Expression, purification, and evaluation for anticancer activityof ribosomal protein L31 gene (RPL31) from the giant panda(Ailuropoda melanoleuca)

Xiu-Lan Su • Yi-Ling Hou • Xiang-Hui Yan •

Xiang Ding • Wan-Ru Hou • Bing Sun •

Si-Nan Zhang

Received: 25 March 2012 / Accepted: 7 June 2012 / Published online: 20 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Ribosomal protein L31 gene is a component of

the 60S large ribosomal subunit encoded by RPL31 gene,

while ribosomal protein L31 (RPL31) is an important

constituent of peptidyltransferase center. In our research,

the cDNA and the genomic sequence of RPL31 were

cloned successfully from the giant panda (Ailuropoda

melanoleuca) using RT-PCR technology respectively, fol-

lowing sequencing and analyzing preliminarily. We con-

structed a recombinant expression vector contained RPL31

cDNA and over-expressed it in Escherichia coli using

pET28a plasmids. The expression product was purified to

obtain recombinant protein of RPL31 from the giant panda.

Recombinant protein of RPL31 obtained from the experi-

ment acted on human laryngeal carcinoma Hep-2 and

human hepatoma HepG-2 cells for study of its anti-cancer

activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphe-

nyl-2H-tetrazolium bromide] method. Then observe these

cells growth depressive effect. The result indicated that the

cDNA fragment of the RPL31 cloned from the giant panda

is 419 bp in size, containing an open reading frame of

378 bp, and deduced protein was composed of 125 amino

acids with an estimated molecular weight of 14.46-kDa and

PI of 11.21. The length of the genomic sequence is

8,091 bp, which was found to possess four exons and three

introns. The RPL31 gene can be readily expressed in

E.coli, expecting 18-kDa polypeptide that formed inclusion

bodies. Recombinant protein RPL31 from the giant panda

consists of 157 amino acids with an estimated molecular

weight of 17.86 kDa and PI of 10.77. The outcomes

showed that the cell growth inhibition rate in a time- and

dose-dependent on recombinant protein RPL31. And also

indicated that the effect at low concentrations was better

than high concentrations on Hep-2 cells, and the concen-

tration of 0.33 lg/mL had the best rate of growth inhibi-

tion, 44 %. Consequently, our study aimed at revealing the

recombinant protein RPL31 anti-cancer function from the

giant panda, providing scientific basis and resources for the

research and development of cancer protein drugs anti-

cancer mechanism research. Further studies of the mech-

anism and the signal transduction pathways are in progress.

Keywords Giant panda � RPL31 � Cloning � Over-

expression � Anti-cancer activity

Introduction

The ribosome is essential for protein synthesis. The com-

position and structure of ribosomes from several organisms

have been determined, and it is well documented that

ribosomal RNAs (rRNAs) and ribosomal proteins (RPs)

constitute this important organelle. Many RPs also fill

various roles that are independent of protein biosynthesis,

called extraribosomal functions. These functions include

DNA replication, transcription and repair, RNA splicing

and modification, cell growth and proliferation, regulation

of apoptosis and development, and cellular transformation.

With the continuous advancement of science and technol-

ogy, the researchers are gradually finding the physiological

functions of ribosomal proteins which play an important

role in human disease and its development [1, 2].

Ribosomal protein L31 (RPL31) was reported to be one

of the constituent proteins of the ribosomal P-site [3].

It is located at or near the vicinity of the peptidyl site of

X.-L. Su � Y.-L. Hou � X.-H. Yan � X. Ding � W.-R. Hou (&) �B. Sun � S.-N. Zhang

College of Life Science, China West Normal University,

Nanchong 637009, China

e-mail: hwr@cwnu.edu.cn

123

Mol Biol Rep (2012) 39:8945–8954

DOI 10.1007/s11033-012-1763-0

ribosomes, which is an important constituent of peptidyl-

transferase center belongs to the eukaryotic ribosomal

proteins which are not conserved in eubacteria but possess

an archaebacterial homolog [4]. In eubacteria, an unrelated

protein, RPL17 occupies the location of RPL31 at the exit

tunnel platform [5]. It has been suggested that proteins not

conserved between eubacteria and archaea/eukaryotes have

arrived by convergent evolution with the main purpose to

fill the cracks between rRNA helices and stabilize the

structure [6]. The crystal structure revealed that RPL31

form a rim around the polypeptide tunnel exit, which

interacts with Zuo1 subunit of RAC(ribosome-as-sociated

complex), physically. Research found RPL31 was down-

regulated in metastatic CRC by oligonucleotide arrays

detecting 50 colon adenocarcinomas and their paired nor-

mal mucosa [7–9]. RPL31 was also recently identified as a

contact site for the SRP (signal recognition proteins)

receptor and the ribosome-associated complex. Since

RPL31 plays such an important role and its primary

structure and function in giant panda has not been defined,

it is significant to clone and analyze the RPL31 gene of the

giant panda and its significance lies not only in the pro-

tection of the giant panda, but also in the therapy for

several kinds of human hereditary diseases.

