Cloning, Improved Expression and Purication of Invasion Plasmid Antigen D (IpaD): An Effector Protein of Enteroinvasive Escherichia Coli (EIEC)

The widespread increase in broad-spectrum antimicrobial resistance is making it more dicult to treat gastrointestinal infections. Enteroinvasive Escherichia coli is a prominent etiological agent of bacillary dysentery, invading via the fecal-oral route and exerting virulence on the host via the type III secretion system. IpaD, a surface-exposed protein on the T3SS tip that is conserved among EIEC and Shigellae, may serve as a broad antigen for bacillary dysentery protection. For the rst time, we present an effective framework for improving the expression level and yield of IpaD in the soluble fraction for easy recovery, as well as ideal storage conditions, which may aid in the development of new protein therapies for gastrointestinal infections in the future. To achieve this, uncharacterized full length IpaD gene from EIEC was cloned into pHis-TEV vector and induction parameters were optimised for enhanced expression in the soluble fraction. After anity-chromatography based purication, 61% pure protein with a yield of 0.33 mg per litre of culture was obtained. The puried IpaD was kept the secondary structure with predominant α-helical structure at 4°C, -20°C, and -80°C using 5% sucrose as cryoprotectants during storage which is the fundamental parameter for protein-based therapeutics. insoluble part were separated by centrifugation. The soluble fraction was collected in the supernatant portion by centrifuged at 20,000 rpm for 30 min at 4°C, and the insoluble part containing inclusion bodies was collected as pellet at bottom of the tube. The pellet was washed two times with a lysis buffer to remove any contaminant of soluble part. 8 M urea was added to the pellet to solubilize the insoluble inclusion bodies and boiled for 15 min, harvested by centrifugation. Protein expression and solubility were analysed by SDS -PAGE using ImageJ Software following two formulas.


Introduction
The Global Enteric Multicenter Study (GEMS) reports bacillary dysentery as one of the leading causes of morbidity and mortality in young children under 5 years of age in developing nations such as India, Bangaladesh, Sri Lanka, Nepal, Bhutan, and Myanmar ). According to the Global Burden of Disease 2016 study, it is ranked as the eighth leading cause of mortality, responsible for more than 1·6 million deaths worldwide Troeger et al. 2018). Enteroinvasive Escherichia coli (EIEC), a pathotype of E. coli, is the leading cause of in ammation and ulceration of the intestinal epithelium in humans, followed by bloody and mucoid diarrhoea. The pathogenicity mechanism for both EIEC and Shigella is similar, hence the physiological and biochemical differentiation of the infection caused by them is quite challenging (Lan et al. 2004; Van den Beld & Reubsaet 2012; Van den Beld et al. 2019). Thus, the evaluation of the actual burden of EIEC induced bacillary dysentery is challenging. The EIEC infections are generally sporadic, however few well studied cases of outbreak such as the 1970s outbreak in United States (Marier et al. 1973), the 2012 outbreak in Italy (Escher et al. 2014) and two linked outbreaks of United Kingdom in 2014 (Newitt et al. 2016) reiterate that causative agents of bacillary dysentery is not limited to the species of Shigella genus, but EIEC signi cantly partakes in such events.
Diarrhoea has been a long-standing priority for the World Health Organization, yet no vaccine for bacillary dysentery currently exists (Hosangadi et al. 2019). Thus, it is one of the leading causes of antibiotic prescription and consumption among children in low-and middle-income countries, leading to resistance against the major third-generation antibiotics (Eltai et al. 2020;Rogawski McQuade et al. 2020). Further, the limited understanding of EIEC and antimicrobial therapy necessitates the development of effective vaccines against diarrhoeal pathogens, which may contribute to the ancillary bene ts such as reducing antibiotic exposure and resistance (Nguyen et al. 2005). The mechanism of virulence in many Gram-negative pathogens is facilitated by the type III secretion system (T3SS). It serves as a conduit for transfer of bacterial effector proteins into the host cell facilitating the host invasion and infection by the bacteria. IpaD, a conserved 37 kDa hydrophilic protein present on T3SS needle tip controls translocator and effector protein secretion in EIEC is the rst bacterial protein that interacts with the host cell (Espina et al. 2006). The immunogenic nature of IpaD con rmed by immune pro ling, together with its conserved nature across the species of the genus Shigella and EIEC makes it promising vaccine candidate (Martinez-Becerra et al. 2012; Ndungo et al. 2018;Turby ll et al. 1998).
Numerous studies have focused on understanding the immunoprotective behaviour of IpaD as vaccine candidate. Jahantigh et. al. reported that IpaD shows a highly protective humoral response in guineapig as IgA titter level signi cantly increased upon nasal administered of chitosan nano brous membrane containing Nterminal region of (Jahantigh et al. 2014). Another study with N-terminal IpaD loaded trimethylated chitosan nanoparticles induced increase in IgG and IgA levels in guinea pigs and exhibited protective behaviour against Shigella infection (Akbari et al. 2019). These studies clearly enphasize the importance of IpaD protein based vaccine conjugated to different carriers. It is also to note that in order to design and develop protein based formulations, it is critical to optimize the parameters for enhanced expression and recombinant puri cation of IpaD in E. coli. But to date, there is no report on optimization of parameters for expression, puri cation and storage to ensure structural and functional stability of protein, which are crucial for the development of any biopharmaceutical formulation.
In the present study, we report the molecular cloning of IpaD into pHis-TEV vector, optimization of expression parameters such as inducer concentration and temperature to improve the yield of IpaD protein. Here, we also report effective puri cation strategies of IpaD by using simple Ni-NTA a nity chromatography. To the best of our knowledge, this study is the rst attempt to clone IpaD gene of EIEC and subsequent successful expression and puri cation of the protein in E. coli BL21 (DE3) strain was achieved. At the optimized IPTG concentration of 0.5 mM, resulted in ∼37% IpaD expression with ∼67% protein distribution in the soluble fraction-a relatively high quantity. After Ni-NTA a nity chromatography puri cation 61% pure protein was obtained. We further studied the optimal temperature and buffer for storage so that structural and functional integrity of the protein remains unaffected for its potential application as biopharmaceuticals. Our results demonstrate that the puri ed IpaD can be stored at 4°C, -20°Cand -80°C with no structural loss, con rmed by CD analysis.

