An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Abstract

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his 6 -tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his 6 -tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his 6 -tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his 6 -site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his 6 -tagged target protein.Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his 6 -tag. KeywordsHigh-throughput; Structural genomics; Maltose-binding protein; TVMV protease; Ligationindependent cloningThe burgeoning genomic information now available makes vast numbers of proteins accessible for structural and functional studies, and many large-scale projects have developed automated protocols for amplifying, cloning, and expressing genes, and for screening proteins for desirable properties [1][2][3][4][5]. Similar strides have been made in streamlining protein purification, but production of sufficient material for detailed structural and functional characterization remains labor-intensive and time-consuming [3,4,6,7]. Typically, purification is facilitated by fusing proteins to affinity tags, most commonly a his-tag, which allows purification by immobilized metal-ion affinity chromatography (IMAC,[8]). Additional tags are often attached to improve proteins' solubility, such as maltose-binding protein (MBP) [2][3][4]9,10]. In typical protein production pipelines, the resulting fusion proteins are first screened for ☆ This manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No.W-31-109-ENG-38 with the US Department of Energy. The US government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. The US Government's right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript solubility, then purified by semi-robotic p...

ALKBH1-Mediated tRNA Demethylation Regulates Translation

“…Cloning, Expression, and Purification of ALKBH1-The human ALKBH1 gene (GenBank Accession NM_006020.2) with deletion of the N-terminal 36 amino acids was subcloned into a pMCSG19 vector by ligation-independent cloning (LIC) to generate the plasmid pMCSG19-His-ALKBH1 (Donnelly et al, 2006). Human ALKBH1 was then expressed in a BL21 (DE3) E. coli strain containing the plasmid pRK1037.…”

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

“…For crystallization, a plasmid, pRedJ T , was used to produce a truncated RedJ, RedJ T (5-residue N-terminal truncation and 19-residue C-terminal truncation). The redJ T construct was PCR-amplified under standard conditions from pMMA1 and ligated into pMCSG7 to give pRedJ T (26). The forward primer was 5Ј-TACTTCCAATCCAATGCCCTGC-TCTCCCAGCGTTCC-3Ј, and the reverse primer was 5Ј-TTATCCACTTCCAATGCTAGAGTTCGGTGCCCA-GGTG-3Ј.…”

Crystal Structure of Bacillus anthracis Transpeptidase Enzyme CapD

“…The DNA sequence of plasmid pXO2 (GenBank accession NC_007323) was used to design primers to amplify the CapD open reading frame by PCR with the following primer pair: 5Ј-TACTTCCAATCCAATGCGTTCAACA-AAATAAAAGACAGT-3Ј and 5Ј-TTATCCACTTCCAAT-GTCATTTATTTGATTTCCAAGTTCC-3Ј. The amplified product was directly cloned in the ligation-independent cloning vector pMCSG19 (25). This primer design avoids cloning of the putative signal peptide of 28 residues (predicted by the SignalP 3.0 server).…”