Laryngeal carcinoma is a common head and neck

malignancy with high incidence as it accounts for

approximately 2.4 % of new malignancies worldwide

every year [9, 10]. Despite extensive application of many

different treatment modalities, the prognosis for patients

with laryngeal carcinoma especially at late stage remains

poor. The overall 5-year survival rate is about 73–92 % for

early disease stages and 50–64 % for advanced disease

stages [10]. While HepG-2 (Hepatocellular carcinoma,

human) cells are epithelial in morphology, which secrete a

variety of major plasma proteins; e.g., albumin, transferrin

and the acute phase proteins fibrinogen, alpha 2-macro-

globulin, alpha 1-antitrypsin, transferrin and plasminogen

[11]. Thus, more efforts are needed to develop novel

approaches and strategies for the treatment of this disease.

The giant panda (Ailuropoda melanoleuca) is one of the

oldest and rarest species in the world, known as ‘‘national

treasure of China’’, belonging to national level of endan-

gered animal. Previous studies on the giant panda have

mainly concentrated on the macro level, such as breeding

and propagation, ecology, genetic diversity, parentage, and

so on. Recently, researches on functional genes of giant

panda are becoming a hot issue, especially in gene cloning

and functional investigation such as RPS14, RPS15,

RPS19, RPLP1 and so on [12–18]. Our team has been

committed to research functional genes of giant panda.

And, the latest research shows that the recombinant protein

RPL23A exhibited anti-cancer function on the Hep-2 cells

[19]. There were some research in human about RPL31,

but, so far, there is little report about RPL31 gene and

protein RPL31 of the giant panda in the literature, espe-

cially RPL31 recombinant protein anti-cancer activity

research.

In this study, the primers are designed according to the

related information of RPL31 gene of some mammalians,

including Homo sapiens, Bos taurus, Pongo abelii, Mus

musculus and Rattus norvegicus. Then, the RT-PCR and

PCR technique was used to amplify and clone the cDNA

sequence of the RPL31 gene from the total RNA extracted

from the muscle tissues of the giant panda. The sequence

characteristics of the protein encoded by the cDNA were

analyzed and compared with those reported mammalian

species, after sequencing of cDNA sequence of the RPL31.

We constructed a recombinant expression vector contained

RPL31 cDNA and over-expressed it in Escherichia coli

using pET28a plasmids. Under the optimized expression

conditions, we got a lot of recombinant protein of RPL31

from the giant panda, which then was purified by Ni che-

lating affinity chromatography. Recombinant protein

obtained from the experiment acted on Hep-2 cells and

HepG-2 cells, then observe these cells growth depressive

effect. Consequently, our study aimed at revealing the

recombinant protein RPL31 anti-cancer function from the

giant panda, providing scientific basis and resources for

the research and development of cancer protein drugs anti-

cancer mechanism research.

Materials and methods

Materials

Muscle tissues were collected from a dead giant panda at the

Wolong Conservation Center of the giant panda, Sichuan,

China. The collected skeletal muscle was frozen in liquid

nitrogen and then used for RNA isolation. The human lar-

yngocarcinoma line Hep-2 cells and human hepatoma

HepG-2 cells were purchased from the deparement of bio-

chemistry and immunology, North Sichuan Medical Col-

lege, China. Total Tissue/cell RNA Extraction Kits were

purchased from Waton company, Shanghai, China. Reverse

transcription kits were from Promega Company, Beijing,

China. Gel Extraction Mini Kits were purchased from

OMEGA Corporation, Kanpur, India. PMD-18 T Vector

Systems and restriction enzyme BamHI and HindII were

got from TaKaRa Bio Group, Dalian China. DNA poly-

merases were purchased from Sangon Co, Shanghai, China.

Host bacteria E. coli DH5a were stored in Key Laboratory

of Southwest China Wildlife Resources Conservation.

