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Human signal peptide had advantage over mouse in secretory expression
时间：2010-08-24 09:11:27 来源： 作者：
Introduction The expression of protein can be classified into secretory expression and constitutive expression. It is well known that signal peptide is a very important component and a critical factor in the secretory protein expression (von Heijne 1990). Signal peptides have been applied in gene engineering and medical&pharmaceutical industries. The procedure of protein secretion is very complicated. Firstly, cytoplasmic signal peptide recognition particle (SRP) recognizes the signal peptide during the secretory expression of protein (Keenan et al 2001; Gundelfinger et al 1983; Siegel and Walter 1988). Then, the signal peptide–SRP complex is anchored to the endoplasmic reticulum (ER) membrane. After that, the signal peptide is subsequently transferred from SRP to the integral membrane glycoprotein which is a signal sequence receptor (SR) on ER membrane close to the translocon, the first gate to the secretory pathway (Miller et al 1995; Schwartz and Blobel 2003). After being transferred to the translocon, the precursor protein will be translocated with the help of translocon cotranslationally or post-translationally (de Keyzer et al 2003; Rapoport et al 1996; Walter and Johnson 1994). The signal peptide will then be cleaved off from the pro-protein by certain signal peptidase during the co-translational translocation (Paetzel et al 2002) to form mature proteins, which are secretory and released into ER lumen (Lemberg and Martoglio et al 2004). Signal peptide has a classical structure with positively-charged amino acid residues at the N-terminal (n-region, 1-5 aa), a hydrophobic core in the middle (hregion,7-15 aa) and a more polar region with non-polar small amino acid residues at positions 1 and 3 at the C-terminal (c-region, 3-7 aa) (Tuteja 2005; Heinrich et al 2000; Lee et al 2000; Plath et al 1998). Although there are many research articles about nerve growth factor, articles about the signal sequence in its expression gene is scarce by now. The signal peptide from mouse nerve growth factor has been testified to mediate the secretory expression of beta-endorphin in vitro and in vivo (Beutler et al 1995; Finegold et al 1999). A counterpart of the signal sequence of mouse nerve growth factor exists in human genome. But no researches about the counterpart from human have been reported, and there was no chance to compare the efficiency of these two parallel signal peptides. In the present study we firstly got the signal sequence of human nerve growth factor from human genome DNA, and then made a fusion gene by ligating human signal sequence and human beta-endorphin sequence with a furin recognition sequence between them. Finally, we found that the signal sequence of human nerve growth factor was able tomediate the secretory expression of beta-endorphin in NIH3T3 cells as its counterpart from mouse does and the efficiency of human signal peptide was higher than that of mouse signal peptide. MATERIALS AND METHODS Strain propagation and cell culture Escherichia coli strain DH5-α (Gibco-BRL, UK) was transformed with recombinant plasmid vecotors in LB culture media. NIH3T3 cells, obtained from the American Type Culture Collection (Manassas, VA, USA), were transfected with recombinant eukaryotic expression vectors and maintained as subconfluent stocks in Dulbecco’s Modified Essential Medium (DMEM, Gibco-BRL, UK) containing 10% fetal bovine serum (PAA, Germany). PCR template preparation The whole blood was taken from the peripheral vein of a volunteer into a tube containing EDTA. Transfer 300-μl aliquots of the whole blood into a microcentrifuge tube, and then add 900 μl of 20 mM Tris-Cl (pH 7.6) to the tube. Mix the content by inverting the tube and incubate the solution at room temperature for 10 min. Centrifuge the tube at maximum speed for 20 seconds at room temperature. Discard all but 20 μl of those supernatant. Resuspend the pellet of white cells in the small amount of supernatant left in tube. Add 600 μl of ice-cold cell lysis buffer and homogenize the suspension. Add 3 μl of proteinase K solution to the lysate to increase the yield of genomic DNA. Incubate the digest for at least 3 hours at 55℃. Cool the digest to room temperature and then add 3 μl of 4 mg/ml DNase-free RNase. Incubate the digest for 60 minutes at 37℃. Allow the sample to cool to room temperature. Add 200 μl of potassium acetate solution and mix the content of the tube by vortexing vigorously for 20 seconds. Centrifuge the sample at maximum speed for 3 minutes at 4℃. Transfer the supernatant to a new microcentrifuge tube containing 600 μl of isopropanol. Mix the solution well and then recover the precipitate of DNA by centrifuging the tube at maximum speed for 1 minute at room temperature. Remove the supernatant by aspiration and add 600 μl of 