Characterization of the overexpressed recombinant human α11 integrin I domain in Escherichia coli for functional analysis

doi:10.21203/rs.3.rs-5290381/v1

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Characterization of the overexpressed recombinant human α11 integrin I domain in Escherichia coli for functional analysis

https://doi.org/10.21203/rs.3.rs-5290381/v1

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Integrins are crucial adhesion receptors involved in cell-matrix and cell-cell interactions. Integrin α11β1 has been linked to tumor progression, metastasis, angiogenesis, fibrotic diseases, and wound healing. The I domain of integrin α11 plays a vital role in ligand binding, integrin activation, and cell signaling. In this study, we focused on expressing the I domain of integrin α11 in E. coli C41 (DE3) using lysis buffers like Triton X-100, sarkosyl, and urea. We also examined the effect of osmolytes on protein stability. A solid-phase assay assessed the binding of the recombinant I domain to type I collagen. Our findings showed that expression condition changes did not affect the I domain's solubility. We successfully obtained a soluble I domain using sarkosyl lysis buffer and purified it via nickel affinity chromatography, optimizing its stability with 40 mM mannitol. The binding of the I domain to type I collagen is concentration-dependent. This study outlines a method for expressing, purifying, and stabilizing the recombinant I domain of integrin α11, which may facilitate further investigations into this protein.

Biological sciences/Biochemistry

Biological sciences/Biotechnology

Integrin α11β1

Recombinant I domain

Prokaryotic expression system

Sarkosyl

Osmolytes

Solid-phase assay

As a type I transmembrane adhesion receptor, integrins are crucial in cell anchoring, tissue, and organismal architecture ^1,2. They are involved in various processes, including embryonic development, angiogenesis, cell proliferation, adhesion, and migration. Integrin dysregulation is linked to the development of various disorders, including altered angiogenesis, inflammation, and susceptibility to infections. Under specific conditions, treatment techniques may directly target integrins or their binding ligands ^3–6. Integrins comprise α and β subunits, each containing multiple domains connected by flexible linkers. Both subunits have single-spanning transmembrane domains and short cytoplasmic tails¹. The extracellular part of integrin α subunits consists of four or five extracellular domains that come together to form a seven-bladed β-propeller, a thigh domain, and two calf domains. Of the 18 α subunits, 9 have an I domain (or A domain). Four of these integrins with α-I domains are collagen receptors, namely α1β1, α2β1, α10β1, and α11β1 that mediate interaction with collagens ^2,7,8.

The α11-I domain of integrin α11β1, a key player in the formation of tumors and the ability of specific carcinoma cells to metastasize ^6,9–11 has been linked to the GFOGER (O=hydroxyproline) sequence in interstitial collagen. It prefers attaching to type I collagen over other collagens ¹². The expression of the α11-I domain in E. coli allows for detailed structural and biophysical characterization. It can be utilized in various biochemical and biophysical assays, such as ligand-binding studies and surface plasmon resonance (SPR) experiments. These assays can help us understand the interactions between the A domain and its extracellular matrix ligands. They can also be used to find potential inhibitors or modulators of integrin α11 function. Additionally, they can serve as an antigen to produce monoclonal or polyclonal antibodies that target specific epitopes on the integrin α11. These antibodies are valuable for studying integrin alpha 11 expression, localization, and function in various biological systems, including in the context of cancer and other disease ^13,14

Many proteins expressed in E. coli are highly insoluble, and suboptimal conditions during expression, purification, and storage may impact protein structure, leading to loss of activity in protein aggregates. ¹⁵. The coaggregation of expressed protein with bacterial outer membrane components may occur through processes similar to the self-aggregation of folding intermediates. Recovering protein from the inclusion body requires high quantities of chaotropic chemicals, such as urea, guanidinium chloride (Gdn-HCl), and sodium dodecyl sulfate (SDS). These chemicals destroy the protein structure, leading to decreased protein activity. As a result, refolding of the inclusion body protein into bioactive forms is problematic, resulting in poor recovery and increased production costs. ¹⁶. Therefore, adjusting the cell lysis buffer conditions, such as pH, ionic strength, or the presence of an additive or mild detergent, could significantly impact the solubility of the produced proteins¹⁵. N-lauroyl sarcosine, commonly known as the sarkosyl detergent, is a relatively mild chaotropic that influences the aggregation process by disrupting lipid bilayers. It can maintain proteins in a soluble form at low concentrations and is particularly useful for dissolving membrane proteins and specific other hydrophobic proteins ^17,18.This study demonstrated a practical and reliable approach for producing highly pure, soluble, and active integrin α11 ectodomain. The recombinant production of integrin α11 I-domain in E. coli C41 (DE3) was achieved using a Sarkosyl-based lysis solution for protein isolation and subsequent purification using nickel affinity chromatography. The study confirmed that the expressed protein retained their native structures and exhibited appropriate bioactivities, thereby enhancing the availability of the protein for various research applications and potentially advancing the development of integrin alpha 11-targeted therapeutics, providing a solid foundation for future research and development in the field.

