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VECTOR SYSTEMS MoBiTec GmbH, 2009 Vector Systems from MoBiTec Page 1 Contents Page NICE Expression System for Lactococcus lactis 2 Bacillus megaterium Expression Systems 3 Bacillus subtilis Expression
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VECTOR SYSTEMS MoBiTec GmbH, 2009 Vector Systems from MoBiTec Page 1 Contents Page NICE Expression System for Lactococcus lactis 2 Bacillus megaterium Expression Systems 3 Bacillus subtilis Expression Vectors 6 pbactag Tagging Vectors 8 Yeast Expression Vectors 9 porf-clone Vector System 10 ppicholi Shuttle Vector System 12 Mammalian Expression Vectors 13 CMV Expression Vectors 13a Fusion Protein Cloning System PheBo 14 Fusion Protein Cloning System pax 16 BRP Plasmids and Competent BRP Cells 18 PCR Cloning Vector p3t 20 Poly(His)-tag Cloning Vector peg-his1 21 Exontrap Cloning Vector 22 ssdna-production and Expression in pmex 24 Promoter-Trap Vector pbbr RESO (Broad-Host-Range) 25 Broad-Host-Range Vectors pbbr122 and pbhr1 26 Suicide Vector pcorrectclone 27 Multiple Cloning Site Vector pmcs5 28 Standard Cloning Vectors 30 EosFP - Green to Red Photoconvertible Fluorescent Protein 32 Related Products Endoproteases, ideal for Removal of Fusion Tags MobiTEV Protease 33 IgA Protease, HRV3C Protease 34 Kex2 Protease, Pro39 Protease 35 Factor Xa Protease, HS-Nuclease 36 MobiSpin Columns for DNA Purification 37 For detailed information: NICE Expression System for Lactococcus lactis Page 2 The Effective & Easy-to-Operate NIsin Controlled Gene Expression System Expression of membrane proteins Tightly controlled gene expression allows production of toxic proteins Secretion of proteins into the medium Less endogenous and no exogenous proteases No inclusion bodies Endotoxin-free food grade expression system Simple fermentation, scale-up and downstream processing Product Description L. lactis is increasingly used in modern biotechnology for the production of recombinant proteins for food, feed, pharma and biocatalysis applications. Essential is the easy genetic accessibility of L. lactis. Protein expression is controlled by Nisin. Areas of application Production of homologous and heterologous proteins for food, feed, pharma and biocatalysis applications Production of prokaryotic and eukaryotic membrane proteins Production of exo-polysaccharides Production of ingredients through metabolic engineering: e.g. alanine, folate, diacetyl Preparation of L. lactis as a biocatalyst by expression of a suitable enzyme as e.g. dehydrogenases and in situ co-factor regeneration High throughput screening for enzyme evolution or en- zyme comparison. Electron microscope image of Lactococcus lactis The effective & easy-to-operate Nisin Controlled gene Expression system (NICE ) for Lactococcus lactis is perfectly suited for a food grade protein expression. The patented NICE system was developed by NIZO food research BV. NICE is a trademark of NIZO food research BV. Patent EP , EP , EP VS-ELV NICE pnz8008 Reference plasmid with gusa gene 10 µg VS-ELV NICE pnz8148 Lactococcus lactis expression vector, NcoI site 10 µg VS-ELV NICE pnz8150 Lactococcus lactis expression vector, ScaI site 10 µg VS-ELV NICE pnz8149 Lactococcus lactis expression vector, food grade, NcoI site 10 µg Shipped at RT, store at 4 C VS-ELV NICE pnz9530 Lactococcus lactis nisrnisk vector, in Stain NZ ml VS-ELS NICE Lactococcus lactis expression Strain NZ ml VS-ELS NICE Lactococcus lactis expression Strain NZ3900, food grade 1 ml VS-ELS NICE E. coli host Strain MC ml Shipped on dry ice, store at -80 C VS-ELK NICE Nisin kit, 1 g nisin, concentration: 2.5%, (balance sodium chloride and denatured milk solids), 1 ml 5% acetic acid 1 kit Shipped at RT, store at 4 C Bacillus megaterium Expression System Page 3 High Yield T7 RNA Polymerase Gene Expression System for Bacillus megaterium Tightly regulated and effi ciently inducible xyla operon/t7 RNA polymerase Strong T7 RNAP promoter with unique