AB Vector - Transmembrane proteins

  Transmembrane proteins
 
  Transmembrane proteins traverse the lipid membrane one or more times and consist of a portion translocated into the endoplasmic reticulum (ectodomain) and a portion exposed to the cytosol. They often have an N-terminal hydrophobic signal peptide sequence, which facilitates protein translocation into the endoplasmic reticulum (ER). However, some transmembrane proteins do not have a canonical N-terminal signal sequence and use alternative mechanisms for their translocation. Many transmembrane proteins are N-glycosylated on Asn-X-Ser/Thr motif in the ER, and disulfide bond formation is also accomplished in the ER. Folding of cytosolic domain(s) is facilitated by molecular chaperones present in the cytosol. N- or C- terminal tags can be provided to facilitate the membrane protein affinity purification and immunodetection. We offer a variety of vectors to facilitate these processes.
 
  Mix and match features provided in the vectors
 

pAB-bee™
encodes powerful signal sequence

pAB-bee™-8xHis encodes powerful signal sequence and His-tag, which facilitates purification by immobilized metal affinity chromatography



ProFold™-ER1 provides Calreticulin, a major molecular chaperone facilitating glycoprotein folding, and PDI which facilitates the correct formation of disulfide bonds






ProFold™-C1 provides Hsp40 and Hsc70, major cytoplasmic molecular chaperones


pVL-GFP can provide GFP tag to facilitate rapid overexpression screening, detergent extraction and purification pAB-6xHis-MBP can provide C-terminal MBP tag to reduce protein aggregation
  The above features can be mixed and matched in various combinations, while supporting a high level of target protein expression. They can also be combined with "plain vectors" which only provide a high level of protein expression (pVL1393, ProEasy™, ProGreen™, ProFold™-0). A plasmid transfer vector is always combined with a baculovirus DNA vector, and some combinations are available as Kits. Click on TECHNOLOGY for more information on baculovirus expression system. Go to BACULOVIRUS TUTORIAL for simple on-line instructions on baculovirus transfection, propagation of recombinant baculoviruses and recombinant baculovirus protein expression studies.
 
  Examples
 
  1.Target protein has a large ectodomain, a weak signal sequence, is known to be glycosylated, and has multiple cysteines. Insert gene encoding mature target protein into pAB-bee™ plasmid vector in lieu of the powerful insect signal sequence and co-transfect with ProFold™-ER1 linearized baculovirus DNA.
  2.Target protein has no N-terminal signal sequence. Its ectodomain is known to be glycosylated and has multiple cysteines. Insert gene encoding entire ORF of the target protein into pVL1393 plasmid vector and co-transfect with ProFold™-ER1 linearized baculovirus DNA.
  3.Target protein has a strong N-terminal signal sequence. Its ectodomain is known to be glycosylated and has multiple cysteines. Insert gene encoding the entire ORF of the target protein into pVL1393 plasmid vector and co-transfect with ProFold™-ER1 linearized baculovirus DNA.
  4.Target protein has a strong N-terminal signal sequence, a large cytosolic tail, and a small ectodomain. Insert gene encoding entire ORF of the target protein into pVL1393 plasmid vector and co-transfect with ProFold™-C1 linearized baculovirus DNA.
  5.You have little experience with recombinant baculoviruses. Get a quick update on recombinant baculovirus technology at Materials and methods. Use ProEasy™ linearized baculovirus DNA in combination with any plasmid transfer vector of choice, as it is the easiest method to construct recombinant baculoviruses.
 
  Signal sequence in transmembrane proteins
 
  A hydrophobic signal peptide sequence which is followed by a signal peptidase cleavage site is often present on the N-terminus of transmembrane proteins. However, some signal peptides are less efficient than others. The cleavage sites also may differ in their efficiency. You can evaluate your target protein for the presence of a signal peptide and its cleavage site using the TargetP program (Emanuelson O. et al., J.Mol.Biol., 300: 1005-10016, 2000). The program is user friendly and is available at http://www.cbs.dtu.dk/services/TargetP/
 
  An inefficient signal peptide or its incomplete cleavage is likely to result in the misfolding of a significant portion of the expressed protein and in an accumulation of inclusion bodies or protein degradation. In such cases the inefficient native target protein signal sequence can be substituted for a signal sequence which is optimized for a particular protein expression system.
 