CW0009 Ni-Agarose His-tag Protein purification kits were

purchased from Beijing Ealysino Biological Technology

Co, Beijing, China. Bradford Protein Assay Kits were

8946 Mol Biol Rep (2012) 39:8945–8954

123

purchased from Majorbio Biotech Co, Shanghai,

China.Penicillin/streptomycin (penicillin 10,000 units/mL,

streptomycin 10,000 lg/mL) and Dulbecco’s minimal

essential medium (DMEM) reagent were purchased from

Gibco BRL (Grand Island, NY, USA). Fetal bovine serum

was obtained from Sijiqing Co. (Huangzhou, China).

DNA and RNA isolation

A total of 500 mg muscle tissue from giant panda was

ground in liquid nitrogen to a fine powder, and the powder

was suspended completely in 15 mL lysis buffer containing

10 mM Tris–HCl, pH 8.0, 100 mM EDTA and 0.5 % SDS.

After treatment with proteinase K (100 mg/mL, final con-

centration) at 55 �C for 3 h, the mixture was then cooled to

room temperature and mixed with an equal volume of

saturated phenol (pH 8) before being centrifuged at

5,000g at 4 �C for 20 min. The supernatant was pooled and

then mixed with an equal volume of 1:1 (v:v) phenol–

chloroform and then centrifuged as above and the super-

natant collected, from which the DNA was precipitated by

ethanol. The DNA obtained was then dissolved in TE

buffer and kept at -20 �C.

Total RNAs were isolated from about 400 mg muscle

tissue using the Total Tissue/Cell RNA Extraction Kits

(Waton Inc., Shanghai, China) according to manufacturer

instructions. The total RNAs extracted were dissolved in

DEPC (diethyl pyrocarbonate) water, and kept at -70 �C.

Primer design and RT-PCR

The polymerase chain reaction (PCR) primers were

designed by Primer Premier 5.0, based on the mRNA

sequence of RPL31 from H. sapiens (NM_000993),

B. Taurus (NM_001025341), P. abelii (NM_001131976),

M. musculus (NM_053257) and R. norvegicus (NM_0225

06). The specific primers of cDNA sequences were as

follows:

RPL31 forward primer: 50-TTCCATCTTCGGCCCTG-

CAGA-30;RPL31 reverse primer: 50-CTTTATTTGACCATCAG-

CAG-30.

Total RNAs were synthesized into the first-stranded

cDNAs using a reverse transcription kit with Oligo dT as

the primers according to manufacturer instructions (Pro-

mega, USA).

Twenty microliters of the first-strand cDNA synthesis

reaction system was included in 1 mg total RNAs, 5 mM

MgCl2, 1 mM dNTPs, 0.5 mg Oligo dT15, 10 U/mL

RNase inhibitor, and 15 U AMV reverse transcriptase, and

incubated at 42 �C for 60 min. The first-strand cDNA

synthesized was used as a template. The total reaction

volume for DNA amplification was 25 lL. Reaction mix-

tures contained 1.5 mM MgCl, 200 lM of each of dATP,

dGTP, dCTP and dTTP (Promega Co., Beijing, China),

0.3 lM of each primer, 5.0 units Taq plus DNA poly-

merase (Sangon Co., Shanghai, China). DNA amplification

was performed using an MJ Research thermocycler, Model

PTC-200 (Watertown, MA, USA) with a program of 4 min

at 94 �C, followed by 30 cycles of 1 min at 94 �C, 0.5 min

at 48 �C and 1.5 min at 72 �C, and then ended with the

final extension for 10 min at 72 �C. After amplification,

PCR products were separated by electrophoresis on 1.5 %

agarose gel with 1X TAE (Tris–acetate-EDTA) buffer,

stained with ethidium bromide and visualized under UV

light. The expected fragments of PCR products were har-

vested and purified from gel using a DNA harvesting kit

(Promega Co., Beijing, China), and stored at -20 �C.

Cloning and identifying the cDNA sequence of RPL31

The harvested PCR products were ligated into a pMD19-T

vector at 4 �C for 8 h. The recombinant molecules were

transformed into E. coli competent cells (DH5a), and then

spread on an LB-plate containing 50 lg/mL ampicillin,

200 mg/mL IPTG (isopropyl-beta-D-thiogalactopyrano-

side), and 20 mg/mL X-gal. Plasmid DNA was isolated and

digested by PstI and ScaII to verify the insert size, or PCR

technology was used. Plasmid DNA was sequenced by

Huada Zhongsheng Scientific Corporation (Beijing, China).

Cloning the genomic sequence of RPL31

The polymerase chain reaction (PCR) primers were

designed by Primer Premier 5.0. The specific primers of

genomic sequence were as follows:

RPL31 forward primer-1: 50-TTCCATCTTCGGCCCTG

CAGA-30;RPL31 reverse primer-1: 50-CTTTGGCCCAGACGGAT

TTG-30.RPL31 forward primer-2: 50-TCCAGATGTGCGCATT

GACA-30;RPL31 reverse primer-2: 50-CTTTATTTGACCATCAG

CAG-30.