Materials
All chemicals were used in this study was analytical grade and used as received without any further puri cation.
All solutions were prepared in milli-Q ultrapure water of resistivity not less than 18.2 MΩ cm -1 .

Bacterial strains and plasmid
The bacterial strain Enteroinvasive Escherichia coli was gifted from NICED, Kolkata. All plasmid and expression vector used in this study were listed in  (Hazen et al. 2016). To collect the cell pellet, the culture was centrifuged at 13000 rpm for 5 minutes. The alkaline lysis procedure was used to isolate the plasmid DNA (Feliciello & Chinali 1993).Primers were generated using the IDT oligo analyzer tool and synthesized by IDT Technologies (India) for the ampli cation of the IpaD gene. To insert ampli ed IpaD gene into the pHis-TEV plasmid Vector, forward and reverse primers (Table No. 2) were constructed containing EcoRI and XhoI restriction endonuclease enzyme sites respectively. Gradient polymerase chain reaction was performed using the thermal Cycler (Biorad T100) to optimise the annealing temperature. A gradient temperature of 57.5°C to 61.5°C was set. The reaction was initiated by heating the reaction mixture at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s, and elongation at 68°C for 1 minute, and nally extension at 72°C for 10 minutes. Using the DNA agarose gel Electrophoresis Apparatus (Tarsons), the PCR product was analyzed in a 0.8% agarose (Sigma Aldrich) gel run at 70 V for 1 hour in 1X TAE (Tris-acetate-EDTA) buffer and bands were visualized using the UV Transilluminator (Himedia). The PCR product was puri ed using a Qiagen PCR Puri cation kit according to the manufacturer's instructions. The Nano-Drop Spectrophotometer (Eppendorf) was used to determine the DNA concentration.

Molecular cloning into expression plasmid and veri cation of the insert
The puri ed PCR product, i.e. IpaD gene and pHis-TEV plasmid vector were digested with EcoRI and XhoI restriction enzyme in digestion buffer (New England Biolabs) at 37°C for 2 hours as directed by the manufacturer and analysed on a 0.8% agarose gel. The T4 DNA ligase enzyme (New England Biolabs) was used to ligate the digested gene and plasmid vector, which was done at 16°C overnight as per the manufacturer's protocol. The resulted ligated product (pHis-TEV-IpaD) was transformed into Escherichia coli-DH5α competent cell following standard CaCl 2 heat shock transformation protocol (Li et al. 2010) and the full construct of pHis-TEV containing IpaD gene was shown in (Fig. 1A). The positive colony was con rmed by double digestion analysis using EcoRI and XhoI restriction enzyme and colony PCR which were analyzed in a 0.8% agarose gel. The separated gene and vector fragmented part were visualized using the Gel Doc (Biorad) system and analyzed by Image Lab (Biorad) Software. The colony was maintained Luria Bertani agar (Himedia) plate containing ampicillin (100µg/ml) (Himedia) as selectable marker. For DNA sequencing, plasmid DNA was isolated from the positive colony using Qiagen Plasmid isolation kit as per manufacturer protocol. The sequencing was done by Integrated DNA Technologies (India) by the Sanger Sequencing method. NCBI Blast was used to analyse the sequence results.

