Cloning And Overexpression Of NS1 Protein Biology Essay

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Influenza virus is globally pathogenic important. It possesses a lipid-bounded segmented genome which encodes at least one biochemically-distinct protein. Its subtype A can be classified according to antigenic differences. NS1 protein is defined as nonstructural protein in the virus. It is known as a multifunctional virulence factor. It only can be detected in the infected cell. In this study, the NS1A gene was successfully cloned into the BamHI/SacI cleaved-pET-32c(+) vector and subsequently electro-transformed to the E.coli BL21(DE3) expressing host. The recombinant NS1A gene has shown identical counterpart with the synthetic NS1A gene and the 3D protein structure was predicted through bioinformatics method. Better protein expression was found at 37°C under 5mM lactose induction in E.coli. Protein expressivity in soluble and insoluble fraction was not greatly detected in E.coli. 20% ammonium sulfate saturation was sufficient to concentrate and partially purify the target NS1A protein. The ammonium sulfate precipitated NS1A recombinant protein has characterized a significant immno-response to the polyclonal antibody in the Western blot. A 37kDa NS1 protein was detected to react with the H1N1 NS polyclonal antibody.

Key Words: Influenza A virus; H1N1 subtype; NS1 protein; Cloning; Overexpression; Purification


Influenza virus is highly pathogenic and it is usually considered as the causative agent of zoonotic respiratory disease. Its transmission is observed upon either interspecies (Webster et al., 1992) or intra-species. Reassortment may occur in this virus (reviewed by Hampson and Mackenzie, 2006; Gibbs et al., 2009). Its infection is usually associated with cellular alteration, apoptosis and host mortality (Schultz-Cherry et al., 2001).

The influenza viruses, which can be classified into types A, B and C, are included into the family of Orthomyxoviridae (Bouvier and Palese, 2008). The influenza A has shown identical pathogenic potential with influenza B and it extensively assesses pandemic or epidemic threat (reviewed by Pushko, 2009). Recently, HIN1 strain has established its cyclic alternation and reassortment in human. Presumably, the influenza infection is caused by direct and intimate interaction between human and swine. Evidence has shown that the H1N1 subtype remains circulating in the world since its first detected outbreak in 1918. Subsequently, its resurgence was documented in 1950 and 1977 (reviewed by Cox, 1998 and Nicholson et al., 2003).

Influenza A virions can appear as spherical shape with 80 to 120nm in diameter (Donatelli et al., 2003; reviewed by Pushko, 2009) or 300nm in length for filamentous form (Suri, 2007). Within the lipid-bound virion, there are eight negative sense single stranded ribonucleic acids (RNA) which are distinguished in length to encode eleven proteins. Influenza A virus nonstructural protein, NS1A protein, which is encoded by segment 8, consists of 230-237 amino acids. The NS1A protein is only can be detected during infection. It is multifunctional, involving significantly in the protein-RNA (Qiu and Krug, 1994) and protein-protein interaction (Xia et al., 2009). Its two functional domains, which are dsRNA-binding domain (RBD) and effector domain (ED), are essential for intracellular and extracellular interaction. This protein is unique and plays a role either as the inhibitor or activator through the association of other internal activator factors or viral proteins in the virus life cycle. In addition, it involves not only in the antiviral response but also in the post transcriptional activity in its host (Lin et al., 2007). Its involvement in cellular signaling pathway also considered important.

To gain more insight of its role in viral life cycle, the cloning and expression of NS1 protein as well as the crystallographic study were carried out purposely for further characteristic-identification and functional-analysis. Regarding the recent proteomic studies, the pET expression vectors are widely used for NS1 gene cloning works. Typically, the NS1 fusion protein was detected at 26kDa but larger molecular weight (Birch-Machin et al., 1997) was also reported. In addition, Ma et al. (2009) has detected the NS1 protein expressing in both soluble and insoluble fraction.

