A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters

Abstract

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

A Novel and Fast Purification Method for Nucleoside Transporters

“…Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Melford Ltd. (Chelsworth, UK). The vectors pL53 and pL55 employed in this study are as described in our previous report (Ma et al, 2015 ), and the same strategy was used to generate pL54.…”

“…Membrane preparation was performed as previously published (Ma et al, 2015 ). Briefly, after being resuspended at 6 mg/ml (wet cell weight) in phosphate-buffered saline (PBS), the cells were disrupted at 30 kpsi using a TS series cell disruptor (Constant Systems Ltd., UK).…”

“…All experiments were performed at 4°C unless stated otherwise. The standard purification was described in Ma et al ( 2015 ) and the optimized purification was carried out as follow. Membrane proteins (100 mg) were solubilised in purification buffer for 1 h. The non-solubilised material was removed by centrifugation (100,000 g for 1 h).…”

“…The inconvenience of using a His-tag directly as an N-terminal fusion of a membrane protein exposing its N-terminus in the periplasm is that the presence of the His 8 positive tag can alter the protein topology (Von Heijne, 2006 ). To avoid this problem, we have used MBP as a linker between the purification tag (8xHis) and the membrane protein of interest, hence keeping the topological stability of the target as well as the flexibility of His-tag purification procedures (Ma et al, 2015 ). In addition, a specific proteolytic cleavage site is added between the MBP and the target protein.…”

“…This optimized purification procedure has been successfully applied to other NTs (SI-Figure 1 ). In addition, we developed an improved expression system by changing the promoter and the linker described in our earlier study (Ma et al, 2015 ). Compared to our previous approach, the optimized method is faster and more cost effective.…”