cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor1

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CHEM. RES. CHINESE U. 2006, 2 2 ( 2 ) , 145-149

cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor * LI Ming-tang I , JIANG Yong' , WANG Shu-mei' , LI Yong-mingl , WANG Fang' , HOU Xia' , YANG Hong' and MA Tong-hui' * * 1. Membrane Channel Research Laboratory, Northeast Normal University, Changchun 130024, P. R. China ; 2. Department of Anesthesia, Second Teaching Hospital, Jilin University, Changchun 130041, P. R. China Received Dec. 31 , 2005 Molecular cloning of the porcine leukemia inhibitor factor( pLIF) has not been reported. A full-length cDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli , as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLIFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation. Keywords Leukemia inhibitor factor; Porcine ; Cloning; Recombinant protein ; Embryonic stem cell ; Pluripotency Article ID 1005-9040( 2006) -02-145-05

Introduction

characterization of gene functions in vivo[61.In the past

The leukemia inhibitory factor ( LIF) is a multifunctional cytokine belonging to the interleukind ( IL6) family"]. It was first found as a myeloid leukemic cell proliferation suppressor and differentiation inducer cytokine"]. The natural form of LIF is a 38 to 67 kDa

20 years, thousands of knock-out and knock-in mouse

glycosylated protein, widely expressed in both adult and embryonic eutherian mammalsr2]. Numerous in vitro and in vivo studies have indicated that LIF exhibits various important biological functions, like, inducing the effect in myeloid leukemic cell differentiation, maintaining embryonic stem ( ES) cell pluripotency, and the effects in hippocampal development and in blastocyte implantation, and so on[3-51. One of its most fascinating roles is its action in inhibiting mouse embryonic stem ( ES ) cell differentiation in vitro , thus maintaining the ES cells in a pluripotent status. This gives rise to a new technology in life science gene targe-

mice, despite the fact that significant efforts have been made in developing stable pluripotent ES cells from pigs, rabbits, sheep, cattle and h ~ m a n s [ ~ - ~ ~ I .

ting, which makes it possible to modify or disrupt a single gene in ES cells and get germ line transmitted mutant mouse colonies by embryonic manipulation, for the

models have been successfully generated and used as powerful tools to study gene functions. The challenge is that so far there have been no successful examples in germ-line transmission in model animals, other than

Studies across eutherian mammals have shown that LIF and its gene share a high level of similarity among species. Many endeavors have been made to maintain ES or embryonic g e m ( EG) cells of other species with mouse LIF or mouse embryonic feeder cells, but none have been successful. Recent studies have shown that the mouse LIF alone is not enough to maintain chicken EG in a pluripotent or even in an undifferentiated status"21. Other cytokines such as the chicken stem cell factor, the bovine basic fibroblast growth factor, human IL-11 and insulin-like growth factor-1 may also be nee-

ded.

* Supported by the National Natural Science Fund for Distinguished Young Scholars( No. 3032501 1 ) , the National Natural Science Foundation of China( Nos. 30470405 and 30570864) , Distinguished Young Scholars Fund of Jilin Province( No. 20030112) and Excellent Young Teachers Program of MOE, P. R. China. * * To whom correspondence should be addressed. E-mail: mathl08@ nenu. edu. cn

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The organ physiology of a pig is very similar to that of a human in many aspects. Thus a pig can be an ideal model for studying human physiology and disease mechanisms. Stable pluripotent porcine ES cells have not been successfully established so far. Many reasons might account for the failures. Considering the reality that none of the studies have used porcine LIF in establishing porcine ES or EG cells, it can be presumed that porcine LIF could be much more effective in maintaining porcine ES or EG cells in the pluripotent status than its mammalian counterparts. However, the gene encoding porcine LIF has not been cloned. In this article, are repored cDNA cloning, prokaryotic and eukaryotic expression , and purification and functional characterization of porcine LIF are discussed.

