Recombinant protein production data after expression in the bacterium Escherichia coli

Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.


Type of data
Pictures. Images of gel electrophoresis.

How data was acquired
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Data format Raw Experimental factors
Fusion proteins: GST, CusF, and SmbP. Inducer concentration, time and temperature during protein expression. Metal affinity chromatography with silver ions Ag(I). Periplasmic or cytoplasmic protein expression.

Experimental features
Production levels of soluble protein and inclusion bodies, protein purity.

Data source location
San Nicolas de los Garza, Nuevo Leon, Mexico Data accessibility Data is within this article

Value of the data
The data shows that the production of soluble recombinant proteins in the bacterium Escherichia coli can be improved by varying the fusion protein, time, temperature, and concentration of inducer during protein expression.
The data indicates that recombinant proteins, exemplified here with the red fluorescent protein (RFP), can be expressed in the cellular periplasm when tagged with full-length CusF and SmbP.
Protein purification data shows that CusF-tagged proteins may be purified using immobilized metal affinity chromatography with Ag(I) ions instead of the most common Cu(II) or Ni(II).

Data
Fig . 1 shows the SDS-PAGE analysis comparing soluble and insoluble protein content for the proteins LovR and SHY2 tagged with two different protein tags and expressed at different conditions. Additionally, the electrophoretic analysis in Fig. 2 compares the content of inclusion bodies.
Recombinant proteins tagged with the protein tags CusFp and SmbPp, containing their signal sequences, are exported to the cell's periplasm. Fig. 3 shows an image of E. coli BL21(DE3) cells after RFP expression and the electrophoretic analysis of the periplasmic lysates. Fig. 4 shows pictures of the synthesized silver chromatographic media before and after incubation with the E. coli lysate expressing green fluorescent protein tagged with CusF. Fig. 5 shows the SDS-PAGE analysis of the purification steps, it shows the protein content in the flow-through (the lysate after incubation with the Ag(I) resin), and two elutions steps with 160 mM methionine.

DNA constructs
Full-length CusF (CusFp, for periplasmic expression) was amplified with primers 5 0 -AGTCAGTCA-CATATGAAAAAAGCACTGCAAGTCG-3 0 (NdeI, forward) and 5 0 -ATGCATGCAGGTACCCTGGCTGACTT-TAATATCCTGTAA-3 0 (KpnI, reverse). The 50 μL reaction comprised 10 ng of template DNA, 60 pmol of each primer, 1.5 μL of 10 mM dNTPs mix, and 2 units of Vent DNA polymerase (New England Biolabs) in 1 Â ThermoPol reaction buffer. The thermocycler conditions were 95°C for 2 min; 30 cycles of 95°C-1 min, 59°C-1 min, 72°C-1 min; and a final extension at 72°C for 10 min. Amplification of CusF lacking the signal sequence (for cytoplasmic expression) was done with forward primer 5 0 -AGTCAGTCACATATGGCTAACGAACATCATCATGAAAC-3 0 (NdeI) and the same reverse primer and thermocycler conditions as before. pET30a vector was linearized with NdeI and KpnI, and the CusF insert was ligated into it following digestion with the same enzymes. The gene for GST was cloned with the same restriction sites for comparative studies. Target proteins were cloned using NcoI and XhoI restriction sites, in order to place the enterokinase recognition sequence between CusF and the target protein for tag removal. The target proteins expressed in the cellular cytoplasm (using the CusF construct) were LovR from Caulobacter crescentus [2], the Arabidopsis thaliana protein Short Hypocotyl  2 (SHY2) [3], and the S65T mutant of Green Fluorescent Protein (GFP) [4]. Red Fluorescent Protein [5], was expressed in the periplasm with the CusFp construct. A second RFP construct was tested for expression in the periplasm but tagged with SmbPp as previously described [6].

E. coli periplasm lysis
Expression of recombinant proteins in the cellular periplasm has certain advantages since it contains less proteases [8], and provides the proper conditions for the formation of disulfide bonds [9]. If the protein of interest is tagged with the CusFp or SmbPp constructs that include the signal sequences, then the target protein is exported to the periplasm. An osmotic shock procedure was performed to obtain the periplasmic fraction. Cells from a 1 L culture were resuspended in 20 mM Tris-HCl, 30% sucrose, 2.5 mM EDTA, pH 8.0 (5 mL/g of cell pellet) and incubated on ice for 1 h. Cells were centrifuged at 10,000 rpm for 15 min at 4°C, the supernatant was recovered and saved, and the sediment was resuspended in 20 mL ice-cold 5 mM MgSO 4 buffer for 1 h. Cells were centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatant was also considered as periplasmic fraction, the two supernatants were pooled, and analyzed by SDS-PAGE.

Purification of CusF-GFP with immobilized Ag(I) affinity chromatography
CusF is a metal-binding protein with high binding affinities to copper and silver ions, especially Cu (I) and Ag(I) [10]; although it has been demonstrated that binds Cu(II) as well [11]. This last feature has allowed the purification of CusF-tagged proteins with immobilized Cu(II) affinity chromatography [1]. Nevertheless, the first metal ion tested to purify CusF-tagged proteins was Ag(I); this metal ion was immobilized on a functionalized acrylamide resin with glutaraldehyde and thiourea. Polyacrylamide beads (Bio-Gel P2, from BIO-RAD) were functionalized with silver ions as previously described [12]. First, a 25% glutaraldehyde solution was polymerized for 24 h at 50°C. After hydration of the Bio-Gel P2 beads with a 50 mM sodium phosphate buffer (pH 8.0) for 24 h, the polymerized glutaraldehyde solution was added to the wet resin (gel) in a ratio of 20 mL/1 g of dry gel. They were incubated at 37°C/120 rpm for 24 h, afterwards the resin was filtered and rinsed with deionized water. For every 1 g of initial dry gel, 20 mL of 1.0 M thiourea solution was added to the glutaraldehyde-activated gel resin and the slurry was incubated at 37°C/120 rpm. After 24 h of reaction, the thiourea solution was removed by decantation and the gel was filtered and washed with deionized water again. The final step involved the addition of silver nitrate in a ratio of 0.9 mL of 1.0 N AgNO 3 for 1 g of dry gel and the solution was incubated at 37°C. After 24 h the resin was washed with deionized water to remove all the unbounded silver. Cells expressing CusF-GFP were harvested by centrifugation at 4°C and then resuspended in ice-cold lysis buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). Cells were lysed using a bead-beater and 0.1 mm glass beads (both from BioSpec Products); the lysate was clarified by centrifugation at 4°C, 15,000 rpm for 20 min. 1 mL of the Ag(I) resin was incubated with the 5 mL of CusF-GFP lysate 30 min at room temperature. The resin was washed thoroughly with lysis buffer. Since two methionines and one histidine are used by CusF to bind Ag(I) [13], elution steps were performed using a buffer with a high methionine concentration (50 mM sodium phosphate, 300 mM NaCl, 160 mM methionine, pH 8.0). Elution fractions were analyzed by SDS-PAGE.