Effects of signal peptide on secretory expression of SARS-CoV-2 S1,RBD and RBD dimer proteins in Expisf9 insect cells / 中国生物制品学杂志

@#Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.

Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host / 生物工程学报

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.

Effects of different signal peptides on the secretion of human-mouse chimeric CMV-IgM / 生物工程学报

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.

Optimization of enterokinase secretion in Pichia pastoris / 生物工程学报

Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences. Therefore, enterokinase has been widely used as a tool enzyme in the field of biomedicine. Currently, the expression level of enterokinase in Pichia pastoris is low, which hinders related practical applications. In this study, the effects of six different signal peptides SP1, SP2, SP3, SP4, SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied. Compared with α-factor, SP1 significantly increased the secretory expression of enterokinase (from 6.8 mg/L to 14.3 mg/L), and the enterokinase activity increased from (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±489) U/mL by co-expressing the endogenous protein Kex2. Moreover, the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Efficacy of difunctional short peptide-conjugated chitosan mediated miR-140 gene transfection in repairing articular cartilage defect of rabbits / 中华创伤杂志

Objective To investigate the effect of nucleus localization signal linked nucleic kinase substrate short peptide-conjugated chitosan (NNSCS)-mediated human miR-140 gene local transfection on the repair of articular cartilage defect in rabbits.Methods Eukaryotic expression plasmid GV268-miR-140 was constructed,and then negative controls GV268 and GV268-miR-140 were respectively combined with NNSCS to form NNSCS/GV268 and NNSCS/GV268-miR-140 complexes.Eighteen healthy male New Zealand white rabbits were randomly divided into transgenic group (Group A),negative control group (Group B),and sham operation group (Group C),with 6 rabbits per group.Both Groups A and B were prepared for the total cartilage damage model of femur trochlear,and Group C only exposed the articular surface of the femur trochlear.One week after operation,Group A was treated with NNS CS/GV268-miR-140 complex,Group B was given NNS CS/GV268 complex,and Group C was given equal amount of isotonic saline,twice a week for 7 weeks.The experimental animals were sacrificed at the end of the eighth week after operation.Real time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-140,Sox9,Aggrecan and Hdac4 in the defect area.HE staining,safranine O/fast green staining,and Aggrecan immunohistochemical staining were used to evaluate cartilage repair in the defect area.Results RT-qPCR showed the expression of miR-140 in Group A (3.16 ± 0.37) was significantly higher than that in Group B (1 ± 0.24) and in Group C (1.24 ± 0.18) (P ＜ 0.05).The miR-140 expression in Group A obviously up-regulated the expression of SOx9 gene (4.38 ± 0.66) compared with Group B (1.04 ± 0.04) and Group C (1.19 ± 0.3),(P ＜ 0.05).The miR-140 expression in Group A obviously up-regulated the expression of Aggrecan gene (3.63 ± 0.58) (P ＜0.05) compared with Group B (1.21 ± 0.14) and Group C (1.34 ± 0.13).The miR-140 expression in Group A obviously down-regulated the expression of Hdac4 (0.37 ±0.06) compared with Group B (0.81 ± 0.06) (P ＜ 0.05).According to results of HE staining,safranine O/fast green and Aggrecan,cartilage repair was evident in Group A,while fibrous tissue proliferation and inflammatory cell infiltration were seen in the defect region in Group B,showing no cartilage repair.Conclusions NNS CS can carry exogenous genes into chondrocytes and the genes can abundantly express locally.High expression of miR-140 might significantly improve the repair of articular cartilage defect in vivoby up-regulating expressions of Aggrecan and Sox9 as well as down-regulating Hdac4 expression.

Comparison of Signal Peptides for Two Hemicellulase Secretory Expression / 中国生物工程杂志

Xylanase and mannanase are two hemicellulases,which are widely used in many fields.To improve the expression of thermostable xylanase DSB and thermostable mannanase ManA in Pichia pastoris,three endogenous signal peptides of Pichia pastoris(Scw1 1,Dse4 and Exg1) were chosed.Their capability to mediate the secretion of DSB and ManA with that of the Saccharomyces cerevisiae α-factor were compared.In shake-flask cultivation,three endogenous signal peptides and α-factor efficiently mediated the secretion of DSB and ManA,but the secretion efficiency has obvious difference.As for DSB,the expression efficiency of α-factor was much higher than three endogenous signal peptides.But as for ManA,the expression efficiency of Dse4 was equal to α-factor and much higher than Scw11 and Exg1.Therefore,α-factoris the most efficient signal peptide for DSB expression and Dse4 or α-factor are the most efficient signal peptide for ManA expression in Pichia pastoris X33.Moreover,the intracellular activities of DSB and ManA by α-factor are higher than Scw1 1,Dse4 and Exg1,and the intracellular activity of ManA was higher than DSB (the molecular weight of ManA was larger than DSB).Thus,when ManA were expressed in Pichia pastoris,different signal peptide,such as Dse4,could be used for improving the secretion efficiency.A basis for identifying more available signal peptides and screening for the optimal signal peptide for the target protein was provided.

