Production, purification, and quality assessment of borrelial proteins CspZ from Borrelia burgdorferi and FhbA from Borrelia hermsii

Abstract

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH_15-20 and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1.

Keywords: CspZ; Factor H; Factor-H like 1; FhbA; Protein quality control; Surface plasmon resonance.

Fig. 1

Protein expression and purification visualization on SDS-PAGE gels, Western Blot and HPLC. a Migration patterns of soluble extract (lane 2) and purified and dialyzed CspZ protein (lane 3), analyzed on a 12% SDS-PAGE gel stained with Coomassie blue (left) and by western blot detected using horseradish peroxidase (HRP)-conjugated anti-histidine tag antibody (right). b Migration patterns of soluble extract (lane 2) and purified FhbA protein (lane 3) were analyzed using a 12% SDS-PAGE gel stained with Coomassie blue (left) and a western blot detected using HRP-conjugated anti-histidine tag antibody (right). c HPLC chromatogram of purified CspZ (main peak). d HPLC chromatogram of purified FhbA (main peak)

Fig. 2

a, b Autocorrelation functions (ACF). Fitted models for multimodal sample (regularization or size distribution fit) (pink) and experimental data (blue) are represented .c, d Percentage of intensity as a function of the hydrodynamic radius (nm). Left – CspZ measurements. Right – FhbA measurements

Fig. 3

SEC-MALS analysis. Peaks and molecular masses are represented with numbers and black line. In red, light scattering; in green, UV; in blue, refractive index. a CspZ measurements. b FhbA measurements

Fig. 4

Far-UV CD spectra of (continuous line) CspZ and (dotted line) FhbA. Data were obtained at 20 °C, in PBS, with protein concentrations of ca. 0.1 mg/mL. Calculated helical contents from these spectra are (53.2 ± 1.4) % and (55.9 ± 1.3) %, respectively

Fig. 5

CspZ stability as a function of the storage conditions over time using one-dimensional 1 H NMR spectra approach. a Stability of CspZ at 4 °C at t₀ (i.e. reference), 15 days and 1 month. b Stability of CspZ at −80 °C at t₀ and after defrosting at 1 month and 3 months

Fig. 6

SPR sensorgrams of the interaction between FH, rFHL-1, or rFH_15-20 and FhbA or CspZ. The ligand was immobilized on the surface of a CM5 sensor chip as described in the Materials and Methods section. Experimental sensorgrams are in black, and fitted sensorgrams are in red. a Analysis of the interaction between CspZ and immobilized rFHL-1. The interaction between CspZ and immobilized rFHL-1 was analyzed by injecting CspZ samples at the following concentrations: 0.5, 2.8, 6.6, 9.5, 14.2, and 23.7 nM. b Analysis of the interaction between FhbA and immobilized rFHL-1. FhbA samples were injected at 0.5, 1.1, 2.2, 4.5, 6.6, 13.4, and 22 µM. c Analysis of the interaction between CspZ and immobilized rFH_15-20. CspZ samples were injected at 2.5, 5, 7.5, and, 10 µM. d Analysis of the interaction between FhbA and immobilized rFH_15-20. FhbA samples were injected at 5.8, 11.6, 29, 58, 87, and 116 nM. e Analysis of the interaction between CspZ and immobilized FH. CspZ samples were injected at 0.5, 3, 7, 10, 15, 25, and 37.5 nM. f Analysis of the interaction between FhbA and immobilized FH. FhbA samples were injected at 1, 5, 10, 25, 50, 75, and 100 nM