Multi-chaperone systems
	Our priority is the development of multi-chaperone systems to facilitate production of therapeutically relevant proteins. We developed the Hsp90 multi-chaperone system, which facilitates production of steroid receptors and Hsp90 client protein kinases. Multi-chaperone systems for GPCRs (G-protein coupled receptors) are currently in development.
	Hsp90 multi-chaperone system
	Hsp90 is a molecular chaperone which facilitates folding of a large number of therapeutically relevant proteins, such as steroid/nuclear receptors, Hsp90 client protein kinases and viral polymerases. A comprehensive list of Hsp90 client proteins can be found on Prof. Picard's lab web site http://www.picard.ch/downloads/Hsp90interactors.pdf Hsp90 works in ensemble with several other proteins. Hsp40, Hsp70/Hsc70, Hop, and p23 are required in addition to Hsp90 for activation of steroid receptors (Pratt and Toft, Exp Biol Med, 2003, 228:111-33), Fig 1. As shown on the figure, we provide these human molecular chaperones using a single recombinant baculovirus, FoldHelper™-905c. Chaperones are expressed to a high level and are visible as prominent bands on Coomassie-blue stained gels of crude extracts of insect cells infected with FoldHelper™-905c. Another molecular chaperone, Cdc37 could be required in addition to Hsp90, Hsp40, Hsp70/Hsc70 and p23 for activation of Hsp90-dependent protein kinases (MacLean and Picard, Cell Stress Chaperones, 2003, 8:114-9). Cdc37 can be provided using a separate recombinant baculovirus FoldHelper™-37, which enables its expression to a high level.
	Fig. 1. Protein expression profiles of conventional and multi-chaperone baculovirus systems. Spodoptera frugiperda Sf9 cells were infected either with NC (Cat# C13) or FoldHelper™-905c recombinant baculoviruses, harvested at 60 h post infection, lysed in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, and the insoluble fraction was removed by centrifugation at 30,000g for 15 min. Soluble proteins were separated in reducing 4-12% SDS-PAGE in MES buffer and stained with Coomassie blue.
	Co-infections of insect cells with recombinant baculoviruses expressing ligand binding domains (LBDs) of steroid receptors and FoldHelper™-905c result in a marked improvement in the production of receptors in soluble form and in a higher yield of purified proteins (Fig. 2A). Proteins expressed in the 5-chaperone system were associated with human Hsp90 and Hsp70 even after extensive washes during the purification, whereas similar insect molecular chaperones either failed to associate with the LBDs or associated weakly and were removed during purification (Fig. 2B). Preparations of steroid receptors containing these chaperones are desirable since Hsp90 is required for high affinity ligand binding by steroid receptors (DeFranco et al., J Steroid Biochem Mol Biol., 1998, 65:51-8). Furthermore, Hsp90 and Hsc70 are sufficient for re-activation of stripped glucocorticoid receptor (Rajapandi et al., J Biol Chem. 2000, 275:22597-604), which quickly becomes inactive in purified form.
	Fig. 2. Yields of steroid receptors produced in a conventional versus 5-chaperone baculovirus system. Ligand binding domains (LBDs) of mineralocorticoid (MR), glucocorticoid (GR), estrogen alpha (ERa) and progesterone (PR) receptors were expressed as GST fusion proteins using recombinant baculoviruses. Insect cells were either infected with recombinant baculoviruses expressing steroid receptors without chaperones (conventional system), (-), or co-infected with the same recombinant baculoviruses and FoldHelper™-905c (5-chaperone expression system), (+). GST-LBD fusion proteins were purified from the soluble protein fraction using affinity chromatography on glutathione beads, separated in 10% SDS-PAGE, stained with Coomassie blue, and bands corresponding to the steroid receptor's LBDs were quantitated using densitometry. Hsp90 and Hsc70 molecular chaperones co-purified with the steroid receptors are marked by triangles. A. Densitometry data. B. 10% SDS-PAGE, Coomassie blue staining.
	Improvement in the production of soluble proteins typically correlates with an improvement in the production of proteins in biologically active form. More importantly, appropriately produced receptors are likely to exhibit native ligand-binding properties, which could allow for making fine distinctions between similar ligands and similar receptors, and overcome the notorious poor selectivity of drugs targeting steroid receptors.
	Improving production of difficult proteins
	We believe that currently only a fraction of the most promising proteins can be produced in their native, biologically-relevant form, allowing protein structure-based drug design, compound library screening, lead-optimization, and development of protein or antibody-based therapeutics.