sequence Stable, high yield protein production up to 8-fold increased in comparison to common xylose inducible expression Easy transformation by use of pretransformed B. megaterium protoplasts Control vector with GFP-sequence included in the kit Product Description This system combines the benefi ts of the tightly regulated and strong T7 RNA polymerase expression system and alkaline protease free Bacillus megaterium. Our system is based on two parallel-replicating plasmids: pt7-rnap and pp T7 (Gamer et al. 2009). In addition to the t7 rnap gene under control of the strong xyla promoter pt7-rnap contains the genes of ampicillin and chloramphenicol for easy selection in E. coli (Amp R ) and B. megaterium (Cm R ). pp T7 is responsible for the T7 RNAP-dependent expression of the target gene. Downstream of the T7 promoter it comprises a multiple cloning site with ten unique restriction enzyme cleaving sites. Additionally the plasmid carries two resistances to ampicillin (for E. coli) and tetracycline (for B. megaterium). For your convenience we offer B. megaterium protoplasts pretransformed with pt7-rnap in the kit, so that you just have to insert your gene of interest into pp T7 and transform the pretransformed protoplasts with this plasmid. For control purposes the GFP-expressing vector pp T7 -GFP is included in the kit. Reference Gamer, M., Fröde, D., Biedendieck, R., Stammen, S., und Jahn, D. (2009). A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl. Microbiol. Biotechnol 82, BMEGT702 Bacillus megaterium protoplasts, strain MS941, pretransformed with pt7-rnap 5x500 µl BMEGT701 Bacillus megaterium high yield T7 gene expression kit, includes pretransformed protoplasts BMEGT702 (5x500µl), pp T7 cloning vector and pp T7 -GFP control vector (vectors lyophilized, 10 µg each) 1 Kit BMEGT710 Bacillus megaterium pp T7 cloning vector, lyophilized 10 µg BMEG50 Bacillus megaterium protoplasts, strain MS941 5 x 500 µl Shipped at RT, protoplasts and kit shipped on dry ice. Store lyophilized vectors at 4 C, reconstituted vectors at -20 C, protoplasts at -70 C. Vectors are E. coli / B. megaterium shuttle vectors. Bacillus megaterium Expression System Page 4 Expression Vectors for B. megaterium Stable protein expression, high yield Xylose operon: tightly regulated and efficiently inducible by xylose (up to 350-fold) Polylinker downstream of promoter allows versatile cloning No indication of proteolytic instability even up to 5 hours after induction, since alkaline proteases (such as e.g. in B. subtilis) are not produced Endotoxins are not found in the cell wall 3 Suited for industrial large scale protein production Compatible with all B. subtilis vectors Product Description Our Bacillus megaterium kit is a new easy-to-handle system for stable protein expression with high yield. It is not only suited for industrial large scale protein production, but offers also an interesting alternative to the standard host E. coli. The kit comes with the E. coli/bacillus megaterium shuttle vector pwh1520 and B. megaterium protoplasts ready for transformation. B. megaterium has proven to be an excellent host for the expression of non-homologous DNA. Over other bacilli strains it has the advantage, that none of the alkaline proteases are present. This fact enables cloning and expression of foreign proteins without degradation. In addition, there are no endotoxins found in the cell wall. Protein yields are exceptionally good, also if inexpensive substrates are used. Mutarotase (Mro) and glucose dehydrogenase (Gdh) e.g. were accumulated to 20% and 30% of the total soluble protein, respectively. Using the tightly regulated xylose operon the genes were 130- to 350-fold induced without proteolysis. The illustration shows the time dependence of the induced protein expression in hours after induction versus enzyme activity. A polylinker downstream of the promoter allows versatile cloning in pwh1520. All B. subtilis vectors are compatible with B. megaterium as