Previously, the NS1 purity was obtained through Ni-NTA purification (Wang et al., 2008), in addition, it was documented that the NS1A proteins were purified by chitin affinity chromatography (Ma et al., 2009) or glutathione S-transferase affinity column (Birch-Machin et al., 1997). Ward et al., (1994) has tried the induction by copper sulfate (CuSO4) on NS1 protein, designing to enhance the expressed NS1 gene to release the toxin in the infected cell. Concluded from the result, the toxicity could affect the cell growth but it was considered vital in yielding nuclear localization signal.

* Author for Correspondence Dr. Chan Geik Far


Material and methods

Construction of recombinant plasmid pET32c-NS1A

E. coli BL21(DE3)-pET-32c and DH5α-TOPO-NS1 were purified using the QIAprep Spin Miniprep Kit. The isolated NS1A gene was ligated into BamHI/SacI-cleaved pET-32c using YEA T4 DNA Ligase from yT&A Cloning Vector Kit. Ligation was carried out at 22°C for 20 min then followed by 65°C for 10 min. The plasmid was electro-transformed into E.coli.

Clone Identification and determination

The colony PCR was performed using the fresh colony as the template then amplified by T7 promoter (100μM) and T7 terminator (100μM). PCR reaction involved 95°C for 5mins, and followed by 25 PCR cycles of 30s at 95°C, 30s 55°C and 1min at 72°C, additionally elongated at 72°C for 7min. The presence of insertion in the selected clones was verified by BamHI and SacI restriction enzyme double digestion respectively then further sequenced using the universal primer, T7 terminator. Few bioinformatics tools were applied to validate and characterize the target protein.

Expression and purification of NS1A recombinant protein

The cells were induced with 1mM IPTG or 5mM lactose when the bacterial growth reached an OD600nm of 1.5. The cells were harvested by centrifugation at 5000 rpm for 30min after 4-hour-induction. Enzymatic-ultrasonication was used for cell disruption. Centrifugation at 12,000 rpm at 4°C for 30 minutes was applied to separate the homogenates components into cytoplasmic fraction (supernatant) and inclusion bodies (IB) fraction (cell pellet). The cell pellet was resuspended by using denaturing lysis buffer containing urea (100mM NaH2PO4, 10mM Tris-HCl, 8M urea, pH7.4) and incubated at 4°C for overnight.

Ammonium sulfate precipitation has been applied to partially purify the NS1A recombinant protein. 0.113g/ml of ammonium sulfate (20%) was added slowly into the sample. The sample was kept stirring for at least 1 hour. The harvested precipitate was resuspended with 1X sterile PBS solution.

SDS-PAGE and Western blot analysis

Purified NS1A fusion protein was analyzed in SDS-PAGE. The electrophoresed proteins were transferred to a nitrocellulose and subsequently blocked with 1% BSA. The membrane was probed with polyclonal antibody to influenza A H1N1 NS at 1:1250 in PBS, and the antibody-antigen complex was detected with secondary antibodies, goat anti-rabbit IgG (H+L) HRP conjugate in 1:5000 dilution. 3-times-washing with PBS-Tween 20 was applied on the membrane after each process of incubation. After that, the membrane was subjected to colour development.


NS1A Gene Cloning and sequencing analysis

Colony PCR result as shown in Figure 1 established that the positive clones, which appear at approximately 1400bp, were assumed as positive clones carrying gene of interest. The correct orientation of pET-32c (+) vector and NS1A gene were identified by BamHI and SacI restriction enzyme double digestion, which respectively gave 5901bp DNA fragment to the pET-32c (+) vector and 698bp to the NS1A gene (Fig 2).

Figure 1: Screening the transformed colonies by colony PCR. Lane 1: GeneRulerâ„¢ 1kb DNA Ladder; lane 2-15: clone 91-clone 104. The clone 92 (lane 3), clone 97 (lane 8), clone 100 (lane 11) and clone 104 (lane 15) were detected showing band at 1400 bp.