The genomic sequence of the RPL31 gene was amplified

using Touchdown-PCR with the following conditions:

94 �C for 30 s, 55 �C for 45 s, 72 �C for 4 min in the first

cycle and the annealing temperature deceased 0.5 �C per

cycle; after 20 cycles conditions changed to 94 �C for 30 s,

45 �C for 45 s, 72 �C for 4 min for another 15 cycles, and

then ended with the final extension for 10 min at 72 �C. The

fragment amplified was also purified, ligated into the clone

vector and transformed into E. coli competent cells. Finally,

the recombinant fragment was sequenced by Sangon.

Mol Biol Rep (2012) 39:8945–8954 8947

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Cloning the expression fragment sequence of RPL31

The PCR fragment corresponding to the RPL31 polypeptide

was amplified from the RPL31 cDNA clone with the forward

primer, 50-CAGGATCCATGGCTCCCGCGA-30(BamHI)

and reverse primer, 50-CGAAGCTTAGTTCTCGTCCA-30

(HindII), respectively. Primers were synthesized by Shang-

hai ShengGong biotechnology Co., LTD.

Taking the restructuring plasmid linked to cDNA of as

templates, the new synthesized sequence containing

restriction enzyme cutting site as primer, PCR was per-

formed at 94 �C for 3 min; 30 cycles of 30 s at 94 �C, 45 s

at 52 �C, 1 min at 72 �C, and 10 min at 72 �C.

Construction of the expression vector

and overexpression of recombinant RPL31

The amplified PCR product was cut and ligated into cor-

responding site of the pET28a vector (Stratagene, USA).

The resulting construct was transformed into the E. coli

BL21 (DE3) strain (Novagen, USA) and used for induction

by adding IPTG at an OD600 of 0.6 and culturing further

for 4 h at 37 �C, using the empty vector transformed

BL21(DE3) as a control. The recombinant protein samples

were induced after 0, 1, 1.5, 2, 2.5, and 3 h and then sep-

arated by SDS-PAGE and stained with Coomassie blue

R 250. Bacteria liquid were induced after 3 h is largely

collected and centrifuge at 4 �C, 5,000 r/min for10 min,

weigh its wet weigh. Add Binding Buffer (50 mM Tris–

Hcl, 300 mM Nacl, 10 mM imidazole, pH 8.0) into the

bacterial to suspension, according to 5 mL/g the weight of

the collected bacteria. Add PMSF to the mixture until the

ultimate concentration to 1 mM. Add lysozyme to the

mixture until the ultimate concentration to 1 mg/mL, and

incubate in the ice for 30 min. Transfer the tube with the

samples in the ice, ultrasonicate the mixture. SDS-PAGE

analysis of the sonicated bacterial cells showed that the

expressed product mostly existed in the form of inclusion

body.

Purition of recombinant protein RPL31

Acquired genetic engineering recombinant protein has a

tag which is comprised of six histidine (His-tag), so nickel

chelating affinity chromatography is available. Centrifuge

the ultrasonic product to collect sediments, then suspend

them with Soluble Binding Buffer (20 mM Tris–Hcl,

0.5 M Nacl, 10 mM imidazole, pH7.9) until the inclusion

body is clean. Centrifuge and suspend the sediments with

Inclusion Body Binding Buffer (20 mM Tris–Hcl, 0.5 M

Nacl, 5 mM imidazole, urea 8 M, pH 7.9) in the ice, until

the inclusion body is thoroughly dissolve. Centrifuge and

transfer the above supernatant to chromatography column

with nickel. Stand for 2 min after the above supernatant is

completely transferred so that the six His-tag and nickel in

the padding can combine fully. Inclusion Body Binding

Buffer of 15 times column volume is used to flush column,

so as to wash the uncombined protein. Collect the excur-

rent liquid. Then, inclusion Body Elution Buffer of 5 times

column volume is used to wash the combined protein.

According to the amount of column volume, collect the

outflow liquid. Then, SDS-PAGE was used to detect the

effect of purification.

Purity test of recombinant protein RPL31

The concentration of recombinant protein was determined by

Bradford Protein Assay Kits. To further purification the elu-

tion protein, dialysis is available. After 48 h of desalination,

purification protein with 10 % glycerol is stored at -20 �C.