  Relevant products
 
Plasmid transfer vectors                             Click on the product name for technical information!
Cat # Product Specification Unit size Price*
B1 pVL1393 High level of target protein expression 50 μl, 0.5 μg/μl $295.00
B3 pAB-bee Provides target proteins with powerful honeybee melittin signal sequence to      facilitate translocation into the ER
Recommended for high level expression of secreted and transmembrane proteins
50 μl, 0.5 μg/μl $495.00
B3H pAB-bee™-8xHis Provides target proteins with powerful honeybee melittin signal sequence to      facilitate translocation into the ER
Recommended for high level expression of secreted and transmembrane proteins
N- or C-terminal 8xHis tag for affinity purification
50 μl, 0.5 μg/μl $495.00
B7 pAB-6xHis-MBP™ High level expression of proteins with cleavable N-terminal 6xHis MBP tag
High level expression of proteins with C-terminal MBP tag
MBP tag improves protein solubility and yield
50 μl, 0.5 μg/μl $495.00
B8 pVL-GFP™ High level expression of proteins with C-terminal GFP tag 50 μl, 0.5 μg/μl $395.00
 
Linearized baculovirus vector DNA
CsCl purified and digested with Bsu 36.I                  Click on the product name for technical information!
Cat # Product Specification Unit size Price*
A1 ProGreen™ High level of target protein expression
GFP marker to monitor virus propagation
25 μl, 2.5 μg
5 transfections
$295.00
A3 ProFold™-C1 Overexpressed Hsp40 and Hsc70 chaperones facilitate target protein folding in the      cytoplasm
GFP marker to monitor virus propagation
High level of target protein expression
25 μl, 2.5 μg
5 transfections
$695.00
A4 ProFold™-ER1 Overexpressed calreticulin facilitates glycoprotein folding in the ER
Overexpressed PDI facilitates folding of cysteine-rich proteins
GFP marker to monitor virus propagation
High level of target protein expression
25 μl, 2.5 μg
5 transfections
$695.00
A9 ProFold™-0 The best value baculovirus vector
High level of target protein expression
25 μl, 2.5 μg
5 transfections
$195.00
A10S ProEasy™ The easiest generation of recombinant baculoviruses
Ideal for HTP applications
High level of target protein expression
25 μl, 2.5 μg
5 transfections
$599.00
 
Controls                                                                    Click on the product name for technical information!
Cat # Product Specification Unit size Price*
C13 NC (negative control) Recombinant baculovirus stock Extracts of cells infected with NC is an ideal negative control as there is no      polyhedrin or target protein expression
NC extracts are run side by side in SDS-PAGE with extracts of cells infected with      recombinant baculoviruses
Recombinant protein is detected as a prominent band which is absent in NC      extracts
1 ml, 108 p.f.u./ml $195.00
C14 GC (green control) Recombinant baculovirus stock Positive control baculovirus in experiments where GFP fluorescence is used to      monitor virus propagation, optimize cultivation conditions, or titrate of virus stocks 1 ml, 108 p.f.u./ml $195.00
C15 GCP (green control plasmid) Plasmid transfer vector DNA Positive control for transfection with linearized GFP- baculovirus DNA. Green      fluorescence of cells is indicative of successful transfection.
50 μl, 0.2 μg/μl $145.00
 
  *Go to PRODUCTS to order online. Alternatively, email an order as pdf attachment to info@abvector.com, or send it by Fax to 1-760-454-2465 or call 1-866-683-2867 to order by phone.
 
  If you have not worked with baculovirus expression system, also referred to as baculovirus expression vector system or BEVS, you can get a quick update at TECHNOLOGY. Go to BACULOVIRUS TUTORIAL for simple on-line instructions on baculovirus transfection, propagation of recombinant baculoviruses and recombinant baculovirus protein expression studies.
 
  Click on PRODUCTS to view the entire list of recombinant baculovirus-related products.


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