Figure 2: Clone identification by BamHI and SacI restriction enzyme. Lane 1: GeneRulerâ„¢ 1kb DNA Ladder; lane 2-4: pET32c-NS1 plasmid isolated from clone 97, clone 100 and clone 104.

Significant identical part was found between NS1A recombinant protein and Influenza virus A/California/04/2009 H1N1 segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes (complete cds; gi: 227809838; gb: FJ966086.1). 3D protein structure was predicted using the comparative modeling (Fig 3). The hydrophobicity profile was generated by its corresponding deduced amino acid sequence (Fig. 4).

Figure 3: Predicted structure of NS1A recombinant protein.

Figure 4: The deduced amino acid sequence and hydrophobicity profile of recombinant NS1A protein.

(created by Kyte-Doolittle Protein Hydrophobicity Plots)

Expression analysis of NS1A recombinant Protein

The crude lysate was harvested from insoluble and soluble portion of cytoplasmic fraction. The SDS-PAGE analysis result has established that the expression products contained a band of fusion protein at approximately 45 kDa (indicated y red arrows, Fig 5). The recombinant NS1A protein was found not greatly expressed in E. coli under 1mM IPTG induction.

Figure 5. SDS-PAGE profile of expressed NS1A fusion protein extracted from E. coli clones after 1mM IPTG induction at 37°C.

Lane 1: MW marker (SDS-Page Molecular Weight Standards, Low Range, Bio-Rad Catalog No.: 161-0304); lane 2, 6 and 10: cytoplasmic fraction of uninduced clone 97, clone 100 and clone 104; lane 4, 8 and 12: inclusion bodies fraction from uninduced clone 97, clone 100 and clone 104; lane 3, 7 and 11: cytoplasmic fraction of induced clone 97, clone 100 and clone 104; lane 5, 9 and 13: inclusion bodies fraction of induced clone 97, clone 100 and clone 104.

Partially purification and immunogenicity analysis of the recombinant protein

The NS1A recombinant protein from 5mM lactose-induced crude insoluble extract of IB fraction was precipitated using 20% ammonium sulphate (Fig. 6). The precipitated NS1A recombinant protein has characterized significant antigenicity to the H1N1 NS polyclonal antibody in Western blot analysis. The molecular weight of expressed protein has shown at Fig. 7 was 37kDa.

Figure 6: Overexpression result and 20% ammonium precipitation on inclusion bodies fraction of NS1A recombinant protein expressed in E.coli after 5mM lactose induction at 37°C.

Lane 1: MW marker (SDS-Page Molecular Weight Standards, Low Range), lane 2: crude lysate before buffer exchange, lane 3: crude lysate after buffer exchanged, lane 4: 20% ammonium precipitated supernatant, lane 5: 20% ammonium precipitated pellet.

Lane 1: MW marker (SDS-Page Molecular Weight Standards, Low Range), lane 2: crude lysate before buffer exchange, lane 3: crude lysate after buffer exchanged, lane 4: 20% ammonium precipitated supernatant, lane 5: 20% ammonium precipitated pellet.

Figure 7: Colloidal gold staining and Western blot analysis of NS1A fusion protein after ammonium sulfate precipitation.

Lane 1: MW marker (Precision Plus ProteinTM KaleidoscopeTM prestained standards); lane 2: crude lysate from inclusion bodies fraction harvested from 5mM lactose induction, lane 3: crude lysate after buffer exchanged with binding buffer (100mM NaCl, 50mM Tris-HCl, pH 7.4), lane 4: 20% ammonium sulfate precipitate.


According to the protein-protein sequence alignment output data, the sequences recombinant NS1A protein has revealed significant protein homology to Influenza virus A/California/04/2009 H1N1 segment 8 nuclear export protein (NEP) and nonstructural protein 1 (NS1) genes (complete cds; gi: 227809838; gb: FJ966086.1). In addition, the generated protein structure has indicated that the NS1A protein was folded and formed into β-sheets bundle and covalently linked with other secondary structures by non-structural coils with conformational change at its loop or turn region.