Cell culture

Human laryngeal carcinoma Hep-2 and human hepatoma

HepG-2 cell lines were grown in RPIM 1,640 medium

supplemented with 10 % heat-inactivated fetal bovine

serum, 100 IU/mL penicillin, 100 lg/mL streptomycin and

10 mM HEPES, pH 7.4. Cells were kept at 37 �C in a

humidified 5 % CO2 incubator.

Test the effect of purified recombinant protein RPL31

on human laryngeal carcinoma Hep-2 cells and human

hepatoma Hep G2 cell activity by MTT method

These cells were seeded into 96-well microculture plates at

appropriate densities to maintain the cells in an exponential

phase of growth throughout the duration of the experiment.

Human laryngeal carcinoma Hep-2 and human hepatoma

HepG-2 cells were exposed to RPL31 protein at 6.58, 3.29,

1.65, 0.82, 0.41, 0.33, 0.13 and 0 lg/mL for 24 h and each

concentration was evaluated in six separate wells, respec-

tively. At the end of the exposure, 20 lL of MTT was added

to each well and the plates were incubated for 2–4 h at 37 �C.

Then, 150 lL of DMSO was added to each well and the

plates were surged for 5 min. The optical density (OD) was

read on a plate reader (BIO-RAD Co., USA) at two wave-

lengths of 490 nm. Media-alone as well as control wells, in

which PMBE was absent, were included in all experiments.

The degree of inhibition of cell proliferation was calculated

using the following formula: Growth inhibition (%) = (OD

control—OD treated)/OD control 9 100.

The morphologic observation of cells

96-well microculture plates which seeded human laryngeal

carcinoma Hep-2 and human hepatoma HepG-2 cells were

8948 Mol Biol Rep (2012) 39:8945–8954

123

observed by inverted microscope, respectively. The change

of these cells conformation was photoed and recorded.

Data analysis

The sequence data were analyzed by the GenScan software

(http://genes.mit.edu/GENSCAN.html). Homology research

of the giant panda RPL31 compared with the gene sequences

of other species was performed using Blast 2.1 (http://www.

ncbi.nlm.nih.gov/blast/). Open-reading frame (ORF) of the

DNA sequence was searched using the ORF finder software

(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein struc-

ture of the RPL31 sequence cloned was deduced using the

PredictProtein software (http://cubic.bioc.columbia.edu/predi

ctprotein/). Multiple sequence alignment was performed with

the software DNAstar Lasergene and DNAMAN 6.0. The pI/

Mw of protein was analyzed by DNAMAN 6.0. Prediction of

tertiary structure of recombinant protein RPL31 was simu-

lated by the SWISS-MODEL software (http://swiss-model.

expasy.org/).

Results and discussion

Analysis of the cDNA of RPL31 from the giant panda

About 400 bp of the cDNA fragment was amplified from

the giant panda with the primers RPL31 forward and

RPL31 reverse. The length of the cDNA cloned was

419 bp. (Fig. 1).

As determined by BLAST analysis, the nucleotide

sequence RPL31 cloned from the giant panda shares a high

homology with those of H.sapiens, B.taurus, P.abelii,

M.musculus and R.norvegicus (93.65, 91.80, 92.59, 90.74

and 92.33 %, respectively). Comparison of the deduced

amino acid sequences of giant panda with those of these

species showed that the RPL31 of giant panda is highly

homologous with that of P. abelii (99.20 %), and is 100 %

identical with the others. This striking pattern of evolu-

tionary conservation is reasonable, as ribosomal protein

genes are a group of highly conserved housekeeping genes

[20]. Physical and chemical analysis showed that the

molecular weights of the putative proteins among the five

mammalians are very close and the theoretical pI are

exactly identical, except for P. abelii (Table 1).

Analysis of the genomic sequence of RPL31

from the giant panda

A DNA fragment of about 8,000 bp was amplified with

primers-1 and primers-2. The length of the DNA fragment

cloned is 8,091 bp. Comparison between the cDNA

sequence and the DNA fragment sequence of the RPL31

amplified from giant panda was performed by software

Lasergene. The result indicated that the cDNA sequence is

in full accord with three fragments in the DNA fragment,

which manifests that the DNA fragment amplified is the

genomic sequence of the RPL31 from giant panda (Fig. 2).

The genomic sequence of the RPL31 has been submitted to

Genbank (accession number:HQ318081).

A comparison of the nucleotide sequences of the geno-

mic and cDNA sequences indicated that the genomic

sequence of RPL31 possesses four exons and three introns,

which is also supported by restriction mapping of the

genomic and cDNA sequences. Compared with some

mammals, including H. sapiens(NC_000002), B. taurus

(NC_007309), P. abelii (NC_012592), M. musculus (NC_

000067) and R. norvegicus (NC_005108), the length of the

genomic sequence, the first, second and third introns,

and the 50- and 30-untranslated sequences are different

(Table 2). The variations in lengths of the introns deter-

mine the lengths of the RPL31 genes.