Experimental 1 Cloning of Porcine LIF and Construction of Expression Plasmids To clone pLIF cDNA, the total RNA was extracted from the spleen tissues of a freshly slaughtered threeweek piglet and mRNA was purified by using an Oligotex@ mRNA Mini kit ( QIAGEN). The first strand cDNA was synthesized from the purified mRNA by using a Superscript First Strand cDNA synthesis kit ( Invitrogen) . PCR amplification was carried out on a PTC loo@Peltier Thermal cycler ( Beckman Coulter ) . Two pairs of specific primers used for RT-PCR, to amplify the cDNA fragment encoding mature and full-length pLIF protein, were designed according to a genomic sequence ( GenBank Accession NO. A5296176 ) containing full-length pLIF coding sequences , homologous to human LIF. The primer sequences for amplifying the cDNA fragment encoding mature pLIF protein were sense : 5 ’-CCCATATGG’ITCTGCACTGGAAACACGG3‘ with an engineered NdeI restriction site as underlined ; antisense : 5 ’-CCCTCGAGCTAGAAGCCCGCTAGCACAG-3’ with an engineered XhoI restriction site as underlined. For eukaryotic expression, the cDNA fragment of the full-length pLIF coding sequence was amplified by using primers: sense: 5 ’ CCCAAGClTATGAAGGTC’ITGGCGGCAGG-3’ with an engineered Hind site ( underlined) ; antisense : 5 ’GCTCTAGAGAAGGCCCGGGCTAGC AC AG-3 ’ with an engineered Xba 1 site ( underlined ) . PCR reactions were performed by using Vent polymerase ( New England Biolabs) for 30 cycles of denaturing ( 30 s, 94 “c ) , annealing( 30 s, 60 “c ) and extension( 1 min, 72 “c ) and a final extension( 6 min ,72 “c ) . The PCR products were purified by using a QIAquick@PCR purification kit( QIAGEN) . The purified PCR fragment en-

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coding mature pLIF was digested with Nde I /Xho I and subcloned into the prokaryotic expression vector PET 3 1b ( T7 promoter, Novagen ) , to make plasmid PET31b-pmLIF. The PCR fragment encoding fulllength pLIF was digested with Hind II /Xba I and subcloned into the eukaryotic expression vector pcDNA3. 1-myc-HisA( CMV promoter, Invitrogen) , which added a cMyc tag and a His tag, in frame and in tandem, at the C-terminus of the pLIF to make expression plasmid pcDNA3. 1-pLIF-MycHis. Finally, a sequence encoding the mature form of pmLIF with MycHis tags, pmLIF-MycHis , was PCR-amplified by using the pcDNA3. 1-pLIF-MycHis plasmid as a template and primers : sense : 5 ’-CCCATATGG’ITCTGCACTGGAAACACGG-3 ‘ ; antisense : 5 ’-CCCTCGAGTCAATGGTGATGGTGATGACCGG-3’. The pmLIF-MycHis fragment was subcloned into the PET31 b vector at Nde I / Xho I to make expression of plasmid PET31b-pmLIFMycHis.

2 DNA Sequencing The recombinant plasmids pET3 1b-pmLIF , PET31b-pmLIF-MycHis and pcDNA3. 1-pLIF-MycHis were purified by using a QIAprep@ spin Miniprep kit (QIAGEN) , and used for automated sequence analysis on a CEQ8000 DNA sequence analyzer( Beckman Coulter) according to the manufacturer’s menu. The sequences were analyzed thoroughly from both directions.

3 Expression of Recombinant pLIF For prokaryotic expression, chemically competent E. coli BL21( DE3 ) cells ( Novagen ) were transformed with the PET31b-pLIF or the PET31b-pmLIF-MycHis plasmid and selected on ampicillin agar plates. Single colonies were cultured at 37 “c in 3 mL of a sterilized Luria-Bertani medium, containing 100 pg/mL ampicillin, for 3 4 h until the cell density reached OD 0.6. Then, the final concentration of IPTG was adjusted to 0. 1-1.0 mmol/L and the cultures were continuously shaken at 37 “c for different time periods( 1 4 h ) . For the SDS-PAGE analysis, 1 mL of the culture was pelleted by centrifugation at 5000 g for 10 min , and the cell-pellet was resuspended in 90 pL of distilled water and lysed by adding 30 pL of 4 x sample loading buffer. The samples were resolved on a 12% SDS-PAGE and stained with Coomassie brilliant blue. For the eukaryotic expression, CHO cells were stably transfected with the pcDNA3. 1-pLIF-MycHis plasmid, by using the LIPOFECTIN@reagent in accordance with the manufacturer’s instructions. After selection by Geneticine@ (Invitrogen) at a final concentration of 0.5 mg/mL for 10-12 d , single cell colonies were picked, expanded

