Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2

Highlights

• PCV2 capsid protein can only be expressed in those E. coli cells that carry extra tRNA copies for rare codons.

• Full length recombinant capsid protein is highly unstable when expressed as histidine tagged protein.

• N-terminal truncated capsid protein is still highly immunogenic and can detect PCV2 positive serum.

Abstract

The present study was aimed at comparing different E. coli strains in expressing the capsid protein of Porcine Circovirus 2 (PCV2). Full length capsid protein could be expressed only in Rosetta-gami 2 (DE3) pLysS strain using pET32b (+) vector. This confirmed that only those strains which possess tRNAs for rare codons can express the full length capsid protein. Purification of full length capsid protein could not be achieved even after several attempts using native and denaturing conditions. Subsequently, an attempt was made for expression of N-terminal truncated capsid protein using the same expression system. Truncated capsid protein was successfully expressed, purified and characterized by western blotting. The truncated capsid protein was also shown to be efficacious in testing serum samples using an optimized indirect ELISA, wherein a diagnostic sensitivity of 88.89% and specificity of 90.82% was obtained as compared to commercially available GreenSpring® porcine circovirus (PCV2) ELISA test kit. Thus, the expressed truncated capsid protein appears to be a promising diagnostic agent for PCV2. The comparative analysis suggests that cluster of arginine residues at N-terminal of capsid protein not only affects its expression in some E. coli strains but also its purification by Ni-NTA chromatography, when expressed as a histidine tagged fusion protein.

Introduction

Porcine circovirus type 2 (PCV2), a member of the Circoviridae family, is a single stranded DNA virus having circular genome [1]. They are found to be associated with a wide variety of disease syndromes in pigs, collectively called porcine circovirus associated diseases (PCVAD) or porcine circovirus diseases (PCVD) [2,3]. They are ubiquitous organisms and found in almost all parts of the world.

The genome size of PCV2 is only about 1767–1768 nucleotides in size and encodes 3 major ORFs [4]. ORF1 encodes for protein involved in replication of the virus [5] and ORF3 was shown to have a role in apoptosis in vitro and was also found to be associated with the pathogenicity of the virus in mice during an in vivo study [6]. ORF2 protein or capsid protein encoded by ORF2 is the only structural protein of the virus and determines the antigenicity of the virus [7]. This protein consists of several immunodominant epitopes and reacts strongly with serum of PCV2 infected animal [8,9]. Therefore, the capsid protein has been targeted by many workers for in-vitro expression, either for the development of vaccines or its use as a diagnostic agent [[9], [10], [11], [12]].

Prokaryotic expression system is usually the most preferred expression system because it is simple and relatively inexpensive than eukaryotic systems. However, the expression of capsid protein in E. coli have had its fair share of difficulties. It is well documented that ORF2 of PCV2 contains a nuclear localization signal (NLS), which consists of multiple arginine residues encoded mostly by codons that are rarely used in E. coli [13]. These properties make the full length capsid protein of PCV2 a difficult protein to be expressed in prokaryotic cells. The NLS of the virus has a role in formation of stable virus like particles (VLPs) by the capsid protein [14]. Thus, it might be sometimes necessary to express the full length capsid protein, particularly when the ultimate aim is development of vaccines. Truncated capsid protein can serve the purpose when the ultimate aim is development of diagnostics. Since different E. coli strains differ in their properties from one another, which can have an effect on expression of heterologous proteins, the efforts of this study were directed towards the expression of capsid protein in different E. coli strains. Expression and purification of recombinant protein was confirmed using SDS-PAGE and western blotting. Western blot analysis using anti-PCV2 polyclonal serum and monoclonal anti-his antibody was done to confirm its specificity as the capsid protein was expressed as a fusion protein having histidine tag. Also, a truncated version of the same protein was expressed, purified and characterized using exactly the same system. An indirect ELISA was optimized and its efficiency to detect anti-PCV2 antibody was compared to a commercially available indirect ELISA kit. The outcome of this study will aid the future works in selecting the correct strain and method for recombinant capsid protein production, either for vaccine or diagnostic development.