70% ethanol to the DNA pellet. Invert the tube several times and centrifuge the tube at maximum speed for 1 minute at room temperature in a microcentrifuge. Remove the supernatant by aspiration and dry the DNA pellet in air. Redissolve the pellet of DNA in 100 μl of TE (pH 7.6). Vectors production To get the signal sequence of human nerve growth factor, two primers were synthesized: forward primer 5’-CCG AAG CTT TTC CAG GTG CAT AGC GTA ACC ATG TCC ATG TTG TTC TAC AC-3’ and reverse primer 5’-CGC TTG CTC TTG TGA GTC CTG TTG A-3’. Two microliters of the genome DNA was used as a template for 50-μl PCR reaction which was catalyzed by Primer-STAR polymerase (TAKARA). The cycling parameters consisted of one cycle of 95℃ for 3 min, followed by 30 cycles of 95℃ for 0.5 min, 58℃ for 0.5 min, 72℃ for 0.5 min, followed by a single 5-min cycle at 72℃ for extension. Electrophoresed products of 392 bp were excised and purified from the gel using the QIAquick gel extraction kit (Qiagen). The DNA sequence of human beta-endorphin was obtain from a plasmid pTCNE (previous work) with the following two primers: forward primer 5’-TCA ACA GGA CTC ACA AGA GCA AGC G-3’ and reverse primer 5’-CGC GAA TTC ATT ACT CGC CCT TCT TGT AGG C-3’ by another PCR reaction, whose cycling parameters is the same as the previous one and was done as the previous with 0.5 microliters of pTCNE as a template. Electrophoresed products of 132 bp were excised and purified as before. The mixture of the product of above two PCR reactions was treated as the substrate for a third PCR reaction, which consisted of one cycle of 95℃ for 3 min and subsequent 30 cycles of 95℃ for 0.5 min, 58℃ for 0.5 min, 72℃ for 1 min followed by a single 5-min cycle at 72℃ for extension. The excised and purified PCR products of 499 bp were subcloned into the pcDNA-3.1(+) vector (Invitrogen) by HindIII and EcoRI to generate pcDNA-3.1-hEP. The other vector pcDNA-3.1-mEP was constructed by subcloning products of another PCR reaction, which had pTCNE vector as the template and included one cycle of 95℃ for 3 min and then 30 cycles of 95℃ for 0.5 min, 58℃ for 0.5 min, 72℃ for 1 min followed by a single 5-min cycle at 72℃ for extension, into pcDNA-3.1(+) with HindIII and EcoRI. Positive clones were screened out by PCR reaction from those bacteria clones. PCR products in pcDNA-3.1-hEP and pcDNA-3.1-mEP were verified by automated DNA sequencing with T7 SequencingTM Kit (Pharmacia Boitech, Sweden) (Sambrook et al 1989). Three vectors, pcDNA-3.1-hEP, pcDNA-3.1-mEP and pcDNA-3.1(+), were extracted and purified by Qiagen Plasmid Mini Kit (Qiagen), and DNA concentration was assessed by spectrophotometry at 280 nm. Cell and transfection NIH3T3 (ATCC) cells were cultured in high glucose DMEM containing 10%FBS at 37℃ with 5% CO2 in six-well plates (Corning). The cultured NIH3T3 cells were transfected with pcDNA-3.1-hEP, pcDNA-3.1-mEP and pcDNA-3.1(+) ( the latter one as the blank control ) respectively using Lipofectamine 2000 reagent (Invitrogen) according to the product manual. One day before transfection, plate 2×105 cells per well in 500 μl of growth medium without antibiotics. Dilute plasmids DNA in 250 μl of Opti-MEM I Reduced Serum Medium (Invitrogen) without serum. Dilute 10 μl of Lipofectamine 2000 reagent in 250 μl of Opti-MEM I Medium. Incubate for 5 minutes at room temperature, and then combine them. Mix gently and incubate for 20 minutes at room temperature. Add 500 μl of the complexes to each well. Mix gently by rocking the plate back and forth for several times. Incubate cells at 37℃ in a CO2 incubator for 48 hours. The transfection rates were evaluated in terms of the percent of those transfected cells to the total cells. Reverse transcription-PCR The NIH3T3 cells transfected with three vectors were collected 48 hours after transfection respectively. Total cellular RNA was isolated from each sample using Trizol solution (Invitrogen) according to the manufacturer’s instruction. The RNA concentration was assessed by spectrophotometry at 260 nm and RNA quality was confirmed by running 5μg of total RNA from each preparation on a 1.2% denatured agarose gel. The first-strand cDNAs were synthesized using Superscript reverse primer were synthesized as follows: 5’-CCG AAG CTT TTC CAG GTG CAT AGC GTA ACC ATG TCC ATG TTG TTC TAC AC-3’ and 5’-CGC GAA TTC ATT ACT CGC CCT TCT TGT AGG C-3’. The PCR reaction was performed in a Mastercycler Gradient thermocycler (Eppendorf Scientific). Two microliters of the first-strand product was used as a template in each 50-μl PCR reaction, whose cycling parameters consisted of one cycle of 95℃ for 3 min and then 30 cycles of 95℃ for 0.5 min, 58℃ for 0.5 min, 72℃ for 1 min followed by a single 5-min cycle at 72℃ for extension. RT-PCR products were electrophoresed on a 1% agarose gel with DNA Marker DL2000 (TAKARA) as a standard to determine the molecular size. Cytological immune fluorescent Vectors-transfected NIH3T3 