well. The B. megaterium system offers unique possibilities for the industrial production of proteins and is of great interest to manufacturers in the biomedical field. In a diagnostic test for AIDS e.g., the HIV coat protein is commercially produced by B. megaterium (Ginsburgh et al., 1989). Time dependence of induced expression of the enzymes Gdh (glucose dehydrogenase) and Mro (mutarotase) in B. megaterium. Enzymatic activity given in U/mg protein. Protein Yield Protein yields vary depending on the protein expressed. Rygus and Hillen (1991) have observed, that e.g. Gdh and Mro accumulated to 20% and 30% of the total soluble protein, respectively. The time dependence of the induced expression of these enzymes is shown in the figure above. Examples Proteins successfully over-produced with this system are: catabolite control protein (ccpa) xylose repressor (XylR) trehalose repressor (TreR) heat shock protein (HPr) from PTS (phosphotransferase sugar transport system) mutarotase (Mro) glucose dehydrogenase (Gdh) β-galactosidase human single-chain urokinase-like plasminogen activa- tor (rscupa) cellulase Our special service for you: B. megaterium protoplasts ready for transformation! A detailed handbook including all protocols is provided with the product. It is also available for download.»one of the most efficient expression systems described in any organism so far!«rygus and Hillen Bacillus megaterium Expression System Page 5 I I I SpeI SmaI BspMI SphI BglII References Rygus, T. and Hillen, W., Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon, Appl. Microbiol. Biotechnol. (1991), 35: Hueck, C. P. et al., Cloning, expression and functional analyses of the catabolite control protein ccpa from Bacillus megaterium, Molecular Microbiology (1995), 16(5): Vary, P., Prime time for Bacillus megaterium, Microbiology (1994) 140: Potential industrial & diagnostical applications: Vary, P., Development of genetic engineering in Bacillus megaterium: an example of the versatility and potential of industrially important bacilli, Biology of bacilli: Applications to Industry, pp. (1992), Ed. by Doi and McGloughlin. Boston: Butterworths-Heinemann Ginsburgh et al., Sporulation promoter spovg controlled expression of PP42 gene of HIV-1 in Bacillus megaterium, Abstr. International Conf. on AIDS, Montreal (1989) Map of pwh1520. Shuttle vector for E. coli/b. megaterium. Tet R (Bac), tetracycline resistance Bacillus; Tet R,Tet R, tetracycline resistance, interrupted; Amp R, ampicillin resistance; xyl R, xylose-dependent repressor; xyla, xylose isomerase, gene incomplete; P A, xyla promoter; MCS, multiple cloning site; ori pbc16, Bacillus origin of replication; pbr ori, ColE1 origin of replication. Host Strains The protoplasts we supply are Bacillus megaterium strains WH320 and MS941. The system has been developed by Prof. Dr. W. Hillen and co-workers at the Institute of Microbiology in Erlangen, Germany. BMEG02 Bacillus megaterium protoplasts ready for transformation (strain WH320) Material is sufficient for 4 transformations plus control experiment. 5 x 500 µl BMEG50 Bacillus megaterium protoplasts, strain MS941 5 x 500 µl Shipped on dry ice; store at -80 C BMEG03 pwh1520 shuttle Vector, original; lyophilized DNA 5 µg BMEG10 pmm1522 shuttle Vector, improved; lyophilized DNA 10 µg BMEG11 pmm1525 shuttle Vector with signal sequence; lyophilized DNA 10 µg BMEG12 phis1522 shuttle Vector, 6xHis-tagged; lyophilized DNA precursor of BMEG20 10 µg BMEG13 phis1525 shuttle Vector with signal sequence; 6xHis-tagged; lyophilized DNA 10 µg BMEG14 pstrep1525 shuttle Vector with signal sequence; Strep-tagged; lyophilized DNA 10 µg BMEG15 pstrephis1525 shuttle Vector with signal sequence; Strep/6xHis double-tagged; lyophilized DNA 10 µg BMEG20 pc-his1622 shuttle vector, C-term. 6xHis-tag; lyophilized DNA 10 µg BMEG21 pc-strep1622 shuttle vector, C-term. Strep-tag; lyophilized DNA 10 µg BMEG22 pn-his-tev1622 shuttle vector, N-term. His-tag,TEV-site 10 µg Not