Does the BGH poly A region in your vectors have a splice donor and splice acceptor (and therefore an intron)? What is the general function of the BGH polyA region?

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Answer

There is no intron in the BGH polyA sequence. The BGH polyA signal (bases 1028-1252 in pcDNA™3.1 vector) is the sequence that allows for polyadenylation of the RNA transcript. It is described in the following reference: The 3’-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J Biol Chem.1992 Aug 15;267(23):16330-4

The reference states that the 3’ untranslated region leading up to the poly adenylation core sequence (AATAAA) contains unique disperse non-consensus elements (consensus elements are poly U and GU-rich tracts) that serve to increase the efficiency of polyadenylation to a high degree, and that a 9-base sequence downstream of the core sequence facilitates efficient cleavage of the RNA strand. This leads to more stable and higher-abundance transcripts.

Answer Id: E3436

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615647417e44185841e6a03a0b8b3b32_FAQ

Do any of the your mammalian expression vectors contain an SV40 intron?

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Answer

We used to sell pcDNA™1 vector and related vectors which contained the SV40 intron and polyA signal fragment downstream of the multiple cloning site. However, the SV40 intron shows "promiscuity", which means that in vectors containing the SV40 intron downstream of an expressed gene, the 3’ end of the SV40 intron tends to splice with sequences within genes cloned upstream of the intron that are near-consensus 5’ splice sites (consensus sequence is MAGGTRAGT), leading to aberrant splice products. In other words, the 3’ end of the intron would often combine and splice out part of the gene of interest (1).

Our expression vectors now use the Bovine Growth Hormone polyadenylation sequence as the polyadenylation sequence for cloned genes of interest, because it is extremely efficient (2). However, the SV40 polyA signal is still included in many of our vectors as a polyadenylation signal for the expressed eukaryotic selection marker (e.g. neomycin, hygromycin, or zeocin resistance genes). There is no intron accompanying the polyA in these vectors.

(1) Huang MT, Gordon CM. The simian virus 40 small-t intron, present in many common expression vectors, leads to aberrant splicing.Mol Cell Biol. 1990 Apr;10(4):1805-10. PMID: 1690852).
(2) Goodwin, EC, Rottman, FM. The 3’-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation.J Biol Chem. 1992 Aug 15;267(23):16330-4. PMID: 1644817

Answer Id: E3952

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b61ed5fcb412fe534d250f476d45aa32_FAQ

Do you have references for Dynabeads® products used in phage display?

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Answer

Here are some references for Phage Display Panning:
1. Hansen MH et al. Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumor. Int. J. Oncol. 2001;19:1303-1309.
2. Kemp EH et al. The melanin-concentrating hormone receptor1, a novel target of autoantibody responses in vitiligo. J. Clin.Invest. 2002;109:923-930.
3. Demartis S et al. A Strategy for the Isolation of Catalytic Activities from Repertoires of Enzymes Displayed on Phage. J.Mol.Biol. 1999;286:617-633.
4. Heinis C et al. Selection of catalytically active biotin ligase and trypsin mutants by phage display. Protein Eng. 2001;14(12):1043-52.
5. Pini A et al. Design and Use of a Phage Display Library. J. Biol.Chem. 1998;273(34):21769-21776.
6. Bracci L et al. Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library. J. Neurochem. 2001;78:24-31.
7. McConnel l SJ et al. Biopanning phage display libraries using magnetic beads vs polystyrene plates. Biotechniques 1999;26:208-214.
8. Wang L et al. Cloning of anti-GAL Fabs from combinatorial phage display libraries: Structural analysis and comparison of Fab expression in pComb3H and pComb8 phage. Mol. Immunol. 1997;34(8/9):609-618.
9. Kontour Z and Walter G. Automation of phage display for high-throughput antibody development. Targets 2002;1(1):30-36.
10. Yeung YA and Wittrup KD. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnology Progress 2002;18(2):212-220.
11. Sawyer C et al. Methodology for selection of human antibodies to membrane proteins from a phage-display library. J. Immunol. Meth. 1997;204:193-203.
12. Demangel C et al. Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody. Eur. J. Biochem. 2000;267:2345-2353.
13. Gebhardt K et al. Adhesive Peptides Selected by Phage Display: Characterization, Applications and Similarities with Fibrinogen. Peptide Research 1996:9(6):269-278.
14. Jenné S et al. High Resolution Mapping of the B Cell Epitopes of Staphylokinase in Humans Using Negative Selection of a Phage-Displayed Antigen Library. J. Immunol. 1998;161:3161-3168.
15. Linse S et al. A Region of Vitamin K dependent Protein S That Binds to C4b Binding Protein (C4BP) Identified Using Bacteriophage Peptide Display Libraries. J. Biol. Chem. 1997;272(23):14658-14665.
16. Samson I et al. Screening a Random Pentapeptide Library, Composed of 14 D-Amino Acids, against the COOH-terminal Sequence of Fructose-1,6-biphosphate Aldolase from Trypanosoma brucei. J.Biol.Chem. 1997;272(17):11378-11383.
17. Danner S and Belasco JG. T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries. PNAS 2001;98(23):12954-12959.
18. Nord K et al. Binding proteins selected from combinatorial libraries of an alfa-helical bacterial receptor domain. Nature Biotech. 1997;15:772-777.

Answer Id: E6055

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72deaeaef05019295d6dc4b3653a0704_FAQ

Instrument Setup & Maintenance: SOLiD BioScope Users Guide (English )

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