cells were grown to form a polarized monolayer on 24 mm coverslips in 6-well plates (Corning). Forty-eight hours later, the coverslips with cell growing were rinsed with PBS for three times (5 minutes each time), fixed by four percent formaldehyde for 15 minutes, and rinsed with PBS again as before. The fixed cells were stained by incubation with the primary antibodies (rabbit polyclonal anti-beta-endorphin antibody (ABcam, ab43825) diluted in 1:200 in PBS with 5% BSA blocking buffer). The coverslips were rinsed for three times (5 minutes each time) in PBS after incubation in blocking buffer, and then exposed to the Cy3-labled secondary antibodies (Cy3-labeled goat anti-rabbit IgG, 1:1000, Sigma) for 1h at room temperature in dark. Cells were stained with Hoechst 33342 (1:1000, Sigma) for 1 minute after rinsed again as before. The rinsed coverslips with PBS were mounted with a drop of 50% of glycerin onto slides and imaged using an Olympus 10×20 IX70 Inverted Fluorescence Microscope. Radio Immuno Assay The culture containing secreted protein was collected 48 hours after the NIH3T3 cells were transfected with those two expression vectors and the control vector, and was centrifuged for 5 min at 5000 × g. The supernatant was transferred into new tubes as the to-be-detected samples. The aprotinin was added to all samples 200 U per milliliter. The concentration of beta-endorphin was measured using a RIA kit (Department of neurobiology, Second Military Medical University, China). The following detection procedure was strictly done according to the manufacturer’s instruction. Pipette 100 μL of Standards, 300 μL of Control and samples into polystyrene tubes respectively. Pipette 400 μL and 300 μL of Assay Buffer into the NSB tubes and B0 tubes respectively. Add 200 μL of Assay Buffer into the Standards’ tubes. Pipette 100 μL of Antiserum into each tube (Except T and NSB). Vortex all tubes. Incubate all tubes for 24 hours at 4°℃. Pipette 100 μL of prepared 125I-Tracer into each tube, including two tubes for Total Activity (T). Vortex all tubes and incubate them for 24 hours at 4℃. Pipette 500 μL of prepared cool Separation Reagent into each tube and vortex. Keep all tubes for 45 min at room temperature. Centrifuge for 20 min at 4000×g. Decant the tubes carefully and count the CPM of all tubes in a Gamma counter for 1 min. The concentration of protein in culture media was calculated according to the parameters read in standard curve, which was drawn on a semilogarithmic paper according to the outcome of standards. RESULTS Construction of Eukaryotic vectors expressing human beta-endorphin We constructed two vectors, pcDNA3.1-hEP and pcDNA3.1-mEP, by inserting the DNA fragments for human beta-endorphin into the eukaryotic expression vector pcDNA3.1(+). The vector, pcDNA3.1-hEP, had a signal peptide of human nerve growth factor, and the other vector, pcDNA3.1-mEP, had a signal peptide of mouse nerve growth factor. The details of construction procedure are given in Materials and Methods. The final form of these plasmids is shown in Fig1. Both two fusion genes were driven by the CMV (cytomegalovirus) promoter. Secretory protein must be mediated by some kind of signal peptide so as to be transported outside of cells, which named secretory expression. Since the receptor for beta-endorphin is on the cell membrane, so if not transported outsides, beta-endorphin cannot function. To implement the secretory expression of human beta-endorphin in these two vectors, we fused DNA sequence of the signal peptide of nerve growth factor from human and mouse, respectively, with human beta-endorphin together. Moreover, to make beta-endorphin smoothly isolating from the precursor including that signal peptide and beta-endorphin, we put a DNA fragment of four amino acids, recognized by proteolytic enzyme furin (Denault and Leduc 1996), between the signal peptide and beta-endorphin sequence as Finegold A.A. did (Finegold et al 1999). Fig1 Schematic representation of the construction of two eukaryotic expression vectors (A) pcDNA3.1-hEP The signal peptide of human nerve growth factor (human NGF-SP) was contained between a promoter and a fragment of polyA. (B) pcDNA3.1-mEP The signal peptide of mouse nerve growth factor (mouse NGF-SP) was contained. Furin: the recognization sequence of furin hydrolytic enzyme beta-EP: human beta-endorphin sequence  Fig2 Expression of beta-endorphin in cultured NIH3T3 cells (A) Detection of mRNA from fusion genes expressing beta-endorphin by RT-PCR M: DNA Marker DL2000 from TARKARA, Lane1: mRNA from pcDNA3.1-hEP, Lane2: mRNA from pcDNA3.1-mEP, Lane3: mRNA from pcDNA3.1(+), Lane4: positive control, Lane5: negative control (B) Immunofluorescence of beta-endorphin Intracellular localization of beta-endorphin was detected by immunocytology in NIH3T3. Nuclei were counter-stained with Hoechst33342. B1: pcDNA3.1-hEP, B2: pcDNA3.1-mEP, B3: pcDNA3.1(+) (both cells transfected with empty vector and control cell showed the same outcome). One representative staining out of several independent experiments is shown. Magnification, ×200.  