those of cat, mink, cow, and
pig, all consisting of a signal peptide of 30 residues and a mature protein of 199 amino
acids. Three putative disulfide bonds were formed between residues 4–11, 58–174, and
191–199 according to their equivalent positions of human PRL (12). A potential
N-linked glycosylation site, as identified in the cat, mink, pig, sheep, and human
PRL, was also retained at position 31 of the panda PRL. Similar to the PRL from other
species, the predicted molecular weight of mature PRL from giant panda is 23 kD.

Sequence Comparison and Alignment

The putative amino acid sequence of mature panda PRL was compared with
those from 10 mammalian, two avian, one amphibian, and four teleostean PRL.
 MOLECULAR CLONING OF GIANT PANDA CDNA 121

Figure 1 cDNA sequence and putative amino acid sequence of PRL from Ailuropoda melanoleuca. The
signal peptides are double lined. The cysteine residues are cycled. The putative N-glycosylation sites are
boxed by open rectangles and the asterisk represents the stop codon.

PRL is well conserved among mammals, birds, and xenopus. Panda PRL displays
the highest identity (98%) to mink PRL with only a two-residue difference, and
95%, 95%, and 92% identities to pig, cat, and possum PRL, respectively, and mod-
erate level of sequence identity to cow (78%), sheep (78%), human (79%), chicken
(78%), duck (79%), and frog (69%) PRL. Less similarity was found between panda
PRL and rodent PRL, with 59% and 62% homology, respectively. The homology
between teleost PRL and mammalian PRL turns out to be relatively low; therefore,
panda PRL shares about 30% identity with those of salmon (31%), tilapia (29%),
and catfish (29%), suggesting a high degree of divergence among PRLs from dis-
tantly related species. Sequence identity between panda PRL and GH is rather
low at the nucleotide level (45%) and amino acid level (23%). As indicated in the
alignment of Figure 2, 29 completely conserved amino acid residues distributed in
four distinct domains of PRL (PD1–PD4) were found among giant panda PRL
and those of the other 15 species.
Following the multiple sequence alignment of PRL mature amino acids from
giant panda and several mammalian species, a phylogenetic tree of PRL (Fig. 3)
was constructed using neighbor-joining methods (NJ). The constructed phylogenetic
tree indicates the closest relationship between giant panda PRL, mink, and cat PRL
consistent with the order of the phylogenetic relationship as members of Carnivora.
122 ZHI-HE ET AL.

Figure 2 Alignment of deduced amino acid of mature PRL from giant panda and other species. The spe-
cies listed are cat (GenBank accession no. U25974), mink (AY249860), pig (X14068), cow (V00112), pos-
sum (AF054634), sheep (M27057), human (NM_000948), rat (NM_012629), mouse (NM_011164),
chicken (J04614), duck (AB158610), frog (L07620) tilapia (M27010, M27011), catfish (AF372653), and
salmon (D00249). Dashes represent maximal similarity. Conserved cysteine residues are indicated by a
black triangle above the sequences. The residues conserved in the PRL from all species are shown in black
boxes. PD1–PD4 indicate four conserved domains of the PRLs between positions 18–32, 58–72, 83–98,
and 160–199. Amino acids that agree with the panda sequence are shaded. Conserved amino acids among
all species are indicated with darkest shading.

Expression of Fusion Protein

To express recombinant PRL in E. coli, the DNA encoding panda mature PRL
was amplified and inserted downstream of the glutathione S-transferase (GST) cod-
ing region of pGEX-4T-1. Cells with 0.6 mM IPTG induction were analyzed on
SDS-PAGE. As shown in SDS-PAGE of Figure 4a, distinct protein bands could
be visualized in samples induced by IPTG, but not in the samples without IPTG
induction or the host strain BL 21. The molecular weight of expressed protein is
the same as predicted size of approximately 49 kD (including 26 kD GST and
23 kD PRL). Solubility analysis (Fig. 4) suggested that the expressed fusion protein
was insoluble, apparently sequestering into inclusion bodies after IPTG induction.
To further confirm GST fusion expression, anti-GST antibody was used to
probe expressed protein by Western blot. As expected, a distinct signal in the pos-
ition of 49 kD was observed in the extracts with IPTG induction, but not in those
without IPTG induction (Fig. 5a). To test the antigenicity of the fused recombinant
panda prolactin, Western blot analysis was performed with use of PRL specific anti-
body. As shown in Figure 5b, expressed GST-panda PRL can be recognized by anti-
human prolactin antibody, suggesting their similar antigenicity.
 MOLECULAR CLONING OF GIANT PANDA CDNA 123

Figure 3 Phylogenetic tree constructed by N-J method based on PRL mature sequences from mammals.
Numbers indicate bootstrap values from 1000 replications. Panda represents Ailuropoda melanoleuca.

