Construction of expression vectors carrying mouse peroxisomal protein gene (PeP) with GST and Flag labels

Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran. Stem Cell Department, Royan Institute, Isfahan Research Campus, Isfahan, Iran. Department of Basic Medical sciences, Islamic Azad University, Khorasgan Branch, Isfahan, Iran; Stem Cell Department, Royan Institute, Tehran, Iran. Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.


INTRODUCTION
Peroxisomes are ubiquitous organelles in almost all eukaryotes that function to rid the cell of toxic substances. Peroxisomes were discovered by pioneer works of Belgian biologist Christian de Duve in 1966(De Duve, 1969. They perform many biochemical functions of lipid *Corresponding author. E-mail: kamranghaedi@yahoo.com. Tel: +98-311-2612900. Fax: +98-311-2605525. metabolism, including the -oxidation of very long chain fatty acids, biosynthesis of plasmalogens and structural ether lipids abundant in the central nervous system, interconversion of cholesterol to bile acids, and glyoxylate transamination (Shimizu et al., 1999;Furuki et al., 2006). Mature peroxisomes are spherical, with diameters between 0.5 and 1.0 micrometer. Each peroxisome is delimited by a single membrane and contains a fine granular matrix (Latruffe et al., 2000).
Peroxisomes are highly responsive organelles, because their size, number, protein composition and biochemical functions vary dramatically depending on the organism, cell type, and environmental milieu. Peroxisomes are essential for normal human development and physiology, as demonstrated by the lethality of the peroxisome biogenesis disorders (PBD) (Wanders, 2004), a group of autosomal recessive diseases including Zellweger syndrome, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy, in which multiple peroxisomal metabolic pathways are dysfunctional because peroxisome biogenesis is compromised (Lazarow and Fujiki, 1985;van den Bosch et al., 1992;Subramani, 1998;Purdue and Lazarow, 2001). Up to now, 35 different peroxins (proteins involved in peroxisome biogenesis) have been identified, of which only 18 are present in human. Most peroxins are peroxisomal membrane proteins or interact through docking sites with the peroxisomal membrane. A complex peroxin interaction network controls biogenesis and division (Pex11p,23p,25p,27p,28p,29p,30 p,31p,and 32p) and allows for the recognition of peroxisome target proteins through specific receptors (Pex5L p, 5Sp, 7p, 18p, 20p, and 21p), for membrane protein assembly (Pex3p, 15p, 16p, Pex19p, and 24p), for the docking of these receptors (Pex13p, 14p, and 17p), for receptor recycling and protein import (Pex1p,4p,6p,8p,9p,22p,and 26p), and for the translocation of proteins to peroxisomal matrix (Pex2p, 10p, and 12p) (Subramani et al., 2000;Lazarow, 2003;Titorenko and Rachubinski, 2004). With respect to biogenesis of peroxisomes and as peroxisomes lack DNA, all peroxisomal proteins are synthesized on cytoplasmic free polysomes and are post-translationally transported to pre-existing peroxisomes (Lazarow and Fujiki, 1985). Two types of peroxisome-targeting signal (PTS) for the matrix proteins are identified: the C-terminal tripeptide-SKL and its conserved variants (PTS1) for most proteins (Baker et al., 2000;Baker and Sparkes, 2005;Gould et al., 1989;Miura et al., 1992) and PTS2, the N-terminal cleavable nonapeptide, -(R/K)(L/V/I)X 5 (H/Q)(L/A)-, present in several proteins such as 3-ketoacyl-CoA thiolase (thiolase) of fatty acidoxidation pathway (Osumi et al., 1991;Singha et al., 2004;Swinkels et al., 1991). Membrane proteins are sorted to peroxisomes by targeting signals distinct from PTS1 or PTS2. Various peroxins have been shown to act as receptors for the different PTSs or as docking sites for these receptors (Roger et al., 2002;Heiland and Erdmann, 2005). In contrast to protein sorting to peroxisomes, much less is known about the mechanism of peroxisome proliferation and the proteins involved in this process. A few proteins (such as Per8p, Pas4p, and Pmp27) have been implicated directly in regulating this process (Tam et al., 2003;Tan et al., 1995;Crane et al., 1994;Marshall et al., 1995).
One of the peroxisomal matrix proteins, termed Peroxisomal Protein (PEP), has been cloned in mouse in 2002. PEP cDNA encodes the protein which its primary Jahantigh et al. 6041 structure is formed by 209 amino acids, with the Cterminal tail (SKI) closely resembling SKL, the consensus sequence for PTS1 (Ferrer-Martinez et al., 2002). Analysis of PEP sequence demonstrates that the only region of PEP similar to a protein with a known function is an FnIII module that includes residues 31 to 114 (Ferrer-Martinez et al., 2002).
All FnIII motifs share three highly conserved residues, which correspond to Trp51, Leu92, and Tyr98 of the PEP amino acid sequence, and the same secondary structure of seven antiparallel-strands (Craig et al., 2004;Li et al., 2004).
Studies have shown that PEP expression in mouse embryo is different in various tissues, while its reason is unclear. Therefore we were interested to construct expression vectors carrying PEP-cDNA with GST and FLAG labels for further analyses. In the present study, PEP-cDNA was inserted downstream of GST and FLAG genes in pGEX6p2 prokaryotic expression vector ( Figure  5A) and pUCD3 eukaryotic expression vector ( Figure  5.B) to used tagged-PEP proteins for further analyses.