Transcriptome analyses of insect cells to facilitate baculovirus-insect expression

The High Five cell line (BTI-TN-5B1-4) isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.

Expression of recombinant h-FGF21 in periplasmic space of Escherichia coli / 药学学报

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator of glucose and lipid, which is safe, effective and independent on insulin. FGF21 is considered as a prospective anti-diabetic drug. The aim of this study was to express recombinant h-FGF21 in periplasmic space of Escherichia coli. The pET27b plasmid was used to create the expression vectors of h-FGF21 with a PelB secretion signal. The ph-FGF21 (periplasmic expression of h-FGF21) was successfully expressed in the periplasm of E. coli BL21 (DE3), and soluble ph-FGF21 was isolated by disruption of the outer membrane. After twice of ion exchange chromatography, the purity of ph-FGF21 was above 95% in an analysis with a gray analysis software. The molecular weight of ph-FGF21 was about 20 kDa in SDS-PAGE and Western blotting analysis. The activity of ph-FGF21 and ih-FGF21 (intracellular expression of h-FGF21) was observed in vitro in the glucose uptake assay in HepG2 cells. The activity was observed in type 2 diabetic db/db mice after short or long-term treatments. The results suggest that the ph-FGF21 has a consistent activity with ih-FGF21 in vitro and in vivo.

Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of Helicobacter pylori urease subunit B in silkworm cells and larvae

This study employed a Bac-to-Bac/Bombyx mori bioreactor to mass-produce immunogenic urease subunit B (UreB) from Helicobacter pylori. The signal peptide bombyxin from B. mori was used to promote secretory expression to improve expression levels and was designed and integrated into the UreB gene to generate the Bacmid/BmNPV/(signal peptide)-UreB baculovirus expression system. To determine whether the bombyxin signal peptide resulted in secretory expression of recombinant UreB (rUreB) and to determine the secretory efficiency, we tested the secretory expression level of rUreB in Bm5 cells using ELISA. To further investigate whether secretory expression affected cell viability, cells were evaluated using 0.4% trypan blue staining, and Bacmid/BmNPV/UreB without the signal peptide served as a control. The above recombinant bacmid constructs were injected to silkworm larvae, and the secretory expression level of rUreB was detected using SDS-PAGE and semi-quantitative western blot analysis. The results indicated that the bombyxin signal peptide directed the secretory expression of rUreB and that this expression improved the viability of Bm5 cells. Moreover, the results showed that the expression level of rUreB was 1.5 times higher with the Bacmid/BmNPV constructs containing the bombyxin signal sequence than those without the signal sequence. These results demonstrate that secretory expression can enhance rUreB expression levels and is likely to aid in the large-scale expression and yield of rUreB in silkworm larvae.

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium.

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/ml) obtained for CGTase activity was very close to the predicted value (9.27 U/ml).

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors / 华中科技大学学报（医学）（英德文版）

Many researchers employed mammalian expression system to artificially express cannabinoid receptors,but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports.In present study,we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system.This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide.In addition,the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95℃),forming a large molecular weight band when analyzed by immuno-blotting.Only denaturing temperatures ≤75℃ yielded a clear band at the predicted molecular weight.Collectively,we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence,and described the requirement for a low sample denaturing temperature in immuno-blot analysis.These findings provide very useful information for efficient mammalian expression and immuno-blotting of mcmbrane receptors.

Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers

The Calotropis procera seed fibers provide an excellent model system to study the genes involved in fiber elongation, fineness and strength. Expansins constitute one of the important gene families involved in plant cell expansion and other cell wall modification processes. Four homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 were isolated from the cDNA library obtained from fast growing Calotropis procera fibers. These homologs represented typical Expansin A family. Each of them had two conserved domains including GH45 like domain and the putative polysaccharide binding domain. The deduced amino acid sequences of the homologs indicated three conserved motifs: i) eight cysteine residues at N-terminus, ii) four tryptophan residues at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the center of the sequence. The presence of N-terminal signal peptide consisting of hydrophobic amino acids and a transmembrane region in all these expansin isoforms suggests their cotranslational insertion into the endoplasmic reticulum and then transportation to the cell wall by secretory pathway. The relative quantification of the four expansins in root, stem, fiber and leave tissues indicated that the transcripts of CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these tissues. The lowest transcription of all the four Expansin A isoforms was observed in elongating roots indicating that root tissue might be having specific expansins other than those confined to air grown organs.