The pET-32c expression vector was selected for this project. This vector possesses a powerful T7 promoter. It was documented that the use of strong promoter could enhance high protein expression (reviewed by Fischer et al., 1993; Thomas and Baneyx, 1996) as well as protein misfolding (Baneyx and Mujacic, 2004). Evidence has shown that such events may lead to formation of inclusion bodies.

The expression result of NS1A recombinant protein was highly efficient at 37°C room temperature under 5mM lactose induction. Evidently, the lactose was detected to be effective inducer than IPTG in this study. It is believed that the lactose utilization rate in E.coli is slower than IPTG. Typically, longer induction by lactose many lead to accumulation of inclusion bodies and protein folding (Howhana and Pornbanlualapa, 2003).

Generally, the recombinant protein expression can be fractionated into soluble and insoluble expression. The cytoplasmic soluble expression frequently leads to formation of inclusion bodies (IB) (Baneyx, 1999), which is usually contributed by high protein synthesis (Rudolph and Lilie, 1996). Therefore, the study concerning both soluble and insoluble protein aggregates can help us to gain better insight of NS1A protein expression in E. coli BL21(DE3). In this study, the expected size of NS1A fusion protein is 37kDa, as the 26kDa-NS1A protein was fused to fusion tags of nearly 11kDa in size.

Finding here has shown that the NS1A recombinant protein did not greatly expressed under induction either by 1mM IPTG or 5mM lactose. NS1 protein extensively forms a hydrophobic network especially at the region of its C-terminus (reviewed by Lin et al., 2007). In addition, NS1A effector domain was strongly favored in hydrophobic interaction (Bornholdt et al., 2008) eventhough few regions were hydrophilic-predominated (Fig. 4). Therefore, its hydrophobic nature may be one the reason why the proteins have difficulty to be solubilized and most likely formed into inclusion bodies.

Ammonium sulfate precipitation was applied for protein purification by altering the protein solubility with the highly hydrophobic portion promptly forms a precipitate. It is ideally used to concentrate and selectively separate the protein; in addition, it could encourage the protein recovery and renature. In this study, the 20% ammonium sulfate saturation was sufficient to concentrate and partially purify the target NS1A protein. The immunodetection analysis has established that the recombinant NS1A protein was retained active after 20% ammonium sulfate precipitation by showing positive signal to the probing polyclonal antibody.


In this study, the main focus involved cloning, overexpression, purification and immunogenicity analysis. The NS1A gene was successfully cloned into the BamH1/SacI cleaved-pET-32c(+) vector and subsequently transformed to the E.coli BL21(DE3) expression host. This study has initially investigated the soluble and insoluble expressions of NS1A recombinant protein in the bacterial cytoplasm. Better protein expression was by 5mM lactose at 37°C. The NS1A recombinant protein was partially purified using ammonium sulfate precipitation at 20% saturation. The precipitated NS1A recombinant protein, which exhibited band at 37kD, has characterized significant antigenicity to the H1N1 NS polyclonal antibody in Western blot analysis. Therefore, it can be concluded that ammonium sulfate precipitation at 20% saturation is a suitable step to partially purify NS1A recombinant protein from the solubilized inclusion bodies fraction. This project already set up a good baseline data for biosensor study to detect preferential binding of a RNA aptamer and its corresponding interaction pathway. In addition, this type of study is required for further investigation of the virus pathogenesis to accelerate vaccine development.


I would like to express my sincere gratitude to my supervisor, Dr. Chan Giek Far, for initiating this research project and for her continuous guidance during the study. A thank you also to all members of Faculty of Biosciences and Bioengineering for guiding and giving me constructive suggestion of this project.