Selectable high-yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Abstract

Recombinant protein expression systems that produce high yields of pure proteins and multi-protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X-ray crystallography and cryo-electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion-tag to generate recombinant proteins using suspension-cultured HEK293F cells. YFP is a dual-function tag that enables direct visualization and fluorescence-based selection of high expressing clones for and rapid purification using a high-stringency, high-affinity anti-GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.

Keywords: FACS; GFP-tag; HEK293F; Topoisomerase 2; YFP-tag; nanobody; recombinant protein expression.

Figure 1

Transfection of YFP‐TOP2α expression construct. (A) Plasmid DNA encoding Top2α with an N‐terminal YFP tag and intervening TEV spacer was designed for expression in human cells. (B) HEK293F cells expressing YFP‐TOP2α (yellow) were stained with DRAQ5 (red) to stain DNA. YFP‐TOP2α localizes to the nucleoplasm. (C) The total number of cells in each culture was measured and plotted for each concentration of PEI used to transfect plasmid DNA. (D) The fraction of YFP‐expressing cells was determined using an automated cell counter and plotted for each concentration of PEI used to transfect plasmid DNA. Colors for plot as indicated in “C”. (E) The total integrated YFP fluorescence was measured and plotted for each culture. (F) The total YFP fluorescence for each culture was determined and normalized to culture volume. Colors for plot as indicated in “E”. (G) The fraction of YFP‐expressing cells was determined using an automated cell counter and plotted for each concentration of transfected DNA.

Figure 2

Isolation of recombinant TOP2α from HEK293F cell lysates. (A) Anti‐GFP nanobody conjugated agarose resin binds tightly to YFP‐TOP2α. Specific elution of TOP2α is accomplished by TEV‐mediated cleavage of the fusion protein. (B) Timeframe for expression and purification of TOP2α using transient expression and YFP‐tag. Cells are transfected on day 0. YFP fluorescence is monitored and recorded 1 and 2 days post‐transfection, and culture media is added to support cell growth during this time. After 3 days, cells are harvested and YFP‐tagged protein is isolated. TEV‐eluted protein is recovered on day 4. (C) Photograph of anti‐GFP/YFP resin after a single pass of YFP‐TOP2α lysate and washing. YFP‐TOP2α is visible as a yellow color. (D) Coomassie blue stained SDS‐PAGE of the resin after 16 h of TEV cleavage shows the removal of TOP2α from the YFP tag is nearly complete. (E) TOP2α eluted from the anti‐GFP/YFP resin was analyzed by SDS‐PAGE stained with Coomassie blue.

Figure 3

FPLC purification of TOP2α. (A) TOP2α from the anti‐GFP/YFP resin was run on a Superdex 200 16/60 size‐exclusion column, and elutes primarily as a monodisperse peak (left). Coomassie blue stained SDS‐PAGE of fractions shows this peak contains purified TOP2α (right). (B) TOP2α from the size‐exclusion column was polished on a Source 15S column (left). Coomassie blue stained SDS‐PAGE of fractions shows this peak contains very pure full‐length TOP2α (right). (C) Analysis of purified TOP2α. SDS‐PAGE of final purified TOP2α sample after concentration and storage at −80°C shows a single band on an SDS‐PAGE stained with Coomassie blue. (D) Final purified TOP2α and TOP2α purchased from a commercial source were compared in kDNA decatenation assays. Both sources of TOP2α show comparable kDNA decatenation activity, requiring ∼0.5 nM to decatenate 50% of kDNA in this assay. (E) SDS‐PAGE of TOP2α proteins used in panel B shows comparable quantities of TOP2α.

Figure 4

Expression of TOP2β (47–1212). (A) Cellular localization of YFP‐TOP2β. Wildtype TOP2β (1–1621) is nuclear while the truncated enzyme core (TOP2β) is cytoplasmic. (B) YFP‐TOP2β (47–1212) fluorescence in HEK293F cell culture after growth under selection with blasticidin for 14 days. FACS was used to isolate medium and high YFP‐fluorescent cell populations. (C) Coomassie blue stained SDS‐PAGE of cell lysates, anti‐GFP resin, and TEV‐eluate from medium and high YFP‐fluorescing TOP2β (47–1212) cell cultures. (D) TOP2β (47–1212) TEV eluates from panel C were analyzed on a S200 10/300 size exclusion column. High YFP‐fluorescing cells produce twice as much protein as medium YFP‐fluorescing cells. (E) TOP2β (47–1212) was purified on a Source 15S column (left). Coomassie blue stained SDS‐PAGE (right) shows that fractions 26–28 contain pure TOP2β. Endogenous wildtype TOP2α and TOP2β are present in fractions 30–33. (F) TOP2β from the Source 15S column was polished on a S200 16/60 column (left). Coomassie blue stained SDS‐PAGE of fractions shows this peak contains very pure full‐length TOP2β 47–1212 (right). (G) Coomassie blue stained SDS‐PAGE of final purified TOP2β. (H) TOP2β (47–1212) show kDNA decatenation activity comparable to that of TOP2α, requiring ∼0.5 nM to decatenate 50% of kDNA in this assay. (I) Recombinant YFP was bound to new anti‐GFP/YFP resin or that which had gone through 20 binding and regeneration cycles. Binding capacity can be assessed visually. Similar quantities of YFP bound to both the new and used resin.