Signal peptide of human nerve growth factor is better than signal peptide of mouse nerve factor in mediating secretory expression of beta-endorphin / 第二军医大学学报

Objective: To clarify whether the signal peptide of human nerve growth factor can mediate secretory expression of beta-endorphin and whether there is difference between the efficiency of signal peptides from human and mouse nerve growth factor. Methods: Two kinds of eukaryotic vectors containing human or mouse signal sequence-mediated secretory expression of beta-endorphin were constructed. The culture supernatant and cells were collected 48 h after NIH3T3 cells were transfected by the two kinds of vectors, and the cover slips with single-layer cells was prepared. The concentration of beta-endorphin in the culture was determined by radio-immunoassay. The total RNA was extracted from cells and mRNA from fusion genes was assayed by RT-PCR. Cells on cover slips were subjected to immunofluorescence staining. Results: RT-PCR showed that the fusion genes were expressed in NIH3T3 cells; the expression of beta-endorphin was mainly in the cytoplasm of NIH3T3 cells. The concentrations of beta-endorphin in the supernatants 48 h after transfection with pcDNA3. 1-hEP and pcDNA3. 1-mEP were (280.33±24.16) pg/ml and (191.04±7.96) pg/ml (P<0.05), respectively, and they were significantly different from that of the blank control group (P<0.01). Conclusion: The signal sequence of human nerve growth factor can mediate the secretory expression of protein and the efficacy of human signal peptide is higher than that of mouse signal peptide.

Establishment and identification of stable cell line secreting humen IgE Cε-4 protein / 中华微生物学和免疫学杂志

Objective To establish a stable cell line secreting human IgE Cε2-4 protein, and in-vestigate the binding capacity of receptor FcεR Ⅰ Methods The E24 gene was derived from SKO-O07 cell line, and was then cloned into pcDNA3.1 (+) (signal peptides were synthesized and fused at the 5'-end of E24 gene) or pCMV-L vector. After transient transfection into 293T cell, the secreted F24 protein was ana-lyzed by sandwich ELISA. The best vector was chosen to be transfected into CHO cells with LipofectAMI-NETM 2000 reagent. After being selected by G418 and subcloned three times by limited-dilution method, two stable cell lines were established. E24 gene was amplified by RT-PCR, and the E24 protein in the superna-tant was identified by ELISA. Besides, the binding capacity of FceR ⅠⅡ was analyzed by flow cytometry method. Results Three mammalian expression vector SP-E24-F3. 1, SP lI-E24-P3.1 and E24-PL were constructed and transient transfected to 293T cells. The output of E24 protein in the supernatant were 19.1, 19.4 and 8.7 μg/ml, respectively. Then the vector SP IX-E24-P3.1 was transfected into CHO cells. Final-ly, two single clones secreting E24 protein were stably obtained. The output of E24 were all at least 25 μg/ml. RT-PCR could detect the E24 gene from one of the two clones. Furthermore, flow cytometry results showed that E24 could bind the receptor in a dose-dependent manner. Conclusion Two stable cell line se- creting E24 protein were obtained, while E24 could specifically bind FcεR Ⅰ.

Construction and Expression of Bivalent Membrane-anchored DNA Vaccine Encoding Sj14FABP and Sj26GST Genes / 华中科技大学学报（医学）（英德文版）

In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sjl4 or pVAC-Sj26 only to get two gene fragments including Sjl4 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES,resulting in another new recombinant plasmid pIRES-Sj26-Sj 14. The expression of Sj14 and Sj26genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and pIRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.

Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/?-factor for Saccharomyces cerevisiae / 中国生物工程杂志

pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

Efficient Secretion of Recombinant PEX in COS7 Cells Directed by MMP-9 Signal Peptide / 中国生物工程杂志

With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Selection of the exogenous signal peptide that conducts the secretion of hepatopoietin / 中国病理生理杂志

AIM:To look for a suitable signal peptide which may effectively conduct hepatopoietin(HPO)secretion,various recombinant eukaryotic expression vectors were constructed.METHODS:Different exogenous signal sequences were spliced with HPO cDNA by PCR,and the spliced genes were cloned into eukaryotic expression plasmids.The different recombinants were respectively tansfected into COS-7 cells by Lipofectamine 2000 method and the secretion of HPO was analyzed by Western blotting.RESULTS:Western blotting analysis indicated that the signal peptides from interleukin-1 receptor antagonist(IL-1ra)and an artifical signal peptide did not secret HPO directly and effectively,but the signal peptide from murine Ig kappa secreted HPO directly with great efficiency.The molecular weight of the secreted HPO was 30 kD,which means that the secreted HPO existed in homodimer.CONCLUSION:Secreted recombinant expression plasmid is successful constructed.The result may pave the way for the gene therapy of hepatic fibrosis.

Signal peptide sequence of human interleukin-2 influenced hEndostatin gene expression and protein secretion in HepG2 cells / 中国病理生理杂志

AIM: The role of human interleukin-2(IL-2) signal peptide sequence in the effect of human Endostatin (hEndostatin) expression and secretion was investigated in HeG2 cells. METHODS: RT-PCR and Western-blotting were conduct to observe mRNA level difference of hEndostatin gene, its protein expression and secretion level difference between with hIL-2 signal peptide sequence and without it. RESULTS: mRNA level of hEndostatin gene in HepG2 (pBlast-hIL2-hEndo) cells was higher than that in HepG2(pBlast-hEndo)( P