Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors

Abstract

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.

Figure 1.

Maps of the lentiviral vectors used. Schematics are shown of (A) lentiviral construct pCVL-A, (B) non-UCOE containing construct pCVL-SFFV-muScn-IRES-GFP and (C) minimized UCOE containing construct pCVL-UCOE0.7-SFFV-muScn-IRES-GFP. SP, signal peptide; UCOE, ubiquitous chromatin opening element; SFFV, spleen focus-forming virus enhancer/promoter; muScn, murine Siderocalin; IRES, internal ribosome entry site.

Figure 2.

Transduction of 293-F cells at varying multiplicity of infection (MOI). (A) GFP MFI (mean fluorescence intensity) is shown for three different doses of pCVL-SFFV-muScn-IRES-GFP virus. (B) Analyses of expression of levels of muScn are shown; 20-µl aliquots from media supernatants from each transduction were separated by SDS–PAGE and stained with Coomassie blue (left) or analyzed by western blot with an anti-Strep II tag antibody (right).

Figure 3.

Comparison of non-UCOE vs. UCOE0.7 containing constructs expressing murine Siderocalin. (A) GFP MFI measured over a 1-month period for pCVL-SFFV-muScn-IRES-GFP. (B) GFP MFI, for the same period of time, for pCVL-UCOE0.7-SFFV-muScn-IRES-GFP. Cells were matched for copy number and data are representative of three replicate experiments.

Figure 4.

Purification and crystallization of murine Siderocalin. (A) Preparative Superdex 75 SEC trace for muScn expressed using the optimized lentiviral construct pCVL-UCOE0.7-SFFV-muScn-IRES-GFP. SDS–PAGE analysis of the peak fraction, before and after PNGase digestion, is shown (inset). (B) Crystals obtained using the PEGs II Suite (Qiagen) sparse matrix screen, condition 63 [0.1 M HEPES (pH 7.5), 10% v/v isopropanol and 20% w/w PEG 4000]. (C) Crystal structure of muScn (red) superimposed on that of huScn (1X89 in black). Electron density from a refined F_o–F_c omit map contoured at 2.0 σ showing the presence of the N-linked glycan and the intrachain disulfide bond.