Nico21 de3 cells

All protein samples used for AFM and ITC as well as Coh used in smFRET were expressed in NiCo21 (DE3) cells (New England Biolabs). Cells were cultured in TB (terrific broth) medium containing 50 μg/mL kanamycin until OD600 reached ~0.6. Protein expression was induced by adding 0.5 mM IPTG to the culture, followed by incubating at 20 °C overnight. Cells were harvested and lysed using sonication. The cell lysate was pelleted and the supernatant was loaded onto a His-tap FF 5 mL column (GE Healthcare) and washed with TBS buffer supplemented with calcium (TBS-Ca, 25 mM Tris, 72 mM NaCl, 1 mM CaCl₂, pH 7.2). Bound protein was eluted using TBS-Ca buffer containing 500 mM imidazole. Eluted protein was further purified using Superose 6 10/300 GL size-exclusion column (GE Healthcare). Protein solutions for long-term storage were concentrated using a Vivaspin 6 centrifugal filter (molecular weight cut-off 5 kDa, GE Healthcare) and stored in 35% (v/v) glycerol at −20 °C. The concentration of the protein stocks were determined to be ~40 µM using UV absorption spectrophotometry.

For recombinant protein production, the Plasmodium berghei Maf1 ortholog (PBANKA_0718500) was selected instead of the P. falciparum ortholog, as it is much smaller and contains a short repeat in place of the P. falciparum asparagine-rich region. Outside this region, the sequences are nearly identical. The P. berghei Maf1 sequence was synthesized with Escherichia coli-optimized codon usage (DNA2.0) with an N-terminal 6×His tag in a T7 promoter expression plasmid. Protein expression was conducted in NiCo21(DE3) cells (NEB; catalog no. C2529). Expression was performed using 1-liter cultures induced at an optical density at 600 nm (OD₆₀₀) of 3.0 with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside)–Terrific broth for 20 h at room temperature. Cells were lysed by sonication, and protein was isolated using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen; catalog no. 30210). The recombinant protein was used to produce antiserum from two separate guinea pigs by Cocalico Biologicals (PA, USA) using the standard protocol. Antiserum was affinity isolated against the recombinant PbMaf1 protein. Western blots were visualized using a Li-COR Odyssey imaging system with 680RD linked anti-mouse (Li-COR; catalog no. 925-68070) and anti-guinea pig (Li-COR; catalog no. 925-68077) secondary antibodies. The 1-10 b PfGAPDH monoclonal (mouse) (66 (link)) antibody was used as a loading control.

A synthetic Bacillus
pumilus lipase gene construct corresponding to UniProt
accession ID W8FKE7 was codon-optimized for expression in Escherichia coli and synthesized by GenScript. The
mature peptide sequence, excluding the signal peptide, was subcloned
into pET22b with cytosolic expression. The expression vector containing
the pLipA gene was then transformed into Nico21(DE3) cells from New
England Biolabs using standard heat shock protocols. Protein production
was performed using ZYP-5052 autoinduction media, with a 5-h shaking
at 37 °C before the temperature was reduced to 17 °C for
overnight production. The protein was purified over three steps utilizing
the encoded hexa-histidine C-terminal tag with an IMAC step, followed
by desalting and then anion exchange with a strong ion exchanger.
A salt gradient was used to elute the protein from the anion exchanger.
For control experiments, the mesophilic B. subtilis lipase A was produced in the same manner.