Production of soluble Borrelia afzelii outer surface protein A in Escherichia coli expression system

Nik Abdul Aziz Nik Kamarudin^1*, Christina Injan Mawang¹, Jama’ayah Mohamed Zahidi², Khairul Mohd Fadzli Mustaffa³, and Mariana Ahamad¹

1. Acarology Unit, Infectious Diseases Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Setia Alam, Malaysia
2. Bacteriology Unit, Infectious Diseases Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Setia Alam, Malaysia
3. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia Health Campus, 16150 Kota Bharu, Kelantan, Malaysia

*Corresponding author: Nik Abdul Aziz Nik Kamarudin. This email address is being protected from spambots. You need JavaScript enabled to view it.. Tel: +603-33627808

CITATION: Nik Kamarudin NAA, Mawang CI, Mohamed Zahidi J, Mustaffa KMF, Ahamad M. Production of soluble Borrelia afzelii outer surface protein A in Escherichia coli expression system. International Medical Research Journal. 2025 May 30;11(1):98–111. https://doi.org/10.63719/imrj.2025.11.01.009

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ABSTRACT
The Borrelia burgdorferi sensu lato complex includes species that pose a growing global health challenge, with increasing reports of severe cases and fatalities, especially those linked to Borrelia afzelii in parts of Asia. Outer surface protein A (OspA), a key lipoprotein essential for tick midgut colonisation, serves as an important target for both diagnostic tools and vaccine development. This study focused on enhancing the production of soluble recombinant OspA from B. afzelii using an Escherichia coli expression system. The OspA gene from B. afzelii strain BO23 was cloned into the pET-28a(+) vector, incorporating an N-terminal His-tag, and expressed in E. coli BL21 Star (DE3). Expression was optimised by induction with 0.5 mM IPTG for five hours at 37°C in LB broth supplemented with 0.2% glucose, resulting in a yield of 5.8 mg/L of soluble OspA. Affinity chromatography achieved a purity level exceeding 90%, and the protein’s immunoreactivity was confirmed using Western blot and Dot blot assays with a commercial monoclonal antibody against OspA.

KEYWORDS: Lyme disease, outer surface protein A, zoonotic, Borrelia