IpaD protein expression optimization
Optimization of inducer concentration Escherichia coli-BL21 (DE3) cells containing pHis-TEV-IpaD plasmid was grown overnight in Luria Bertani Broth supplemented with ampicillin (100 μg/ml). Five asks containing fresh LB media were inoculated with overnight grown culture (1:100) and incubated at 37°C, 150 rpm, until the OD 600 reached between ~0.7-0.8. The cultures were induced individually by ve different isopropylthio-β-galactoside (IPTG) Concentrations, respectively 0.05mM, 0.25mM, 0.5mM, 1 mM and 2 mM for 18 hours at 15 C with 150 rpm. After induction, cells were harvested by centrifugation at 8000 rpm for 10 min at 4°C then the resultant pellet was resuspended in lysis buffer (20 mM Tris (Himedia) (pH 8.0), 500 mM NaCl (Himedia),10 mM imidazole (Himedia) and 5% sucrose (Himedia) in a 1:100 lysis buffer:culture volume ratio. Then cells were disrupted by sonication (5 cycles, 15second pulse with 1-minute interval). To prevent protein degradation, 1mM Phenyl methyl sulfonyl uoride (PMSF, Sigma Aldrich) and 1mg/ml lysozyme (Himedia) were added before sonication. The soluble and insoluble part were separated by centrifugation. The soluble fraction was collected in the supernatant portion by centrifuged at 20,000 rpm for 30 min at 4°C, and the insoluble part containing inclusion bodies was collected as pellet at bottom of the tube. The pellet was washed two times with a lysis buffer to remove any contaminant of soluble part. 8 M urea was added to the pellet to solubilize the insoluble inclusion bodies and boiled for 15 min, harvested by centrifugation. Protein expression and solubility were analysed by SDS -PAGE using ImageJ Software following two formulas.
Whereas, S is the amount of IpaD protein and I is the total protein after induced by IPTG; S'' is the amount of the IpaD in supernatant fraction and P is the amount of total protein in pellet fraction

Optimization of post induction temperature
Escherichia coli-BL21 (DE3) cells harbouring pHis-TEV-IpaD plasmid were grown overnight with antibiotic containing LB media. From the overnight culture, three asks containing fresh media were inoculated and kept at 37 C with 150 rpm until the OD 600 reached ~0.7-0.8. After that, the effect of temperature on the expression of IpaD, culture was induced with different IPTG concentration and kept at three different temperatures such as 10 C, 15 C and 37 C for 18 hours. After the completion of incubation time, cells were harvested and disrupted by sonication, and expression was analyzed by densitometry analysis.

Puri cation of IpaD protein
Escherichia coli BL21 (DE3) containing pHis-TEV-IpaD plasmid was grown for 18 hours at 15° C with 150 rpm after induction with 0.5 mM IPTG. Then induced cells were collected by centrifugation and disrupted by sonication. Consequential supernatants were separated by centrifugation and ltered through a 0.45µm syringe lter unit (Himedia) to the trace amount of debris. Then, IpaD protein was puri ed by Ni-NTA a nity chromatography using the 5 ml His trap column (GE Health Care) as per manufacturer instructions with minor modi cation. Brie y, the column was equilibrated with 10 column volume (CV) equilibration buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 10 mM imidazole) containing 5% Sucrose. The supernatant was passed through the column four times for proper binding, and ow-through was collected. Afterwards, the column was washed twice with 10ml CV wash buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 5% Sucrose) with two different concentration of imidazole (20 mM imidazole, 60 mM imidazole) and wash fraction was also collected. The protein was eluted by applying the 5 ml elution buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 5% Sucrose) with three different concentration of imidazole (150 mM imidazole, 250 mM imidazole, and 500 mM imidazole). Then eluted fractions were immediately diluted with 1:1 ratio dilution buffer (20 mM Tris (pH 8.0), 500 mM NaCl) and were dialyzed to remove out the salt and imidazole against 1X TBS Buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5% sucrose) for 6 hours. The his tagged-IpaD protein was incubated with TEV protease overnight at 4°C (Sigma Aldrich) to remove the His-tag as described in the user manual provided by manufacturer. Further, the IpaD protein was applied to the His-trap column, and the ow-through fraction was collected as puri ed protein.
Protein concentration was estimated using a Bradford reagent (Biorad) and analyzed by SDS -PAGE.

