Vectors for co-expression of an unrestricted number of proteins

Abstract

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five 'pQLink' vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.

Figure 1.

Map of the pQLink vectors and construction of co-expression plasmids. (A) Map of vector pQLinkH and genetic elements of all pQLink vectors. The LINK sequences are shown with lines indicating overhangs generated by restriction digest and T4 DNA polymerase treatment. MCS = multiple cloning site, TEV = TEV protease cleavage site, term. = transcription terminator. (B) Construction of a co-expression plasmid from two pQLink plasmids with two different cDNA inserts, labelled 1 and 2. The resulting plasmid can accept additional inserts, labelled 3 and 4. S = SwaI, P = PacI. (C) The LINK sequences and their digestion and annealing. The lines indicate overhangs generated by restriction digest and T4 DNA polymerase treatment. Two plasmids, identified by upper and lower case nucleotide codes, are digested and annealed, leading to a product with a LINK1 and a LINK2′ sequence, slightly larger than the original LINK2 sequence.

Figure 2.

Co-expression of His-tagged VCP with GST-tagged AMFR fragments. SDS-PAGE and Coomassie staining of whole cells solubilized in SDS-PAGE sample buffer (W) and proteins purified under non-denaturating conditions (P) (A) Proteins were eluted from a glutathione agarose column by TEV protease treatment and analysed by SDS-PAGE and Coomassie staining. Degradation products of the larger AMFR construct were observed. W = whole cellular protein extract, P = purified proteins, M = molecular weight marker. (B) Protein products of co-expression clones of His-VCP with GST-AMFR and GST alone were purified with nickel chelating beads and separated by SDS-PAGE and visualized by Coomassie staining (upper panel) or anti-GST western blot (lower panel, reagents: anti-GST HRP conjugate, GE-Healthcare, and Western Lightning Western Blot Chemiluminescence Reagent, Perkin Elmer).

Figure 3.

Co-expression of four subunits of the human TRAPP complex. (A) Protein expression products and nickel affinity chromatography products of seven clones expressing different subunits were analysed by SDS-PAGE and Coomassie staining. W = whole cellular protein extract, P = purified proteins, M = molecular weight marker. (B) Expression test with pETDuet-1 Bet3:Tpc6 and pQLinkH Bet3:pQLinkN Tpc6 in BL21 (DE3). Total cellular protein was analysed before (0) and after (I) 4-h induction.

Figure 4.

Analysis of TRAPP subcomplexes. (A) Proteins were co-expressed, purified with nickel chelating beads and loaded on a calibrated gel filtration column. (B) SDS-PAGE and Coomassie staining of isolated subcomplexes (marked with an asterisk in A).