	Protein therapeutics are produced either in E.coli, such as interferon and insulin, or in mammalian cells, such as erythropoietin. However, none of the proteins for use in humans are produced using recombinant baculoviruses. There is no specific reason for this except that until now, nobody was eager to "break the ice" with the FDA by introducing the first baculovirus-produced protein. This is likely to change as recent volunteer trials of baculovirus-expressed influenza vaccine have been successful (Treanor et al., JAMA, 297:1577-82, 2007). Due to its unique multi-chaperone baculovirus technology, AB Vector is well positioned to tap into the vast protein therapeutics market. Such multi-chaperone technologies are not available for mammalian cells, and E.coli cells are not suitable for producing the majority of therapeutically relevant proteins.
	Among drug targets, GPCRs, nuclear receptors and protein kinases comprise the most important categories (details). Drugs targeting these proteins constitute more than 90% of total drug sales. The paradox is that about 30% of the R&D effort is spent on protein kinases, whereas drugs targeting nuclear (steroid) receptors generate several times more revenue than drugs targeting kinases. Drugs targeting GPCRs generate more revenue than drugs targeting protein kinases and nuclear receptors combined.
	Some of the key GPCRs, steroid receptors, and protein kinases may have never been available in a form suitable for adequate drug screening due to the inability to produce them in native form or in sufficient quantities. AB Vector's technology will enable their partners to unlock the potential of such promising but "difficult to produce" targets.
	Steroid receptors are difficult to produce in mammalian, baculovirus, yeast, and E.coli systems (Srinivasan, Mol Endocrinol., 1992, 6:857-60). So far, all commercially available steroid receptors and the overwhelming majority of protein kinases have been produced using conventional recombinant baculoviruses. Recombinant baculoviruses are also a system of choice for producing GPCRs, and on the whole could be preferable to other systems (Akermoun et al., Protein Expr Purif. 2005, 44:65-74). AB Vector's multi-chaperone systems is the next generation of baculovirus system, which are specifically designed to produce certain classes of target proteins. Multi-chaperone systems outperform traditional baculovirus system due to overexpression of specific sets of human molecular chaperones and protein folding partners.
	Collaborations
	Please feel free to contact us worldwide at info@abvector.com
	Multi-chaperone systems for G-protein coupled receptors (GPCRs)
	Multi-chaperone expression systems specifically designed for production of certain GPCR classes are currently in development. Such systems will include major molecular chaperones as well as certain protein folding partners specific to GPCRs. Meanwhile, you can follow our recommendations for expression of Transmembrane proteins.
	AB Vector provides recombinant baculovirus stocks for expression of all Histamine receptors, most of the Serotonin and Adrenergic receptors as well as selected Dopamine, Muscarinic and Angiotensin receptors of prime importance. Recombinant baculoviruses expressing GPCRs were constructed using ProEasy™ technology which ensures generation of 100% pure baculovirus stocks.
	Relevant products
