CFTR Protein

Accelagen, Inc.

Accelagen provides comprehensive protein purification services for drug discovery and pre-clinical studies. Accelagen’s protein purification expertise includes purification of full-length CFTR and many nucleotide binding domain (NBD) constructs of CFTR.

CFTR  Production Services

1. Full length recombinant CFTR
   
   With funding and guidance from the CFFT, Accelagen has established in-house systems that enables them to express and purify full-length CFTR.  With technical guidance by Dr. Jack Riordan and his colleagues, Accelagen is able to generate enough biomass essential for purification of CFTR at milligram scale, using Cell Factories to produce in large-scale stably transfected BHK cells.
   
   
2. Recombinant CFTR NBDs
   
   Recombinant NBDs can also be made for functional and structural studies. With technical guidance by former SGX Pharmaceutical scientists, Accelagen has successfully purified the following NBD1 and NBD2 domains of CFTR.
   
   

   Clone name	Termini and Mutations	Comments
   2935c469BSt1p1	387-646[del405-436](M)	Human WT NBD1 without RI
   2935c472BSt1p2	387-646[del405-436, del508](M)	Human NBD1
   2935c404CLt1p1	389-678[F429S, F494N, N620H*, L636E, Q637R](A)	Human NBD1
   2935c392CLt1p1	389-678[F429S, F494N, del508, N620H*, L636E, Q637R](A)	Human NBD1
   2935c371CLt1p1	389-678[F429S, F494N, del508, Q637R](M)	Human NBD1. S579(2BBS)
   5832c684BSt1pi1	1193-1427[Q1280E, Y1307N, E1308A, Q1309A, W1310H, H1402A, Q1411D, 1427ins153](M)	Human NBD2.  MalK dimerization domain (219-371); fusion at C-terminus
   5762c54BSt1pi1	1193-646[del405-436, F494N, Q637R, Q1280E, Y1307N, E1308A, Q1309A, W1310H, H1402A, Q1411D, 1446ins22](M)	Human NBD1-NBD2 fusion. 394-646 [del405-436, F494N, Q637R]+RLI (long)+1193-1446 [Q1280E, Y1307N, E1308A, Q1309A, W1310H, H1402A, Q1411D]
   MmCD00287500	389-673[del508]	Mouse NBD1

   
   
3. Other Available Reagents

• pCDFDuet-1-GroE plasmid
• E. coli BL21(DE3)GroE chemically competent cells

Certain NBD domain proteins, e.g., 5762c54BSt1pi1, are only expressed in soluble form at a significant level in the presence of chaprone co-expression, hence the above plasmid for co-expression or competent cells harboring the chaperone protein are generated and available to researchers.

General Services

Accelagen can provide protein expression in mammalian, baculovirus and Escherichia coli expression systems. Accelagen has protein purification capabilities with extensive selections of chromatography media and flexible configuration of chromatography columns tailored to each protein.

Please contact Accelagen directly regarding more detail about CFTR protein expression and purification capabilities on a fee-for-service basis.

Accelagen, Inc.
6044 Cornerstone Ct W, Ste. C
San Diego, CA 92121
Tel: (858) 678-8618
Fax: (858) 678-8628
Toll-free: (888) 365-8085

University of Alabama at Birmingham

Dr. Christie Brouillette is funded by the CFFT to provide limited amounts of purified NBDs to the CF research community for pilot functional and structural studies. Typically, submilligram to several milligrams of pure NBDs can be provided frozen in a standard buffer containing ATP.

Proteins are expressed in an E. coli expression system according to the methods found in [Atwell et al. (2010)  PEDS 23:375-384 and Lewis et al., (2005) JBC 280:1346-1353]. Plasmids for expression have been obtained from the Arizona Biodesign Institute, which is accessible through their website.

The following NBD proteins are currently available. New proteins are continually added to the list so check for availability. Please contact Christie Brouillette for additional information to request protein.

CFTR Folding Consortium

Additional protein constructs of NBD1 are available through the CFTR Folding Consortium.

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