A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

Abstract

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

Figure 1.

Vector derivations and maps. Derivation of the pOPIN vectors from pTriEx2. PCR fragments were prepared as described in the Materials and methods section and either ligated into the pTriEx2 vector or inserted by In-Fusion™. In cases where the pOPIN vector is not directly derivatized from pTriEx2, the intermediate vector is also shown. Features of the pTriEx2 vector retained in the pOPIN vector suite are: T7/lacO promoter/operator and terminator for inducible expression in E. coli harbouring the λ (DE3) prophage, CMV Enhancer/Chicken β-actin promoter and rabbit β-globin polyA site for efficient expression in mammalian hosts, p10 baculoviral promoter and 5′ UTR/ORF603 and ORF 1629 for efficient expression from/recombination into baculovirus respectively. The high-copy pUC origin of replication and β-lactamase (Ampicillin resistance marker) gene allow high-copy production of the vector in E. coli.

Figure 2.

Expression of Neisseria sp. proteins from either pOPINE (C-His₆) or pOPINF (N-His₆) in B834(DE3) E. coli. Plasmids were transformed into B834(DE3) cells, expression was induced by the addition of IPTG to the media and the resulting expression levels assayed by a Ni-NTA robotic screen followed by SDS-PAGE. The lane labels refer to unique OPPF identifiers (OPPF construct numbers; see Supplementary Table 1) followed by N or C to indicate the position of the His₆ tag in the construct. The lane labelled ‘Low’ contains Low Range Sigma Markers (M3913) and the lane labelled ‘High’ contains High Range Sigma Markers (M3788), molecular marker masses are indicated in kDa. Construct numbers labelled * were selected for scale-up and purification (Table 2).

Figure 3.

Expression of KIAA0319 proteins in HEK 293T cells using the pOPING vector. Domain and multi-domain constructs were screened for expression in HEK293T cells and subsequent secretion into the cell media analysed using SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels refer to unique OPPF identifiers (OPPF contruct numbers: see Supplementary Table 1). The lane labelled BM contains the BenchMark™ ladder (InVitrogen 10747-012), molecular marker masses are indicated in kDa. Construct numbers labelled * were selected for scale-up and purification (Table 3).

Figure 4.

(A) Expression of viral and human protein/protein domains in Rosetta(DE3)LysS E. coli. Plasmids were transformed into Rosetta(DE3)pLysS cells, expression was auto-induced in TB Overnight Express™ and the resulting expression levels assayed by a Ni-NTA robotic screen, followed by SDS-PAGE. The lane labels refer to unique OPPF identifiers (OPPF construct numbers; see Supplementary Table 2) followed by the pOPIN vector name (pOPINF contributes an N-His-3C site fusion to the protein of interest, pOPINJ contributes an N-His-GST-3C site fusion to the protein of interest and pOPINM contributes an N-His-MBP-3C site fusion to the protein of interest). eGFP refers to enhanced GFP and ‘null’ refers to vector alone (i.e. vector with no target genes inserted), pOPINJ and pOPINM will however express N-His-GST (29.5 kDa) and N-His-MBP (44.3 kDa), respectively. The lane labelled ‘Low’ contains Low Range Sigma Markers (M3913) and the lane labelled ‘High’ contains High Range Sigma Markers (M3788), molecular marker masses are indicated in kDa. (B) Expression of viral and human protein/protein domains in HEK293T cells. Plasmids were robotically transfected into HEK293T cells and the resulting expression levels assayed by a SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels are identical to panel A except that the standard molecular weight markers are replaced with marked positions of the BenchMark™ ladder (InVitrogen 10747-012), His-tagged molecular mass markers (masses in kDa) on the right. (C) Expression of viral and human protein/protein domains in Sf9 cells. Plasmids were robotically co-transfected into HEK293T cells with linearized A. californica bacmid and the resulting expression levels assayed by a SDS-PAGE and western blotting as described in the Materials and methods section. The lane labels are identical to panel A except that the standard molecular weight markers are replaced with marked positions of the BenchMark™ ladder (InVitrogen 10747-012), His-tagged molecular mass markers (masses in kDa) on the right.