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and analyzed for the pLIF expression by immunoblotting.

an SDS-PAGE 1 xloading buffer. The samples were blotted and detected by ECL as described above.

4 Purification and Refolding of Recombinant pLIF Protein Containing( His) ,-tag E. coli cells transformed with the recombinant plasmid PET3 1b-pmLIF-MycHis were cultured at 37 “c in 3 mL of the fresh LB medium containing 100 pg/mL of ampicillin for 3 h. The freshly grown bacteria were subcultured in 100 mL of the 2 x YT medium until OD reached 0.6. Then IPTG was added until a final concentration of 1 mmoVL was reached and the culture was continuously shaken at 37 “c for 4 h. The cells were harvested by centrifugation at 5000g for 15 min. The cell paste was resuspended in 4 mL of Tris-HC1( 20 mmoVL, pH = 8 . 0 ) . The cells were disrupted, with sonication, on ice and centrifuged at lOOOOg for 10

6 pLIF Biological Activity Assay

min. The pellet containing the inclusion bodies was resuspended in 3 mL of cold 2 moVL urea, 20 mmoVL Tris-HC1, 0.5 mmoVL NaC1, 0.2% Triton-100 at pH = 8.0 and sonicated again as above. It was then centrifuged at 1OOOOg for 10 min. The pellet was dissolved in 5 mL of a buffer( 20 mmoVL Tris-HC1, 0.5 mmoVL NaC1, 50 mmoVL imidazole, 6 moVL guanidine hydrochloride, 2 mmoVL 2-mercaptoethanol , pH = 8.0). The solution was stirred for 30-60 min at room temperature and centrifuged for 15 min at 1OOOOg. The supernatant was loaded on a Hitrap”chelating column ( Amersham Biosciences ) , for purification, according to the manufacture’s protocols.

5 Western Blotting Analysis of Recombinant pLIF Protein The conditioned CHO medium, the total CHO cell lysates and the purified pmLIF-MycHis proteins were electrophoresed on 12% SDS-PAGE and then electrophoretically transferred onto PVDF ( Amersham Biosciences) membranes at 30 volts, overnight, on ice. The membranes were blocked with 5% non-fat milk in a TBS-T buffer for 1h at room temperature and then incubated with mouse anti-cMyc monoclonal antibody (Chemicon) for 1 h. Having been washed in TBS-T, the membranes were incubated with HRP-labeled goat anti-mouse IgG polyclonal antibody ( Sigma) for 1 h at room temperature, and then washed thoroughly with TBS-T. The blots were detected by using an ECLTM Western blotting analysis system ( Amersham Biosciences). To detect pLIF protein secreted by transfected CHO cells, an aliquot of 500 yL of each conditioned medium was lyophilized and redissolved in an SDSPAGE 1 xloading buffer. To detect the total CHO cell pLIF expression, the cells were lyzed by using 50 y L of

pLIF-expressing CHO cells were cultured in an F12 Ham’s medium containing 10% FBS for 24 h. The conditioned medium was collected and passed through 0. 22-ym filters. El4 mouse ES cells were grown in 96-well tissue culture plates, seeded at 1000 cells per well, in 100 y L of the GMEM medium with 15% FBS, containing various dilutions of the CHO cell conditioned medium or the purified pmLIF-MycHis protein. pLIF activity was determined by assaying ES cell differentiation after 5 - 6 d in culture, by using an Alkaline Phosphotase Detection kit ( Chemicon) as described previously[131.