Figure 4 Electrophoresis analysis of PRL expressed products: M, protein molecular weight marker;
1, total protein extracts with IPTG induction; 2, total protein extracts without IPTG induction; 3, total
protein extracts from the host BL21 strain; 4, insoluble part of expressed product by IPTG induction;
5, soluble part of expressed product by IPTG induction. Arrows indicate the position of target recombi-
nant protein.
124 ZHI-HE ET AL.

Figure 5 Western blot analysis of crude expressed protein with and without IPTG induction using GST
antibody (a) and anti-human PRL antibody, (b): 1, 3, total protein extracts without IPTG induction;
2, 4, total protein extracts with IPTG induction.

DISCUSSION
In this present work the pituitary PRL cDNA from giant panda was success-
fully isolated, sequenced, and expressed in E. coli. The frame structure of cloned
PRL protein is completely identical with PRL from cat, mink, pig, and cow. As
expected, the amino acids of mature panda PRL displayed a reasonably high level
of identity with PRL from other species including most mammals, birds, and xeno-
pus, ranging from the highest identities of 98% with mink PRL to lowest identity of
59% with rodent PRL. The percentage of sequence identity mostly reflected the evol-
utionary relationship of organisms except rodent and ruminant PRL with great
sequence divergence (Fig. 2) because PRL from rodent and ruminant are believed
to have a rapid molecular evolutionary rate (13) compared to other species.
Amino acid comparisons among PRL from giant panda and other species dis-
played 29 conserved residues clustering in four distinct domains (PD1–PD4), which
are considered to form determinants for specific binding to receptors and might be
critical for PRL-specific activities (10), suggesting similar secondary structure of
panda PRL. In tertiary structure, PRL is predicted to fold in a four-helix bundle.
Putative helix 1, loop 1, and helix 4 constitute receptor binding site 1, while receptor
binding site 2 is located around helix 1 and helix 3. Studies on human PRL using
alanine scanning and site-directed mutagenesis have determined some key amino
acids that are involved in specific receptor binding of PRL (14) on these two binding
sites, including 12 conserved residues located on binding site 1 (Y169, H173, R176,
R177, H180, K181, Y185, K187, H59, P66, K69) and 2 (G129, A22) on binding site
2. Curlewis et al. (15) revealed these 14 residues were conserved in all the known PRL
sequences from mammals, chicken, and xenopus species. Our result that panda PRL
also possesses these conserved 14 residues was in agreement with the findings of
Curlewis. This finding provides further evidence that these 14 conserved residues
are essential for biological function of PRL in all vertebrates.
As members of the GH=PRL family arising from a common ancestor, GH and
PRL share several similar structural features, such as similar predicted 3D structure
folding to four-helix bundle, and functional features including a binding specificity
for their receptors (14). Although the percentage of identity between panda PRL
and GH both on nucleotide (45%) and amino acids (23%) levels is rather low, as
shown in Figure 6 the complete alignment of these two proteins displayed a distinct
resemblance on protein length and identical amino acids residues including four
 MOLECULAR CLONING OF GIANT PANDA CDNA 125

Figure 6 Comparison of PRL and GH based on their mature amino acids sequence. Similar amino acids
between PRL and GH are represented by shading. Conserved cysteine residues are indicated by black
triangles above two sequences.

cysteines. These identical amino acids located at topologically equivalent positions of

two proteins appeared important to the folding of their tertiary structures and of
importance to their common biological function.
PRL was traditionally thought to be a non-glycoprotein pituitary hormone; how-
ever, a proportion of PRL molecule is found to be glycosylated with a varying degree
in most species (10). Structural and functional studies indicated that glycosylated-PRL
exhibited significant differences in bioactivity, receptor binding, and immunoreactivity
with non-glycosylated PRL (10). Both N-glycosylation depending and O-glycosylation
could be observed in PRL, and the occurrence of N-glycosylation depends on the
presence of an N-glycosylation site in PRL. The N-linked glycosylation site at position
31 of panda PRL sequence demonstrated the existence of potential N-glycosylation
PRL in the giant panda.
Non-glycosylation PRL was allowed to express in prokaryotic cells and recom-
binant bacterial expression of several PRL proteins has been reported (16,17). Here,
to express the panda PRL protein, DNA encoding mature protein was inserted
between EcoR I and Xho I site of pGEX-4T-1 vector. The vector used is a fusion
expression vector designed for maximizing the yield of proteins fused with 30 end
of GST, under the control of tac inducible promoter. The expressed protein is then
easy to separate from GST fusion protein due to a thrombin cleavage site designed
downstream of GST. Because of a high level of fusion expression, recombinant PRL
protein was aggregated into a form of insoluble inclusion bodies losing their biologi-
cal activity. Therefore, solubilizing, purifying, and refolding expressed protein would
be needed to obtain a bioactive PRL protein for further activity studies. Solubilizing
PRL-GST fusion protein with a proper method and obtaining purified PRL sepa-
rated from fused protein are being conducted in our laboratory.