Construction of pGEX6p2-PEP
The coding region of PEP (PEP-cDNA) was inserted into the pGex6p2 vector (Pharmacia Biotech) in order to constructing the GST-PEP fusion protein prokaryotic expression vector, pGex6p2-PEP ( Figure 1). PCR experiments were performed in an Eppendorf Mastercycler gradient thermal cycler that is described in details. Primers used during this study were ordered from Bioneer (Korea) and presented in the Table 1.
The aim of this step is amplification of PEP-cDNA using pEGFP-PEP as a template, with primers introducing BamHI and SalI restriction sites at both of 5 and 3 ends, respectively. 25 l of PCR reactions containing 50 ng template DNA, 5 pmol (100 nM) each of primers, 0.25 µl Pfu DNA polymerase (Fermentas), 0.5 l dNTPs at 10 mM (Fermentas), 2.5 l 10X buffer of Pfu (200 mM Tris-HCl with pH 8.8 at 25°C, 100 mM (NH4)2SO4, 100 mM KCl, 1% Triton X-100, 1 mg/ml BSA and 20 mM MgSO4) was applied for a PCR by the following conditions: 4 min of denaturation at 94°C followed by 35 cycles of amplification (94°C 1min; 65°C 1min; and 72°C 2 min.), and ended to 10 min at 72°C. The amplified product of this step with the length of 647 bp (PEP) was purified by QIAprep Spin Miniprep kit (Qiagen). Purified product was used for digestion with restriction enzymes. Both of pGex6p2 vector and amplified PEP-cDNA were cut with BamHI (TaKaRa)/SalI (TaKaRa) restriction enzymes and were ligated by TaKaRa ligation kit (TaKaRa). Transformation was done immediately using one shot TOP10 competent bacterial E.coli cells (Invitrogen). Insert check analysis on grown colonies was done the next day. Plasmid preparation from bacterial colonies was done by QIAgen plasmid miniprep kit (Qiagen). To confirm insertion of PEP, two strategies were applied: First, PCR on constructed vector with primers which used for PEP amplification. The second one was sequencing. Samples were sent for sequencing through Takapozist Company.

Construction of pUcD3-FLAG-PEP
The coding region of PEP (PEP-cDNA) was tagged with FLAG    Mastercycler gradient thermal cycler that its condition is described in details. Primers used during this study were ordered from Bioneer (Korea) and presented in the Tables 2 and 3.
Step 1: The aim of the first step of PCR is production of PEP and FLAG fragments. PEP-cDNA was amplified using pEGFP-PEP as template, with primers introducing BamHI and ApaI restriction sites at 5 and 3 ends, respectively (Table 2). FLAG gene was also amplified using pUcD3-FLAG-PEX3 (was constructed by Ghaedi et al., 2000 in Kyushu University) as template, with forward primer introducing NotI restriction site in its 5 end and reverse primer containing BamHI restriction site and 9 sequences from 5 end of PEP-cDNA at its 5 site, that it was designed for next step (Table 3 and Figure 2A). 25 l of PCR reactions containing 50 ng template DNA, 5 pmol (100 nM) each of primers, 0.5 µl Pfu DNA polymerase, 0.5 l dNTPs at 10 mM, 2.5 l 10X buffer of Pfu (200 mM Tris-HCl with pH 8.8 at 25°C, 100 mM (NH4) 2SO4, 100 mM KCl, 1% Triton X-100, 1 mg/ml BSA and 20 mM MgSO4) was applied for a PCR by the below conditions: 5 min of denaturation at 94°C followed by 35 cycles of amplification (94°C 1 min; 65°C 1 min and 72°C 2 min), and ended to 10 min at 72°C. The amplified products of this step used as templates with the length of 647bp (PEP) and 77bp (FLAG). The products were purified by QIAprep Spin Miniprep kit (Qiagen) The amplified product was used for the step 2.
Step 2: In second step of PCR, FLAG and PEP fragments were used as templates and FLAG-PEP was amplified using FLAG forward primer and PEP reverse primer that 700bp expected product were generated ( Figure 2B). 25 l PCR reactions contained 3 µl template DNA (FLAG 2.5 µl and PEP 0.5 µl fragments), 5 pmol (100 nM) each of primers 0.5 µl Pfu DNA polymerase, 0.5 l dNTPs at 10 mM, 2.5 ul 10X buffer of Pfu. Conditions of PCR reaction was the same as aforementioned. The product of this step was FLAG-PEP chimeric DNA. Then, both of pUcD3 vector and amplified FLAG-PEP were cut with NotI (TaKaRa) /ApaI (TaKaRa) restriction enzymes and were ligated by TaKaRa ligation kit. Transformation was done immediately using JM105 competent bacterial E. coli cells (Fermentas). Insert check analysis on grown colonies was done next day. Plasmid preparation from bacterial colonies was done by QIAgen plasmid miniprep kit (Qiagen). To confirm insertion of PEP, two aforementioned strategies were applied again

