This paper describes the modification of a ligation-independent cloning (LIC) vector to allow simultaneous expression of interacting proteins and selective purification of functional complexes.
The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8-15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.
For more information about these LIC vectors, please see the 2011 review by Kim, et al.
Stols L, Zhou M, Eschenfeldt WH, Millard CS, Abdullah J, Collart FR, Kim Y, Donnelly MI. "New vectors for co-expression of proteins: Structure of Bacillus subtilis ScoAB obtained by high-throughput protocols." Protein Expr. Purif. 53, 396-403 (2007). PubMed ID: 17363272 | Search SBKB Publications portal
Kim Y, Babnigg G, Jedrzejczak R, Eschenfeldt WH, Li H, Maltseva N, Hatzos-Skintges C, Gu M, Makowska-Grzyska M, Wu R, An H, Chhor G, Joachimiak A. "High-throughput protein purification and quality assessment for crystallization." Methods. 55:12-28 (2011). PubMed ID: 21907284 | Search SBKB Publications portal
Mark Donnelly, Email: email@example.com
Midwest Center for Structural Genomics
Vectors are available from the PSI:Biology-Materials Repository.
Last edited:Fri 09 Mar 2012 - 6 years, 4 months ago