J Virol Methods 2014 Feb;196:56-64.

High-yield soluble expression of recombinant influenza virus antigens from Escherichia coli and their potential uses in diagnosis.

Jang YH, Cho SH, Son A, Lee YH, Lee J, Lee KH, Seong BL.

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea.



Although antiviral drugs and vaccines have been successful for mitigating influenza virus infections, the lack of general technical platform for the timely supply of soluble and highly purified influenza viral antigens presents a serious bottleneck for the subsequent analysis for the effective control of the viral disease. Using the Escherichia coli (E. coli) lysyl tRNA synthetase (LysRS) as a novel fusion partner, this study reports the soluble expression of influenza viral proteins in E. coli host, construction of antibody library against the virus, and detection of anti-influenza antibodies using the expressed viral antigens. When influenza A and B viral proteins were fused with the LysRS, the fusion proteins were expressed predominantly as soluble forms and their production yields were high enough to be amenable to immunization protocols in rabbits for antibody generation. The produced antibodies showed high level binding specificity against the respective viral proteins, with cross-reactivity across heterologous viruses within the same type of influenza viruses. In addition, LysRS-HA fusion protein could bind specifically to anti-HA antibodies in the virus-infected mouse serum in widely accepted two detection methods, Western blot and ELISA. These results present a convenient tool for the production of antibodies specific to the virus as well as the rapid detection of influenza viral infections, ultimately contributing to the control of influenza viruses including highly pathogenic H5N1, pandemic H1N1, or the recent H7N9 influenza viruses.



Although Escherichia coli (E.coli) systems remain the most preferred choice for the production of various heterologous proteins, proteins of eukaryotic or mammalian viral origin, have been hardly amenable to soluble expression in this host. In addition to maltose-binding protein (MBP), N-utilizing substance-A (NusA), and glutathione S-transferase (GST), a number of fusion partners have been advanced to overcome the misfolding of target proteins into insoluble aggregation in bacterial host. However, most of the fusion partners still fail to support soluble expression, underlining the need for an innovative system that ensures solubility and proper folding for target proteins.

In previous studies, we reported a novel type of chaperone that facilitates protein folding by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding (Choi SI et al, PLoS ONE. 2008:3;e2677). Thus, RNA binding affects the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation (Figure 1). The RNA-interaction mediated chaperone (Chaperna) not only gives new insights into de novo folding mechanisms in vivo, but also could be usefully implemented for the expression of aggregation-prone proteins, as exemplified by influenza viral proteins of the present report.


Figure 1. Chaperna technology. Upon binding with RNA, both the RNA-binding protein and the bound RNA prevent inter-molecular interactions among folding intermediates, leading to soluble expression and favoring kinetic network into productive folding of target proteins. Subsequent cleavage of the fusion protein with appropriate protease generates active conformation of the intact target protein.

The present study by Jang et al. extended the Chaperna technology into the development of a convenient and potentially universal platform for rapid production of influenza viral proteins, including transmembrane proteins NA and M2. With a view to generate antigen/antibody library against the influenza viruses, the Chaperna strategy was applied into the soluble expression and purification of influenza internal and surface membrane proteins by fusion to E. coli lysyl tRNA synthetase (LysRS) (Figure 2A). The fusion induced high-yield soluble expression of influenza A and B viral proteins including transmembrane HA, NA, and M2 proteins. The final yields of the fusion proteins were in the range of 1–2 mg/ml from 1 L culture and the solubility was ≥80% for all proteins expressed (Figure 2B and 2C)


Figure 2. Soluble expression of LysRS-fused influenza A or B viral proteins in E. coli. (A) pGE-LysRS vector used for the expression of fusion proteins in E. coli. (B) Expression profiles of ten fusion proteins in soluble or insoluble fraction. (C) Solubility of each of fusion proteins in B estimated by gel densitometer.

Purified viral antigens were used for immunization protocol in rabbits for antibody library construction. The antibodies were examined for their binding specificity against an array influenza virus. In addition, we examined the usefulness of the expressed viral antigens in diagnostic purposes. The viral HA antigen successfully detected specific antibodies in virus-infected mouse sera in Western blot and ELISA analyses, the two most widely-used diagnostic protocols (Figure 3A and 3B). Taken together, these results present significant benefits of the Chaperna strategy for the production of viral antigen or antibody libraries for analytical, diagnostic, therapeutic, and prophylactic purposes in numerous non-clinical and clinical investigations.


Figure 3. Detection of anti-influenza antibodies in virus-infected mice sera using LysRS-fused viral proteins. Mice were infected with influenza virus (A/Puerto Rico/8/34) or mock-infected with PBS and three weeks later sera were collected from the mice. HA-specific antibodies in the mice sera were easily detected by Western blot (A) and ELISA (B) analyses in which the purified viral HA and NA antigens were used as binding antigens.


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