LIC (Ligation-Independent Cloning) vectors

Technology

Cloning

Summary

A paper describing ligation-independent cloning vectors constructed for high-throughput cloning and purification of proteins.

Description

Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a Tobacco Etch Virus (TEV) protease site for removal of tags that facilitate protein purification (His-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by Tobacco Vein Mottling Virus (TVMV) protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here.

We developed a set of superior LIC vectors tailored specifically for automated purification of proteins for crystallization. Details of these vectors can be found at http://bioinformatics.anl.gov/mcsg/technologies/vectors.html. More information can be found in the 2011 review by Kim, et al.

Publication

Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID. "A family of LIC vectors for high-throughput cloning and purification of proteins." Methods Mol. Biol. 498, 105-15 (2009). PubMed ID: 18988021 | Search SBKB Publications portal | PMC Link

Nallamsetty S, Kapust RB, Tözsér J, Cherry S, Tropea JE, Copeland TD, Waugh DS. "Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro." Protein Expr Purif. 38, 108-15 (2004). PubMed ID:15477088

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

Contact

Mark Donnelly, Email: mdonnelly@anl.gov

Midwest Center for Structural Genomics

Availability

Vectors are available from the PSI:Biology-Materials Repository.

Link




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Last edited:Fri 09 Mar 2012 - 4 years, 5 months ago