Prediction and analysis of protein functional sites

in RPL31 protein of the giant panda

Primary structure analysis revealed that the molecular

weight of the putative RPL31 protein of the giant panda is

14.46 kDa with a theoretical pI of 11.21. Topology pre-

diction shows that there are 2 different patterns of func-

tional sites: two Protein kinase C phosphorylation site and

one Ribosomal protein L31e signature in the RPL31 pro-

tein of the giant panda (Fig. 3).

Overexpression of the RPL31 gene in E. coli

The RPL31 gene was over-expressed in E. coli using

pET28a plasmids carrying strong promoter and terminator

sequences derived from phage T7. For this purpose, the

Fig. 1 Reverse transcription polymerase chain reaction products of

the giant panda RPL31. 1, 2: the amplified RPL31. M molecular

ladder DL2000

Mol Biol Rep (2012) 39:8945–8954 8949

123

RPL31 gene was amplified individually by PCR and cloned

in a pET28a plasmid, resulting in a gene fusion coding for

a protein bearing a His-tag extension at the N-terminus.

Expression was tested by SDS-PAGE analysis of protein

extracts from recombinant in E. coli BL21 strains.

The results indicated that the protein RPL31 fusion with

the N-terminally His-tagged form gave rise to the accu-

mulation of an inclusion bodies, as shown at the arrow in

Fig. 4. Apparently, the recombinant protein was expressed

after half an hour of induction and after 3 h reached the

highest level. The expression product obtained could be

used for purification and further study of its function.

Purification of recombinant protein RPL31

Under the optimized expression conditions, we got a lot of

recombinant protein. Through the affinity chromatography,

we obtained purified protein. During affinity chromatog-

raphy, the twice changes of protein solution PH value make

us gain high purity of protein. It means that protein solution

Table 1 Comparison of

nucleotide, amino acid

sequences and

physicochemicalproperty

between the A.melanoleuca and

other 5 mammal species

Items Species

H. sapien B. taurus P. abelii R. norvegicus M. musculus

Cds similarity (%) 93.65 91.80 92.59 92.33 90.74

Aa similarity (%) 100 100 99.20 100 100

Molecular weight (kDa) 14.46 14.46 14.39 14.46 14.46

PI 11.21 11.21 11.33 11.21 11.21

Fig. 2 Nucletide sequence of

cDNA encoding the giant panda

(A.melanoleuca) RPL31 protein

and the amino acid sequence

deduced from its ORF. The

nucleotide and amino acid

sequences are numbered on the

left. The translation is given

under the center of each codon

(asterisk is the termination

codon)

Table 2 Comparison of RPL31genomic sequence among 6

mammal species

Pd A. melanoleuca, Ho

H. sapiens, Bo B. Taurus, Po

P. abelii, Mu M. musculus, Ra

R. norvegicus

Items Length of genomic

sequence (bp)

Number

of introns

Number

of exons

Join sites in the CDS GenBank

accession numbers

Pd 8,091 3 4 1…107, 6085…6210,

7619…7731, 8040…8071

HQ318081

Ho 17,465 3 4 474…580, 1930…2055,

3731…3843, 16770…16810

NC_000002

Bo 5,352 3 4 433…539, 3615…3740,

4855…4967, 5288…5319

NC_007309

Po 3,745 3 4 1…106, 1467…1592,

3280…3392, 3673…3704

NC_012592

Mu 4,061 3 4 299…405, 2164…2289,

3104…3216, 3452…3483

NC_000067

Ra 3,486 3 4 203…309, 2143…2268,

3102…3214, 3419…3450

NC_005108

8950 Mol Biol Rep (2012) 39:8945–8954

123

firstly get through a column in acid conditions, then out-

flow the column by changing the PH value of the effluent

liquid. SDS-PAGE analysis clearly indicated that there are

about 18-kDa polypeptide in the fourth and fifth lane

(Fig. 5).

The sequence of acquired ribosomal protein RPL31 gene

recombinant protein from the giant panda is 157 amino

acid residues. And the molecular weight of recombinant

protein RPL31 was 17.86-kDa and theoretical PI was

10.77. The simulative tertiary structure of recombinant

protein RPL31 from the giant panda is shown in Fig. 6.

Size consistency of the purified protein and acquired

RPL31 recombinant protein suggest that the protein is only

the protein encoded by the RPL31 from the giant panda.

