New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag

BMC Biotechnology

Methodology Article

Wayne F. Anderson¹  Marco Biancucci²  Karla J. F. Satchell³  Jennifer Wong⁴  Jazel S. Dolores⁵  Sarah Grimshaw⁶  Keehwan Kwon⁶ 

[1] Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Ward 6-205, 60611, Chicago, IL, USA;Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Ward 6-205, 60611, Chicago, IL, USA;Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Ward 6-205, 60611, Chicago, IL, USA;Present address: Indiana University, Bloomington, IN, USA;Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Ward 6-205, 60611, Chicago, IL, USA;Present address: Northwestern Memorial Hospital, Chicago, IL, USA;Infectious Diseases Group, J. Craig Venter Institute, 9714 Medical Center Drive, 20850, Rockville, MD, USA;Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA;

关键词: Self-cleavable tag;    Protein purification;    Cysteine Protein Domain;    Ligation-independent cloning (LIC);    InsP6;   

DOI  :  10.1186/s12896-016-0323-4

 received in 2016-11-21, accepted in 2016-12-09,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundRecombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols.ResultsIn this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called “the CPD-tag”. The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline.ConclusionpCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

【 授权许可】

CC BY   
© The Author(s). 2017