In general, recombinant proteins are the most common source of the diagnostic reagents and can be expressed in mammalian, insect and bacterial cells [19–21]. While each of these systems has its advantages , the bacterial expression system is one of the most universally used and has been employed widely due to multiple factors, including its relative inexpensive cost, ease of manipulation and rapid growth rate . However, different codon usage pattern in E. coli can cause diminished the production of heterologous eukaryotic protein. Many E. coli strains, such as BL21 CodonPlus and Rosetta-2 derived from BL21, are optimized to enhance expression of gene sequences that contain codons used rarely by E. coli . Because there are a few rare codons in the US11 gene, three different E. coli strains, including E. coli BL21 (DE3), BL21 CodonPlus and BL21 Rosetta were used to optimize the expression of the fusion protein. As a result, the His-tagged US11 protein could be expressed in all of these three bacteria hosts, but with a slight better expression in E. coli BL21 (DE3) (data not shown). Additionally, different expression parameters were tested to optimize the expression of the US11 protein, demonstrating that the US11 protein gained the highest expression under the condition of 37°C (Figure 2A, lane 7) with 1 mM IPTG (Figure 2A, lane 3) for 5 h (Figure 2B, lane 6).
It is very common that high level expression of recombinant proteins in E. coli result in the formation of insoluble and inactive aggregate known as inclusion bodies . Although the proteins in the inclusion bodies are easy to purify and are protected from the intracellular protease, they are synthesized in the form of misfolding or partial folding polypeptides. Therefore, purified inclusion bodies must undergo the process of protein renaturation. In the present study, denaturation solution was removed by dilution or dialysis, which permitted renaturation of the His-tagged US11 proteins. However, the proteins purified from the inclusion bodies are not suitable for the researches that require the correct protein conformation.
The US11 protein is among the most-abundant viral proteins present in cells late in infection and is packaged in the tegument of the native virions . In this study, anti-US11 polyclonal antibody reacted with one band with apparent molecular masses of 21 kDa in Vero cells infected with HSV-1 for 24 h., which is consistent with previous reports used by monoclonal or polyclonal antibodies against US11 [4, 10–12]. The specificity of our US11 polyclonal antibody was confirmed by both WB and IFA in transfected and infected cells, and it also worked well, which maybe special advantages of our antibody over those antibodies. The subcellular localization of a viral protein determines its function. It is reported that soon after HSV-1 infection, the US11 protein is found in the cytoplasm, either as heterogeneous oligomers or associated with ribosomes or both [26, 27]. Later during infection, the US11 protein accumulates into nucleoli and is also found in RNP fibrils as well as in clusters of interchromatin granules. As expected, our results also demonstrate that the US11 protein localizes in the cytoplasm and nucleolus of the infected cell.
Early studies have demonstrated US11 is a multifunctional protein involved in posttranscriptional regulation of gene expression and in biological processes related to the survival of cells following environmental stress . To further gain insight into the molecular mechanisms underlying the multiple functions of this protein, identification of cellular factors capable of interacting with the US11 protein is necessary. Therefore, the anti-US11 polyclonal antibody may serve as a useful tool for further study of US11 interaction partners by co-immunoprecipitation.