DISCUSSION Here we determined the function of the signal sequence of human nerve growth factor to mediate the secretory expression of protein, beta-endorphin. The signal peptide plays a very important role in the secretory expression of protein. It has been reported that properties of residues at the h/c boundary and +1 position of mature protein can influence the translocation and cleavage of signal peptide (Nothwehr et al 1990; Barkocy-Gallagher and Bassford 1992). Besides that, the signal peptide also can affect the regulation of gene expression (Serruto and Galeotti 2004), and it has an effect on the secretion of protein production (Holden et al 2005; Knappskog et al 2007). After that, we compared the efficiency of two signal sequence from human and mouse nerve growth factor. In eukaryotic cells, secretory and membrane proteins contain a signal sequence essential for protein anchoring to the endoplasmic reticulum (ER), which is the necessary entry into the secretory pathway to the exocytosis of expressed protein. The expressed protein, beta-endorphin, was a kind of “designed” secretory protein and was just a “reporter” protein to indirectly check the function of the signal peptides. Moreover, we will exploit the secretory protein beta-endorphin in other relevant works, which was the second reason to adopt this protein as the checking target. Signal peptide-bearing precursor proteins are initiated into the secretory pathway (Blobel 1980). After insertion into the protein-conduction channel, signal peptides are usually cleaved off from the pre-protein by signal peptidase (Dalbey et al 1997). Although all signal peptide-bearing proteins are by default translocated across the endoplasmic reticulum membrane, the subsequent fate of precursors can be modified by the presence or absence of specific cleavage sites for signal peptidases (Chang et al 1978). Type I signal peptides comprise a cleavage site for signal (leader) peptidase, and the mature polypeptides are released from the membrane (Dalbey and W. Wickner 1985). Type II signal peptides are the substrate for covalent modification with thioether-linked diacylglycerol (Hantke and V. Braun 1973). Our selected signal peptides belong to Type I. It is not only that the level of synthesis and secretion of proteins in cultured mammalian cells is heavily dependent on the choice of signal peptide (Knappskog et al 2007), but also that the optimization of signal peptides can increase the production of heterologous proteins (Ravn et al 2003). Apparently, our expressed beta-endorphin would be mature only if the signal peptide was cleaved off from the “whole” protein, the signal peptide plus beta-endorphin. And the level of beta-endorphin in cell culture medium was highly associated with the efficiency of the adopted signal peptides. It has been reported that the signal peptide of mouse nerve growth factor could mediate the secretory expression of beta-endorphin in vitro and in vivo when the signal sequence of mouse nerve growth factor was ligated in front of beta-endorphin sequence (Beutler et al 1995; Finegold et al 1999). We previously constructed a fusion gene, contained in pTCNE vector, and found that the fusion gene was expressed smoothly in NIH3T3 cells and that the expressed protein was active and had therapeutic function (unpublished data). We noticed that in human genome there exists a counterpart of the signal peptide of mouse nerve growth factor. But, no report was showed about the counterpart in human genome. The function of signal sequence of human nerve growth factor was tested in this report and the efficiency of the signal peptides in mediating the secretory expression of beta-endorphin was compared with that of mouse signal peptide. In the present study, we constructed these two eukaryotic expression vectors, pcDNA3.1-hEP and pcDNA3.1-mEP, to determine whether the signal sequence of human nerve growth factor can function as the signal sequence of mouse nerve growth factor does and to figure out that which sequence has a better efficiency in mediating the secretory expression of protein. The vector, pcDNA3.1-hEP, had a signal sequence from human nerve growth factor and the other vector, pcDNA3.1-mEP, contained a signal sequence from mouse. After comparing these two adopted DNA sequences and their translated amino acids sequences, we found that the similarity of two DNA sequences is up to 83% [Fig4A] and the similarity of two protein sequences reaches about 80% [Fig4B]. In conclusion, this work documents the expression of beta-endorphin polypeptide in cultured mouse primary fibroblast NIH3T3 cells by immunofluorescent. These peptides appear to be localized mainly in cytoplasm, not in cell nucleus (Fig2B). 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