Size exclusion chromatography
IpaD was further concentrated using Amicon ultra lter (Millipore) and puri ed by size exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare). Column was equilibrated using the mobile phase as TBS Buffer (20mM Tris, 150mM NaCl pH-7.4) containing 5% sucrose. The ow rate was maintained at 0.4ml/min, and UV detection was set at 280 nm. 500 µg protein was loaded with injection volume of 500µl. Eluted puri ed was analysed by SDS PAGE.

Circular dichroism analysis
For secondary structure analysis, Far-UV CD was performed using J-815 Spectrophotometer (Jasco). Three Spectra was measured between the 250-200 region with scan speed 50nm/min and bandwidth 1 nm. 1X TBS (20mM Tris, 150mM NaCl pH-7.4) buffer containing 5% sucrose was used as control for the analysis.

Statistical analysis
Statistical analysis was performed by using Student's t test to calculate signi cance of differences. All experiments were accomplished at least three replicates and the data are presented as the mean ± standard deviation (SD).The values of *p < 0.05 represents signi cant, **p < 0.01and ***p < 0.001 represents very signi cant.

Molecular cloning of IpaD
Cloning of full length IpaD gene from EIEC was carried out by PCR ampli cation using the speci c primers ( Table 2). For ampli cation, different annealing temperatures were set based on the melting temperature of primers, and the optimal annealing temperature was determined to be 59.5°C as evident from the agarose gel band intensity which was maximum for lane 3 ( kbp, depicting gene and vector respectively (Fig.1C, Lane 6). The undigested empty vector showed three distinct bands depicting nicked, linear, and supercoiled structures respectively in agarose gel (Fig.1C, Lane 2); whereas, a single band depicting linear structure was present for the double digested empty vector (Fig.1C, Lane 3).
Furthermore, the insertion of IpaD gene was again con rmed by conventional colony PCR. Three colonies were picked randomly as templates and colony PCR was performed. Agarose gel results showed a band at ~ 1kbp for one colony (Fig.S1, Lane 2), which was also con rmed to be positive by double digestion experiments (Fig. 1C).
A positive control was run to validate PCR reaction using the ampli ed product as template (Fig.S1, Lane 5). After getting the nucleotide sequence the blast result shows that our sequence was 99% identical with E. coli strains and 98-96% identical with Shigella subtypes, but with 100% coverage for both types (Fig.S2). Hence, it might be concluded that the insertion of IpaD gene into the pHis-TEV vector was successful.
Optimization of inducer concentration for IpaD protein expression  (Fig. 2D and 2E) using ImageJ software.
The expression level of IpaD with 0.5 mM IPTG concentration was 37% and increased up to 52% for 1.0 mM IPTG concentration (Fig. 2D). However, the protein solubility substantially decreased from 67% at 0.5 mM of IPTG to 40% at 1.0 mM of IPTG concertation (Fig. 2E). According to the results, as depicted in Fig 2D and 2E, induction by other IPTG concentrations less amount of IpaD was expressed and solubility was decreased. So, among the ve IPTG concentration variation 0.5 mM IPTG concentration was chosen for further study because this concentration displayed maximum soluble expression.

Optimization of post induction temperature for IpaD protein expression
Further, to prevent protein aggregation and achieve maximal soluble expression of IpaD proteins in E. coli three different post induction temperatures was varied .To this end, three asks containing LB medium were cultivated under optimized conditions. As shown in Fig 2D and 2E, at induction temperature of 37°C and 10°C, both expression and solubility decreased but signi cantly solubility was increased at 15°C.Therefore, the optimised post-induction temperature was preferred 15°C.