Results and Discussion 1 Cloning of the pLIF cDNA A 606 bp fragment encoding the full-length pLIF and a 567 bp cDNA fragment encoding the mature form of pLIF were successfully amplified by RT-PCR from piglet spleen mRNA. The RT-PCR products were subcloned into prokaryotic expression vector PET31 b or mammalian expression vector pcDNA 3. 1-myc-His A. The recombinant plasmids were sequenced thoroughly from both directions. Fig. 1 shows the cDNA coding sequence and the deduced amino acid sequence of pLIF (deposited in GenBank as accession No. AY585336). The full-length pLIF contains an open reading frame of 606 bp that encodes a 202 amino acid protein. The Nterminus contains a transmembrane signal polypeptide of 13 amino acids as seen in human and mouse LIF[14*151 . There are six consensus N-linked glycosylation sites and six cysteines with potential to form disulfide bonds in the pLIF protein. Fig. 2 shows the amino acid sequence alignment of pLIF with human and mouse LIF. pLIF has 86% identity with human LIF and 84% identity with mouse LIF. The 6 N-linked glycosylation sites and 6 disulfide bond-forming cysteines are conserved in all the three species. 2 Expression and Purification of Mature pLIF Protein in E. coli Expression of the mature pLIF was analyzed in BL21( DE, ) E. coli cells, transformed with plasmids PET3 1b-pmLIF and pET3 1b-pmLIF-MycHis , by IITG induction. After induction for 1 4 h , both pmLIF ( 20 kDa) and pmLIF-MycHis ( 25 kDa ) were expressed similarly, as was seen, when analyzed by 12% SDSPAGE. The expressed pLIF proteins reach the highest level at 3 h after induction, which accounts for about 20% of the total bacterial proteins. To purify the

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60/20 ATG AACI GTC TTG GCG GCA GGA GTT GTG CCC CTG CTG CTG GTT CTG CAC TGG AAA CAC GGG V L H W K H G 120/40 GCA GGG AGC CCC CTT TCC ATC ACT CCT GTC AAT GCC ACC TGT GCC ACA CGT CAC CCA TGT A G S P L S I T P V N ' A T C A T R H P C 180/60 CAC AGC AAC CTC ATG AAC CAG ATC AAG AAC CAG CTG GCG CAG CTC AAC AGC AGT GCC AAC S N L M N 4 I K N Q L A Q L N ' S S A N H 240/80 GCC CTC TTT ATT CTC TAC TAC ACA GCC CAG FGG GPCI CCA TTT CCC AAC AAC TTG GAC AAG A L F I L Y Y T A Q G E P F P N N L D K 300/100 CTG TGT GGC CCC ARC GTG ACC AAC TTC CCG CCC TTC CAC GCC AAC GGC ACC GAG AAG GCC L C G P N ' V T N F P P F H A N' G T E K A 360/120 CGG CTG GTG GAG CTG TAC CGC ATC ATC GCC TAC CTT GGC GCC TCC CTG GGC AAC ATC ACG R L V E L Y R I I A Y L G A S L G N ' I T 420/140 CGG GAC CAA AGG AGC CTC AAT CCT GGT GCT GTG AAC CTG CAC TCC APCI CTG AAC GCC ACG R D Q R S L N P G A V N L H S K L N ' A T 480/160 GCG GAC AGC ATG CGA GGC CTC CTC AGC AAT GTG CTC TGC CGC CTG TGC AAC AAG TAC CAC A D S M R G L L S N V L C R L C N K Y H 540/180 GTG GCC CAT CTG GAT GTG GCC TAC GGC CCC GAC ACC TCG GGC AAcr GAC GTC TTC CAG AAG V A H V D V A Y G P D T S G K D V F Q K 600/200 AAG AAG CTG GGC TGT CAG CTC CTG GGG A& TAT AAG CAG GTC ATC TCT GTG CTA GCC CGG K K L G C Q L L G K Y K Q V I S V L A R 609/202 GCC TTC TAG A F Z

Fig. 1 cDNA sequence and deduced amino acid sequence of porcine LIF. Residue numbers are indicated on the right as nucleotides/amino acids. The signal polypeptide sequence is shown with gray shadow; consensus N-linked glycosylation sites are marked by " * '' ; potential disulphate bond-forming cysteines are underlined. 40