available in the US BMEG23 pn-strep-tev1622 shuttle vector, N-term. Strep-tag,TEV-site 10 µg Not available in the US BMEG24 pn-strepxa1622 shuttle vector, N-term. Strep-tag; Xa site; lyophilized DNA 10 µg BMEG25 pstop1622, shuttle vector, lyophilized DNA 10 µg Shipped at RT, protoplasts shipped on dry ice Store lyophilized vectors at 4 C, reconstituted vectors at -20 C, protoplasts at -80 C All vectors are E. coli / B. megaterium shuttle vectors Bacillus subtilis Expression Vectors Page 6 Expression Vectors for B. subtilis Product Description: Gram-positive bacteria are well known for their contributions to agricultural, medical and food biotechnology and for the production of recombinant proteins. Among them, Bacillus subtilis has been developed as an attractive host because of several reasons: It is non-pathogenic and is considered as a GRAS organism (generally regarded as safe) It has no signifi cant bias in codon usage It is capable of secreting functional extracellular proteins directly into the culture medium (at present, about 60% of the commercially available enzymes are produced by Bacillus species) A large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation and large-scale fermentation has been acquired There are two obstacles reducing the use of B. subtilis: (i) production of a number of extracellular proteases which recognize and degrade heterologous proteins, and (ii) stable vector plasmids. The fi rst obstacle has been largely solved by the construction of protease-defi cient strains. And the second has been completely overcome by introducing plasmids using the theta-mode of replication such as those derived from the natural plasmids pamβ1 and pbs72. Quite recently, the construction and use of four different expression vectors based on the E. coli - B. subtilis shuttle vector pmtlbs72 exhibiting full structural stability was published. The two vectors pht01 and pht43 allow high-level expression of recombinant proteins within the cytoplasm, where pht43 directs the recombinant proteins into the medium. Both vectors are based on the strong αa-dependent promoter preceding the groe operon of B. subtilis which has been converted into an effi ciently controllable (IPTG-inducible) promoter by addition of the lac operator. Derivatives of pht01 are available either with 8xHis tag (pht08), Strep tag (pht09) or c-myc tag (pht10). Pgrac promoter (consisting of the groe promoter; the laco operator and the gsib SD sequence) ColE1 ori: ColE1 origin Amp R : ampicillin resistance lacl: lacl gene (lac repressor) Cm R : chloramphenicol resistance SamyQ: amyq signal sequence Bacillus subtilis Expression Vectors Page 7 The pal Vector cold-inducible vector One further expression vector was constructed containing the cold-inducible des promoter of Bacillus subtilis. pal12 is suited for extracellular synthesis of recombinant proteins. When mid-exponential phase bacterial cells are rapidly transferred from 37 C to 25 C or even a lower temperature, the synthesis of most cellular proteins greatly decreases, while that of cold-shock proteins is transiently upregulated. In Bacillus subtilis, one of these cold-shock proteins is a membrane-bound desaturase (Δ5-Des) encoded by the des gene. This enzyme catalyzes the introduction of a cis double bond at the Δ5 position of a wide variety of saturated fatty acids. It has been shown that the des gene is tightly regulated during cold shock. Production of recombinant proteins started within the fi rst 30 min after temperature downshock to 25 C and continued for about 5 h. References 1. Anagnostopoulos, C. and Spizizen, J. (1961). Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81: Jannière, L., Bruand, C. and Ehrlich, S.D. (1990). Structurally stable Bacillus subtilis cloning vectors, Gene 87: Nguyen, D.H., Nguyen, Q.A., Ferreira, R.C., Ferreira, L.C.S., Tran, L.T. and Schumann, W. (2005). Construction of plasmidbased expression vectors for Bacillus subtilis. Plasmid; 2005 Nov; 54(3): Epub 2005 Jul Phan, T.T.P., Nguyen, H.D. and Schumann, W. (2005). Novel plasmid-based expression vectors for intra- and extracellular production of recombinantproteins in Bacillus subtilis. Protein Expr. Purif.; 2006 Apr; 46(2): Epub 2005 Aug Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A laboratory manual. 