	Multi-chaperone systems Cat # Product Specification Unit size Price* MC90 FoldHelper™-905c Overexpressed p23, Hop, Hsp40, Hsc70 and Hsp90 using a single recombinant      baculovirus GFP marker to monitor virus propagation 1 ml, 108 p.f.u./ml Negotiable H18 FoldHelper™-37 Overexpressed cdc37 to facilitate folding of client protein kinases 1 ml, 108 p.f.u./ml $395.00 Recombinant baculoviruses for expression of steroid receptors Cat # Product Specification Unit size Price* S3 BV-ERa-LBD Recombinant baculovirus stock Expression of Estrogen receptor a LBD 1 ml, 108 p.f.u./ml $399.00 S9 BV-ERb-LBD Recombinant baculovirus stock Expression of Estrogen receptor b LBD 1 ml, 108 p.f.u./ml $399.00 S15 BV-PR-LBD Recombinant baculovirus stock Expression of Progesterone receptor LBD 1 ml, 108 p.f.u./ml $399.00 S21 BV-AR-LBD Recombinant baculovirus stock Expression of Androgen receptor LBD 1 ml, 108 p.f.u./ml $399.00 S27 BV-MR-LBD Recombinant baculovirus stock Expression of Mineralocorticoid receptor LBD 1 ml, 108 p.f.u./ml $399.00 S33 BV-GR-LBD Recombinant baculovirus stock Expression of Glucocorticoid receptor LBD 1 ml, 108 p.f.u./ml $399.00 S35 BV-GR Recombinant baculovirus stock Expression of Glucocorticoid receptor full length 1 ml, 108 p.f.u./ml $499.00 Histamine receptors Cat # Product Specification Unit size Price* GH41 BV-HRH1 Recombinant baculovirus stock Expression of Histamine receptor (HRH1) 1 ml, 108 p.f.u./ml $499.00 GH45 BV-HRH2 Recombinant baculovirus stock Expression of Histamine receptor (HRH2) 1 ml, 108 p.f.u./ml $499.00 GH49 BV-HRH3 Recombinant baculovirus stock Expression of Histamine receptor (HRH3) 1 ml, 108 p.f.u./ml $499.00 GH53 BV-HRH4 Recombinant baculovirus stock Expression of Histamine receptor (HRH4) 1 ml, 108 p.f.u./ml $499.00 Serotonin receptors Cat # Product Specification Unit size Price* GS27 BV-HTR1A Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A) 1 ml, 108 p.f.u./ml $499.00 GS31 BV-HTR1B Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 1B (HTR1B) 1 ml, 108 p.f.u./ml $499.00 GS35 BV-HTR1D Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 1D (HTR1D) 1 ml, 108 p.f.u./ml $499.00 GS37 BV-HTR1F™ Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 1F (HTR1F) 1 ml, 108 p.f.u./ml $499.00 GS39 BV-HTR2A Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A) 1 ml, 108 p.f.u./ml $499.00 GS43 BV-HTR2B Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B) 1 ml, 108 p.f.u./ml $499.00 GS47 BV-HTR2B Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 2C (HTR2C 1 ml, 108 p.f.u./ml $499.00 GS51 BV-HTR5A Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 5A (HTR5A) 1 ml, 108 p.f.u./ml $499.00 GS55 BV-HTR7B Recombinant baculovirus stock Expression of 5-hydroxytryptamine (serotonin) receptor 7B (HTR7B) 1 ml, 108 p.f.u./ml $499.00 Adrenergic receptors Cat # Product Specification Unit size Price* GA3 BV-ADRA1A™ Recombinant baculovirus stock Expression of Adrenergic Receptor alpha 1A (ADRA1A) 1 ml, 108 p.f.u./ml $499.00 GA7 BV-ADRA1B™ Recombinant baculovirus stock Expression of Adrenergic Receptor alpha 1B (ADRA1B) 1 ml, 108 p.f.u./ml $499.00 GA11 BV-ADRA1D™ Recombinant baculovirus stock Expression of Adrenergic Receptor alpha 1D (ADRA1D) 1 ml, 108 p.f.u./ml $499.00 GA15 BV-ADRA2A™ Recombinant baculovirus stock Expression of Adrenergic Receptor alpha 2A (ADRA2A 1 ml, 108 p.f.u./ml $499.00 GA19 BV-ADRA2C™ Recombinant baculovirus stock Expression of Adrenergic Receptor alpha 2C (ADRA2C) 1 ml, 108 p.f.u./ml $499.00 GA23 BV-ADRB2™ Recombinant baculovirus stock Expression of Adrenergic Receptor beta 2 (ADRB2) 1 ml, 108 p.f.u./ml $499.00 Dopamine receptors Cat # Product Specification Unit size Price* GD3 BV-DRD1 Recombinant baculovirus stock Expression of Dopamine receptor 1 (DRD1) 1 ml, 108 p.f.u./ml $499.00 GD5 BV-DRD1 Recombinant baculovirus stock Expression of Dopamine receptor 2, transcript variant 1 (DRD2) 1 ml, 108 p.f.u./ml $499.00 Muscarinic receptors Cat # Product Specification Unit size Price* GM1 BV-CHRM1 Recombinant baculovirus stock Expression of Muscarinic acetylcholine receptor 1 (CHRM1) 1 ml, 108 p.f.u./ml $499.00 GM5 BV-CHRM2 Recombinant baculovirus stock Expression of Muscarinic acetylcholine receptor 2 (CHRM2) 1 ml, 108 p.f.u./ml Inquire about availability Angiotensin receptors Cat # Product Specification Unit size Price* GAT1 BV-AGTR1 Recombinant baculovirus stock Expression of angiotensin II receptor (AGTR1) 1 ml, 108 p.f.u./ml $499.00
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