20

60

Pig : Human: Mouse :

: 74

Pig : Human I Mouse :

: 149

: 74

: 75

: 149 : 150

160

180

200 : 202

Pig : Human: Mouse :

: 202 : 203

Fig. 2 Alignment of the amino acid sequence of porcine, human and mouse LIFs. Residue numbers are indicated on the right side; the conserved six N-linked glycosylation sites( arrow) and six conserved potential disulfde bond-forming cysteines( arrow heads) are indicated.

pmLIF-MycHis protein, inclusion bodies were isolated from IPTG-induced B E 1 cells and dissolved in 6 moVL guanidine hydrochloride solution( pH = 8 . 0 ) , for purification, by affinity chromatography on the HiTrapm chelating column containing NiZ+ -NTA resin, under denaturing conditions. Fig. 3 ( A ) shows the SDS-PAGE analysis of the IFTG-induced total BL21 cell lysate, the inclusion bodies and the affinity-purified pmLIF-MycHis protein stained with Commassie

brilliant blue. The specificity of the purified pmLIFMycHis protein was verified by Western blotting with an anti-cMyc monoclonal antibody [ Fig. 3 ( B) 1.

3 Expression of pLIF-MycHis in CHO Cell Lines pLIF-MycHis-transfected and

mock-transfected

CHO cells were analyzed for the pLIF-MycHis expression after 12, 24, 36, 48 h of post seeding. The conditioned media and the total cell lysates were analyzed by immunoblotting. As Seen in Fig. 4 ( A ) there is a 9

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35.0 kDa25.0 kDa-

mouse LIF, whereas the purified E. coli-expressed mature LIF exhibits higher activity. The expression level of pLIF in CHO cells needs to be improved.

(B) M

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Fig. 3

Purification and verification of bacterially expressed porcine LIF. ( A ) pLIF expression and purification. M: Rainbow protein marker; lane 1 : bacterial extract; lane 2: inclusion body; lane 3: flow through; lane 4: recombinant pLIF purified by Ni2 -NTA affinity chromatography. ( B ) Western blot analysis of the purified pLIF. Lane I : Lysate of BL21 expressing untagged pLIF; lane 2 : affinitypurified pmLIF-MycHis protein. +

single glycosylated band at 37 kDa, detected by the cMyc antibody, indicating that the secreted form of pLIF is fully glycosylated. In the total cell lysates, there are a 37 kDa glycosylated band and a 25 kDa unglycosylated band visualized by immunoblotting , which suggests that some of the intracellular forms of pLIF remain unglycosylated. 1

(A)

,..

2

3

4

..

01

0

I

I

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10 20 30 Dilution of LIF samples

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Fig. 5 Activity of recombinant pLIF. CHO conditioned medium; o : purified pL.IF expressed in E. coli. ; 0 : commercial mouse LIF. A:

Conclusion The porcine leukemia inhibitory factor has been cloned for the first time. The cloned pLIF was successfully expressed in both prokaryotic and eukaryotic systems. The recombinant pLIFs expressed in both systems were able to maintain mouse E-14 ES cells in undifferentiated status. The recombinant pLIFs will be useful in establishing stable pluripotent porcine ES cells for further manipulation.

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References 37 0 kDa

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Western blot analysis of pLIF expressed in CHO cells. ( A ) Expression of pLlF in the conditioned CHO medium. Lane 1 : untransfected sample; lanes 2 , 3 , 4 and 5 : transfected samples collected at 12, 24, 36 and 48 h after seeding. ( B ) pLIF expression in the CHO cell lysate [the same cells as that in ( A ) 1. Lane 1 : untransfected lysate; lanes 2 , 3 , 4 and 5 : transfected lysates prepared from 12, 24, 36 and 48 h cultures post seeding.

4 Bioactivity of Recombinant pLW The biological activity of the recombinant pLIF is determined by its ability to maintain the mouse E-14 ES cells in an undifferentiated status. As shown in Fig. 5 , the activity of the CHO conditioned medium is similar to that of the working concentration commercial

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