Effect of recombinant protein RPL31 on human

laryngeal carcinoma Hep-2 and human hepatoma

HepG-2 cells growth inhibition by MTT assay

In the study, the MTT method was used to detect the

effects of different concentrations of recombinant protein

RPL31 on the proliferation of the human laryngeal carci-

noma Hep-2 and human hepatoma HepG-2 cells in 24 h.

The data indicate that the effect at low concentrations is

better than high concentrations, and the concentration of

0.33 lg/mL has the best rate of growth inhibition, 44 %, in

human laryngeal carcinoma Hep-2. However, there was

few obvious effect to human hepatoma HepG-2 cells

(Fig. 7).

Effect of recombinant protein RPL31 on Hep-2 cells

conformation by inverted microscope

The 96-well plates were placed under inverted microscope,

camera records different concentrations changes for cell

Fig. 3 Comparison of the

amino acid sequence of RPL31

between the giant panda and

five other vertebrate species.

Under line, Ribosomal protein

L31e signature; double underline, Protein kinase C

phosphorylation site; Wavy line,

Polymorphic site Pd

A. melanoleuca, Ho H. sapiens,Bo B. taurus, Po P. abelii, Ra

R. norvegicus, Mu M. musculus

Fig. 4 Proteins extracted from

recombinant E. coli strains were

analyzed by SDS-PAGE gel

stainedwith Coomassie blue R

250. Numbers on the right show

the molecular weight, and the

arrow indicates the recombinant

protein bands induced by IPTG

for 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5

and 4 h (lanes 1–9),

respectively. 10: molecular

marker

Fig. 5 The purified of recombinant proteins RPL31 Numbers on the

left show the molecular weight, Lane 1: molecular marker; Lane 2:

RPL31 proteins extracted from recombinant E. coli strains; Lane 3:

effluent liquid collected from columns; Lane 4–9: the eluent collected

from columns

Mol Biol Rep (2012) 39:8945–8954 8951

123

morphology in order to measure the effect of recombinant

protein RPL31. RPL31 exhibited the high anticancer

activity as can be seen from the cell morphology which was

rounded into group, and even cracking off in pieces in Hep-

2 group while HepG-2 cells (data not shown) displayed no

significant change compared to those in control group

(Fig. 8).

Morphological alteration in the human laryngeal carci-

noma Hep-2 cells with RPL31 also vividly told us RPL31

possess very good inhibiting human laryngeal carcinoma

Hep-2 cells growth or proliferation activity. The prolifer-

ation of human laryngeal carcinoma Hep-2 cells was

restrained by recombinant protein RPL31, while human

hepatoma HepG-2 cells did not change. Maybe there were

some RPL31 receptors on the face of the human laryngeal

carcinoma Hep-2 cells, which combining with recombinant

protein RPL31. Therefore, some substance generated acted

on human laryngeal carcinoma Hep-2 cells, resulting in the

inhibition of human laryngeal carcinoma Hep-2 cell pro-

liferation. So, further studies of the mechanism and the

signal transduction pathways are in progress.

Discussion

Ribosomal protein gene mutations or disturbance in their

expression levels were found in many inherited genetic

diseases such as Diamond-Blackfan anaemia syndrome,

Tuner syndrome, Noonan syndrome, Camurati-Engelmann

disease, BardetBiedl syndrome 4 [21]. The similar results

appeared in ribosomal protein such as carcinoma of breast

[22], prostate [23], uterine cervix [24], esophagus [25],

liver [26] and also in the glioblastoma and multiforme

brain tumors [27]. Ribosomal protein RPL31 gene as one

of the ribosomal protein gene also play an important role in

cell growth or proliferation regulation, cell malignant

transformation tumor progression, invasion, metastasis and

differentiation [28] .