Construction of pGEX6p2-PEP
PCR on pEGFP-PEP as template generated a 647 bp band (PEP), which showed amplification has been carried out correctly and designed restriction sites have been added at the ends of PEP fragment ( Figure 3A). Insert check analysis on colonies which had grown one day after transformation with ligated products, showed that PEP-cDNA has been inserted into the pGEX6p2 vector ( Figure 3B). Plasmid preparation was performed and constructed vectors were extracted from bacterial Figure 2B. Second step of PCR reaction for construction of FLAG-PEP chimeric DNA by using FLAG and PEP fragments as templates. Chimer fragment was cut and was inserted into the eukaryotic expression vector pUcD3.
colonies. To further confirm insertion of PEP into the vector, PCR were applied on constructed vector, with primers which had been used for PEP amplification. This PCR generated a 647bp band as expected for PEP fragment (data not shown). Finally sequencing confirmed that PEP was inserted without any mutation downstream of GST into the vector appropriately.

Construction of pUcD3-FLAG-PEP
PCR on pEGFP-PEP and pUcD3-FLAG-PEX3 as template in separate reaction generated a 647 bp band (PEP) and a 77 bp band (FLAG), respectively, which showed amplification, has been carried out correctly and designed restriction sites have been added at the ends of PEP and FLAG fragments ( Figure 4A, B). Second step of PCR using PEP-cDNA and FLAG tandem as templates generated a 700 bp band that demonstrated amplification of FLAG-PEP fragment ( Figure 4C). Insert check analysis on colonies which grew one day after transformation with ligated products, showed that FLAG-PEP has been inserted into the pUcD3 vector ( Figure 4D). Then by Plasmid preparation, constructed vectors were extracted from bacterial colonies. To further confirm insertion of FLAG-PEP into the vector, PCR were applied on constructed vector, with primers which had been used for FLAG-PEP amplification. This PCR generated a 700 bp band as expected for FLAG-PEP fragment (data not shown). Finally sequencing confirmed that FLAG-PEP was inserted into the vector appropriately and it was also free from mutation.

DISCUSSION
We have sub-cloned PEP-cDNA in prokaryotic and eukaryotic expression vectors to tag it with GST and FLAG  tandems. Previous studies have indicated that PEP protein is a peroxisomal protein (Ferrer-Martinez et al., 2002). The primary structure of PEP protein comprised 209 amino acids, containing the C-terminal tail (SKI) closely resembling SKL, the consensus sequence for PTS1, one of the two peroxisomal targeting signals described to date. PTS1 has been found in approximately half of the peroxisomal proteins, and it directs peroxisomal proteins import from the cytosol to the organelle matrix (Subramani, 1998;Sacksteder and Gould, 2000). PTS1 was first identified in firefly luciferase and a few other proteins with the C-terminal tail sequence S/A/C-K/R/ H-L/M (Sacksteder and Gould, 2000). PEP-cDNA was cloned in pEGFP-C1 vector in our laboratory and transient transfection of CHO cells with pEGFP-C1-PEP clearly demonstrated punctuated pattern as expected for peroxisomes (Tanhaei et al., 2008). The only region of PEP similar to a protein with a known function is an FnIII module that includes residues 31 to 114. In order to see the possible role of this domain in molecular structure of PEP, proteomic analysis of PEP using Flag-PEP in mammalian cell lysates and purification of PeP protein for biochemical analysis and identifying related proteins seems to be necessary. Thus our constructions can be used for the further analyses of the function of this protein.

ACKNOWLEDGEMENT
This study was supported by a grant of Royan Institute to K.G.