Bifunctional hybrid proteins Z-mHoneydew and Z-CyOFP1 based on the Z-domain of Staphylococcus aureus protein A for immunofluorescence assay of immunoglobulins G in blood serum

INTRODUCTION. Traditional methods for detecting serum IgG rely on antibodies chemically conjugated to enzymatic or fluorescent tags. The efficiency of such methods may be limited by steric hindrance during chemical cross-linking. As an alternative, bifunctional hybrid proteins Z-mHoneydew and Z-CyOFP1, based on the Z-domain of Staphylococcus aureus protein A and bright fluorescent proteins mHoneydew and CyOFP1, combining the binding to IgG and intense fluorescence, have been proposed for IgG immunofluorescence analysis; their production excludes the stage of chemical conjugation.

AIM. This study aimed to produce hybrid proteins based on the Z-domain of protein A for the creation of universal fluorescent reagents for the detection of IgG in blood serum.

MATERIALS AND METHODS. The pCCO-Z-mHoneydew and pCCO-Z-CyOFP1 genetic constructs were created by molecular cloning in the pCCO vector. Z-mHoneydew and Z-CyOFP1 proteins (~35 kDa) were expressed in Escherichia coli strain M15. Protein purification was performed by affinity chromatography on Ni²⁺-NTA agarose. The protein properties were studied using spectrofluorimetry, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay.

RESULTS. Genetic constructs pCCO-Z-mHoneydew and pCCO-Z-CyOFP1 under the control of the T5 promoter encode chimeric proteins with the following structure: Z-domain of protein A — flexible linker (Ser-Ser-Ser-Gly-Ser-Ser-Ser-Gly) — fluorescent protein mHoneydew or CyOFP1 — 6×His tag. Soluble proteins were obtained with a yield of ~37 mg/L (Z-mHoneydew) and ~6 mg/L (Z-CyOFP1) with a purity ≥90%. For the Z-mHoneydew protein, excitation and emission maxima were observed at 480 and 536 nm, respectively; a 26 nm blue shift in emission was observed relative to native mHoneydew (562 nm). For Z-CyOFP1, the excitation maximum was recorded at 515 nm; an 18 nm red shift in excitation was observed relative to native CyOFP1 (497 nm), while maintaining the emission maximum. Dose-dependent binding of IgG to the hybrid proteins was demonstrated using ELISA. A linear dependence was observed for Z-mHoneydew in the concentration range of 30–235 ng/μL (R²=0.96) and 15–250 ng/μL (R²=0.97) for Z-CyOFP1, confirming the capacity of the hybrid proteins to bind IgG. Immunofluorescence assay showed the feasibility of detecting IgG, specific to conjugate benzo[a]pyrene-bovine serum albumin, in rabbit serum using hybrid proteins. A linear relationship was observed for Z-mHoneydew in the serum dilution range of 1:10–1:160 (R²=0.92) and 1:10–1:80 (R²=0.98) for Z-CyOFP1, evidencing the suitability of hybrid proteins for detection of specific antibodies in blood serum.

CONCLUSIONS. Hybrid proteins Z-mHoneydew and Z-CyOFP1 retain fluorescent properties and the capacity to bind to IgG, including serum IgG, which is promising from the point of view of developing prototypes of universal immunofluorescent test systems.

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