Puri cation of IpaD protein
Nickel column a nity chromatography was used for puri cation of the IpaD protein containing 6×His in its Nterminal. The steps involved in puri cation of IpaD is depicted by Fig 3A. A prominent protein band of ~40 kDa corresponding to IpaD present in soluble fraction was observed on SDS-PAGE (Fig. 3B, Lane 2). For separation and recombinant puri cation IpaD, the supernatant was allowed to bind by passing it through Ni-NTA column.
Removal of impurities and non-speci c protein was achieved by extensive washing with 20 mM rst and then with 60 mM imidazole which breaks non-speci c binding of protein. (Fig. S3, Lane 4 and Lane 5). In this study, the imidazole concentration in the elution buffer was varied as follows: 150 mM, 250mM and 500mM. It is observed that imidazole concentrations of 250 mM and 500 mM in elution buffer resulted complete elution of bound IpaD (Fig S4, Lane 8, Lane 7). However, at 150 mM imidazole concentration the amount of IpaD eluted was far less (Fig S4, Lane 9). Together, these results suggested that 250 mM imidazole can be used in elution buffer for successful puri cation of IpaD. IpaD was e ciently puri ed as the eluate consisted mainly of a single prominent protein band of ~40 kDa (Fig. 3B, Lane 5). In each puri cation step, percentage of puri cation and yield were calculated by densitometry analysis of SDS-PAGE using Image J (Table 3) (Burgess, 2009).
His tag was removed by TEV protease cleavage as per manufacturer's instruction. SDS PAGE analysis of tagcleaved IpaD does not exhibit any major difference with His tag protein probably due to the small size of the His tag ~3kDa (Fig 3C, Lane 3 and 4). The densitometry analysis showed that the band corresponded to 60.84% with 2.82 mg pure protein from 1000ml culture. Furthermore, the expressed protein was puri ed by sizeexclusion chromatography. The chromatogram showed that the protein was eluted at retention volume 23.70 ml (Fig 4A). The eluted protein was analysed by SDS PAGE (Fig 4B, Lane1). Quantitative analysis was revealed that each litre of culture yields 0.33 mg pure IpaD protein. Finally, western blot analysis with mouse monoclonal anti-IpaD antibody con rmed the puri ed protein as IpaD (Fig 4C, Lane 1).

Puri ed IpaD stability and integrity analysis
Considering all these factors, the puri ed IpaD protein was subjected to dialysis against TBS pH 7.4 and then stored at four different temperatures (4°C, 25°C, -20°C and -80°C) for 15 days in TBS Buffer (20 mM Tris, 150 mM NaCl, pH 7.4) with 5% sucrose as stabilizing agent to determine the ideal storage condition. After 15 days of incubation at different temperatures, the secondary structure analysis by CD spectrum revealed that the samples stored in 4°C, -20°C and -80°C were structurally stable, detailed discussion follows in the subsequent section.

Circular Dichroism Analysis and Secondary Structure determination
To the best of our knowledge, neither the crystal structure of full-length EIEC IpaD protein nor its secondary structure content has been reported to date. Therefore, we studied the secondary structure content of IpaD using far UV Circular Dichroism (CD) spectroscopy. To estimate the secondary structural contents of puri ed IpaD, far UV CD spectroscopic analysis was performed. CD spectrum of IpaD showed a negative peak at 222 nm, and 208 nm con rming the presence of the α-helix; presence of antiparallel β-sheet were con rmed by presence of the negative peak at 218 nm (Fig. 5A) (Green eld 2006). From the BestSel, the secondary structural components of IpaD consisted of 44% α-helix, 25.1% β-sheet, and 11.3% turn for samples stored at 4°C; 35% α-helix, 16% βsheet and 10.2% turn at -20°C; 26.3 % α-helix, 21% β-sheet and 10% turn at -80°C. However at room temperature i.e. 25°C, percentage of α-helix content IpaD protein drastically decreased to 1.9%, β-sheet increased to 36.4% and 15.3% of turns were observed as shown in Fig.5B. The drastic reduction of α-helixcal structures along with high variation in β-sheet and turns clearly indicate that protein was not stable at room temperature. However, the percentage of secondary structural components viz., α-helix, β-sheet and turns were found to be in close approximation for 4°C and -20°C. For -80°C stored IpaD samples, α-helix percentage slightly decreased in comparison to 4°C and -20°C (Fig 5B). CD analysis revealed that IpaD secondary structure predominantly consisted of α-helix along with a good percentage of β sheet and turn. valuable, the stability of the puri ed IpaD on different storage temperatures has great importance. In this study, the aim was to optimise the soluble expression of recombinant IpaD in the E. coli host and achieve a simple puri cation of the soluble form. Two fundamental parameters were tested: the inducer concentration and the induction temperature. No such extensive study on the recombinant expression of IpaD in an E.coli expression system and the storage temperature effect on puri ed IpaD has been reported previously. Therefore to best of our knowledge, this is the rst attempt to develop a broader picture of IpaD production, stability and integrity of the protein on storage, it is crucial to enhance our understanding of the soluble expression, puri cation of IpaD in E. coli and storage condition.