In our research, the cDNA and the genomic sequence of

RPL31 were cloned successfully from the giant panda

(A. melanoleuca) using RT-PCR technology respectively,

following sequencing and analyzing preliminarily. We

constructed a recombinant expression vector contained

RPL31 cDNA and over-expressed it in Escherichia coli

using pET28a plasmids. The expression product was

purified to obtain recombinant protein of RPL31 from the

giant panda. Recombinant protein obtained from the

experiment acted on human laryngeal carcinoma Hep-2

and human hepatoma HepG-2 cells for study of its anti-

cancer activity by MTT method. Then observe these cells

growth depressive effect. The result indicated that the

cDNA fragment of the RPL31 cloned from the giant panda

is 419 bp in size, containing an open reading frame of

378 bp, and deduced protein was composed of 125 amino

acids with an estimated molecular weight of 14.46-kDa and

PI of 11.21. The length of the genomic sequence is

8,091 bp, which was found to possess four exons and three

introns. The RPL31 gene can be readily expressed in

E.coli, expecting 18-kDa polypeptide that formed inclusion

bodies. Recombinant protein RPL31 from the giant panda

consists of 157 amino acids with an estimated molecular

weight of 17.86-kDa and PI of 10.77. Size consistency of

the purified protein and acquired RPL31 recombinant

protein suggest that the protein is only the protein encoded

by the RPL31 from the giant panda. Research of recom-

binant protein L31 anti-cancer activity indicated that it

displayed the relying effect of doses on the degree of

inhibition of cell proliferation. Especially, when the con-

centration of ribosomal protein L31 was 0.33 u g/mL, it

showed the highest degree of inhibition of cell prolifera-

tion, 44 %.

Theoretically speaking, RPL31 recombinant protein as a

biological macromolecule, it is impossible to enter the

cancer cells to inhibit their growth and reproduction. Why

our research showed RPL31 recombinant protein own high

degree of inhibition of cell proliferation on human

Fig. 6 Tertiary structure of recombinant protein RPL31 from the

giant panda

Fig. 7 The effect of recombinant protein RPL31 on the growth of

human laryngeal carcinoma Hep-2 and human hepatoma HepG-2

cells 1–8, the concentration of recombinant protein RPL31 are 0,

6.58, 3.29, 1.65, 0.83, 0.41, 0.33, 0.12 lg/mL, respectively

8952 Mol Biol Rep (2012) 39:8945–8954

123

laryngeal carcinoma Hep-2 cells? Based on past experi-

ence, RPL31 recombinant protein as a biological macro-

molecule, acted on human laryngeal carcinoma Hep-2 and

human hepatoma HepG-2 cells. It should promote the

proliferation of two cancer cells, instead of inhibiting Hep-

2 cells growth or proliferation activity, which seems to be

equivalent to a component of the medium. In this sense, our

research implied that RPL31 recombinant protein played a

similar role as a cell factor not nutrient content. We can

assume that, cell factor have different receptor factor in

different cell surface. At the same time, different cell

receptors factor caused different internal change, which

lead to difference of the result, when it received the stim-

ulation of cell factors. As the experimental results showed

that, RPL31 recombinant protein had the high rate of

growth inhibition on human laryngeal carcinoma Hep-2

cells. For comparison, human hepatoma HepG-2 cells

displayed no significant change when compared to the

control (untreated) cells. This further showed that, RPL31

recombinant protein have the obvious target effect on

inhibition of cancer cells. To further studies of the mech-

anism and the signal transduction pathways, varieties of

tumor cell lines will be used to screening the inhibition

effect of RPL31 recombinant protein. Meanwhile, we

should investigate effect factors in effect cells to explore

the cells influence of the transcription and translation.

Comparison of the deduced amino acid sequences of giant

panda with other species showed that the RPL31 of giant

panda is highly homologous, however, there were some

differences in sequence information of those cDNA frag-

ments. So, we also can select the highest expression of

RPL31 recombinant protein which is most easily been

expressed for industrial production.

More and more research indicated that the high level

express of ribosomal protein is a prognostic factor in some

kinds of tumor [29–34], and several ribosomal proteins

have been identified in high level in cancers in recent years

by applying some high-throughput techniques, such as

gene array technology and subtractive hybridization tech-

nology. This research showed ribosomal protein L31 gene

recombinant protein can effectively inhibit Hep-2 cells

growth or proliferation activity. These effects will con-

tribute to the research of the anticancer mechanism of

ribosomal protein L31 gene recombinant protein and the

development of tumor vaccine for tumor prevention.

Acknowledgments The Key Chinese National Natural Science

Foundation (30570275), Key Discipline Construction Project in

Sichuan Province (SZD0420), Sichuan key discipline zoology con-

struction funds subsidization project (404001), Application Founda-

tion Project in Sichuan Province (2009JY0061), Youth Fund Project

of Educational Committee of Sichuan Province (09ZB088), Founda-

tion Project of Educational Committee of Sichuan Province

(10ZC120). Application Foundation Project in Sichuan Province

(2011JY0135).

Conflict of interest The authors report no conflicts of interest in this

work.

Fig. 8 The effect of recombinant protein RPL31 on the morphology of Hep-2 cells 1–8: the concentration of recombinant protein RPL31 are 0,

6.58, 3.29, 1.65, 0.82, 0.41, 0.33, 0.12 lg/mL, respectively

Mol Biol Rep (2012) 39:8945–8954 8953

123

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