ACKNOWLEDGMENTS
We are grateful to the Chengdu Zoo for providing experimental materials. The
experiments described here would not have been possible without help provided by
everyone at the Reproductive and Genetic Laboratories at the Chengdu Base of
Giant Panda Breeding. This work was supported by the Research Fund of Giant
126 ZHI-HE ET AL.

Panda Breeding (Grant No. 2000-19), the National Basic Research Project, the
Eastbay Zoological Society, Oakland, CA, USA, and the Oakland China Wildlife
Preservation Foundation.

REFERENCES

1. Chen DY. Active salvage of the giant panda won studying its reproductive biology in
China. Life Sci 1997; 9(1):31–33 (in Chinese).
2. Liao MY, Zhang ZH, Zhu MY, Zheng Y, Zhang AJ. cDNA cloning of growth hormone
from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli. Com
Biochem Phys B. 2003a; 135(1):109–116.
3. Liao MJ, Zhu MY, Zhang ZH, Zhang AJ, Li GH, Sheng FJ. Cloning and sequence analy-
sis of FSH and LH in the giant panda (Ailuropoda melanoleuca). Anim Reprod Sci
2003b; 77(1–2):107–116.
4. Feng WH, Zhang AJ, Ye ZY. Study on semen character of giant panda. Acta Theriolo-
gica Sinica 1991; 1:1–9.
5. Chen DY, Zhao XK, Chen L. On the follicular atresia and cyst of the giant panda. Acta
Zoologica Sinica 1988; 34(3):219–221.
6. Feng WH, Li GH. The Saving of Giant Panda, 1st ed; Sichuan Publishing House of
Science and Technology, China, 2000 (in Chinese).
7. Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA. Prolactin (PRL) and its receptor:
Actions, signal transduction pathways and phenotypes observed in PRL receptor knock-
out mice. Endocr Rev 1998; 19(5):225–268.
8. Freeman ME, Kanyicska B, Lerant A, Nagy G. Prolactin: Structure, functional, and regu-
lation of secretion. Phys Rev 2000; 80(4):1523–1631.
9. Niall HD, Hogan ML, Sauer R, Rosenblum IY, Greenwood FC. Sequences of pituitary
and placental lactogenic and growth hormones: Evolution from a primordial peptide by
gene reduplication. PNAS 1971; 68(4):866–870.
10. Sinha YN. Structural variants of prolactin: Occurrence and physiological significance.
Endocr Rev 1995; 16(3):354–369.
11. Murata K, Tanioka M, Murakami N. The relationship between the pattern of urinary
oestrogen and behavioral changes in the giant panda. Int Zoo Yb 1986; 2425:274–279.
12. Cooke NE, Coit D, Shine J, Baxter JD, Martial JA. Human prolactin cDNA structural
analysis and evolutionary comparisons. J Biol Chem 1981; 256(8):4007–4016.
13. Wallis M. The molecular evolution of pituitary growth hormone, prolactin and placental
lactogen: A protein family showing variable rates of evolution. J Mol Evol 1981; 17:1018.
14. Goffin V, Shiverick KT, Kelly PA, Martial JA. Sequence-function relationships within the
expanding family of prolactin, growth hormone, placental lactogen, and related proteins
in mammals. Endocr Rev 1996; 17(4):385–410.
15. Curlewis JD, Saunders MC, Kuang J, Harrison GA, Cooper DW. Cloning and sequence
analysis of a pituitary prolactin cDNA from the brushtail possum (Trichosurus
vulpecula). Gen Comp Endocr 1998; 111(1):61–67.
16. Rentier-Delrue F, Swennen D, Prunet P, Lion M, Martial JA. Tilapia prolactin: Molecu-
lar cloning of two cDNAs and expression in Escherichia coli. DNA 1989; 8(4):261–270.
17. Paris N, Rentier-Delrue F, Defontaine A, Goffin V, Lebrun JJ, Mercier L, Martial JA.
Bacterial production and purification of recombinant human prolactin. Biotechnol Appl
Biochem 1990; 12(5):436–449.
18. Owerbach D, Rutter WJ, Cooke NE, Martial JA, Shows TB. The prolactin gene is located
on chromosome 6 in humans. Science 1981; 212(4496):815–816.