Discussion
To maximize expression of the IpaD gene, modi ed pET21d (pHis-TEV) vector with an innate 6X His-tag at the Nterminal and a TEV protease site allowing TEV protease enzyme directed cleavage of His tag from the recombinant IpaD protein, was used. The PCR ampli ed IpaD gene product was incorporated next to the T7 promoter and lac operator (Fig. 1A), For IPTG concentration of 0.5mM, the level of IpaD expression was 37% and increased up to 52% for 1.0 mM IPTG concentration (Fig. 2D). However, the protein solubility substantially decreased from 67% at 0.5 mM of IPTG to 40% at 1.0 mM of IPTG concertation (Fig. 2E) at 15°C. With increase in the IPTG concentration beyond 0.5 mM, IpaD protein were predominantly localized into the inclusion body i.e. level of solubility was decreased at higher concentration of IPTG (Fig. 2E) solubility was also affected as bacterial growth was barred due to loss of membrane uidity and enzyme activity (Song et al. 2012). Together these results established that the optimum induction temperature was 15°C for the expression of IpaD protein at 0.5 mM of IPTG concertation, among the three temperatures studied. Earlier, it is reported that the induction temperature 15°C-30°C was the suboptimum temperature range for protein overexpression as the solubility and expression level substantially increased as this temperature range supports production of correctly folded polypeptide and decrease the heat denaturation and heat shock proteases (Song et al. 2012). Therefore, together these results suggested that a low IPTG concentration of 0.5 mM and the induction temperature of 15°C were the optimum conditions required for high yield production of appropriately folded protein in soluble fraction.
In the present study, Ni-NTA column was used for the puri cation of expressed IpaD protein based on the interaction between the immobilized divalent metal ion (Ni +2 ) and imidazole group of histidine (Bornhorst & Falke 2000). The pI of IpaD from Expasy server was calculated to be 5.32 (Wilkins et al. 1999). Therefore, pH of the wash buffer and elution buffer pH was adjusted to 8.0 (> pI) to have a net negatively charged protein (Novák protein elution because of its competitive nature towards the metal against histidine, which helps to elute all proteins without di culty (Lee et al. 2008). Hence, the optimization of imidazole concentration in the elution buffer is a critical parameter for any protein puri cation study. Hence, the optimization of imidazole concentration in the elution buffer is a critical parameter for any protein puri cation study. In the present study we have seen that at 250 mM and 500 mM imidazole concentration almost the same amount of protein eluted but previous study reported that imidazole have a negative impact on protein stability (Walter, 2020). So, from this point of view we have chosen 250 mM imidazole concentration in elution buffer. Tag-less IpaD was further puri ed using Ni-NTA column and was eluted with 60 mM imidazole (Nguyen et al. 2019). In the present study we have calculated band intensity and getting a total 2.82 mg with ~61% pure protein after TEV cleavage and the purity pro le was characterized by size exclusion chromatography, con rmed by western blot analysis with anti-IpaD monoclonal antibody. Therefore, from this study we concluded that by following this simple puri cation method IpaD protein was puri ed successfully.
It is essential to determine an optimal storage condition for puri ed proteins as the structural integrity of proteins may be easily compromised due to aggregation during puri cation, shipping and storage process (Jain

Conclusion
In conclusion, this study is the rst attempt to clone IpaD gene of EIEC and subsequent successful expression and puri cation of the IpaD protein in Escherichia coli BL21 (DE3) strain. The EIEC IpaD gene was successfully cloned into the pHis-TEV vector by optimizing annealing temperature. We have also optimised the expression parameters, such as IPTG concentration and induction temperature -the key players for any recombinant production in soluble form and puri cation of protein e ciently using simple techniques. Our results validate improved puri cation as evident from high-yield and high-purity protein quality. Another important fundamental parameter that we addressed herein is the storage temperature, prerequisite of any protein biologics for further applications. Structural analysis of puri ed IpaD protein showed that it e ciently retained its secondary structure when stored at 4°C, -20°C and -80°C, implying that as a protein therapeutics, IpaD could e ciently maintain its structural and functional stability. Therefore these ndings represent a signi cant step towards the soluble production in E. coli, provide optimal puri cation method and storage condition of IpaD, a potential therapeutic candidate for bacillary dysentery.