6. Titok, M.A., Chapuis, J., Selezneva, Y.V., Lagodich, A.V., Prokulevich, V.A., Ehrlich, S.D. and Jannière, L. (2003). Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector, Plasmid 49: PBS001 pht01 vector, lyophilized plasmid DNA 10 µg PBS002 pht43 vector, lyophilized plasmid DNA 10 µg PBS003 pht08 vector, lyophilized plasmid DNA 10 µg PBS004 pht09 vector, lyophilized plasmid DNA 10 µg PBS005 pht10 vector, lyophilized plasmid DNA 10 µg PBS007 pal12, Bacillus subtilis cold-inducible vector for intracellular expression 10 µg Shipped at room temperature (RT). Lyophilized plasmid DNA can be stored at 4 C. Once the DNA has been dissolved in sterile water or buffer we recommend storage at -20 C. PBS020 Bacillus subtilis strain 1012wt 1 ml PBS021 Bacillus subtilis strain 168 Marburg 1 ml PBS022 Bacillus subtilis strain WB800N (for secretion vectors) 1 ml Shipped on dry ice; store at -80 C. pbactag Tagging Vectors Page 8 Epitope- and GFP-tagging Integration Vectors for Bacillus subtilis Helpful for localizing target proteins within specifi c cell compartments Easy purifi cation of fusion proteins produced in B. subtilis by metal affi nity chromatography Allows detection of fusion proteins on immunoblots by commercially available antibodies Tags: FLAG, HA, c-myc, GFP+, YFP and CFP Product Description The six pbactag vectors are based on the backbone of pmutin2 and allow for translational fusions of two different types of tagging sequences, epitope and localization tags, to the 3 end of any chromosomal gene of interest within the B. subtilis chromosome and of any other bacterial species not allowing for replication of pbactag. Transcriptional fusion of the tagging sequences is accomplished by PCR amplifi cation of the 3 terminal part of the Gram-positive gene of interest (about 300 bp), insertion into the tagging vector s multiple cloning site in such a way that (1) it is in frame with the tag sequence to create a fusion protein and (2) that this tagged fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. The construct is then introduced back into the Gram-positive organism that was the source of the gene and the plasmid will integrate into the chromosome by homology with the cloned gene. The pbactag vectors are unable to replicate in B. subtilis independently, however upon insertion of about 300 bp derived from the coding region of the gene to be inactivated by PCR, they can integrate into the respective genes by homologous recombination. Integration of the recombinant pbactag vector into the target gene transcriptionally fuses the different tags to that promoter of the gene, and downstream genes can be controlled by the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter. Selection for erythromycin resistance allows for recovery of these integrants. The six vector plasmids were primarily constructed for use in B. subtilis but can be applied to any bacterial species not allowing replication of the pbr322-based plasmids. On the other hand, expression of downstream genes depends on synthesis of the Lac I repressor protein and proper functioning of the Pspac promoter. The epitope sequences (FLAG, c-myc, and HA) allow detection of tagged proteins within the cell using commercially available antibodies. Furthermore, the FLAG and HA tags can be used to purify the fusion proteins by affi nity chromatography. The Green Fluorescent Protein (GFP) localization tags GFP+ (which produces enhanced fl uorescence), YFP and CFP can be used to localize a protein to a specifi c compartment within the cell. A detailed handbook is provided with the product. It is also available for down
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