2.1. Materials

Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Escherichia coli C41 (DE3) cells were obtained from Novagen in the USA. The pET21a (+) vector was procured from GeneCust in Luxembourg. Peptone and yeast extract were purchased from Difco (Detroit, MI, USA). Other reagents, all of the analytical grade, were provided by Merck (Darmstadt, Germany). Dr. Sadegh Hasannia (Tarbiat Modares University, Tehran, Iran) gifted Collagen type I. Ati-His tag and 3,3’,5,5’-tetramethylbenzidine (TMB) were provided by Sina Biotech Co and DNA Biotech Co (Iran), respectively.

2.2. Synthesis and cloning I domain gene sequence

GeneCust company synthesized the codon-optimized sequence of the I domain, which was derived from the residues 162 to 345 of integrin α11 (UniProtKB: Q9UKX5) and cloned in the pET2-a(+) expression vector (pET21a-I domain). The His6-tag was placed at the C-terminus to purify the protein by Ni-affinity chromatography.

2.3. The overexpression of I domain

The pET21a-I domain construct was introduced into E. coli C41 (DE3) using chemical transformation. The E. coli C41 (DE3) transformed cells were cultured in LB medium supplemented with the appropriate antibiotic at 37°C for 16 hours. The gene expression was induced when the optical density reached around 0.5. This was achieved by treating the cells with 0.1, 0.5, and 1 mM IPTG for 5, 14, and 22 hours at temperatures 18, 25, and 37°C, all at 220 rpm. After induction, the cells were harvested by centrifugation at 4000 ×g for 15 minutes at 4°C and then lysed by sonication in tris buffer pH 7.8 with various additional components (refer to Table 1). The sample's homogeneity was confirmed by utilizing 12.5% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2.4. Purification of recombinant I domain

To remove sarkosyl, supernatant fractions were dialyzed against 50 mM Tris-HCl buffer with 100 mM NaCl (pH 7.8). After dialysis, the solution was applied to a Ni-NTA agarose column pre-equilibrated 50 mM Tris-HCl,100 mM NaCl, and 5 mM imidazole buffer (pH 7.8). Impurities were removed using a Tris-HCl buffer at pH 7.8, 100 mM NaCl, and various concentrations of imidazole. Proteins were eluted using 300 mM imidazole in the same buffer¹⁹.

2.5. Storage conditions assessment

The storage condition for the purified proteins was determined by dialyzing them against different buffers, as listed in Table 2. After dialysis, the samples were stored at -80°C for 48 and 96 hours. Subsequently, the samples were centrifuged at 12,000 xg for 20 minutes at 4°C, and the protein concentration was measured using the Bradford method.²⁰. After centrifugation, an equal volume of each sample was separated and analyzed using SDS-PAGE to investigate the amount of soluble protein. The optimal buffer containing appropriate stabilizing chemicals was chosen to achieve the highest thermal stability.

2.6. Surface hydrophobicity study

The integrin alpha 11 model (ID: Q9UKX5) was used in the Alpha Fill database to visualize and analyze molecular structures and surface hydrophobicity by UCSF Chimera tool (v.1.10.2). The Kyte and Doolittle hydrophobicity scale (ProtScale, ExPASy) was utilized to indicate the hydrophobicity properties of amino acids.²¹.

The surface hydrophobic areas of the I domain were probed at a concentration of 0.1 mg/mL using 50 µM 8-anilino-1-naphthalene-sulfonic acid (ANS) at 25ºC. The excitation wavelength was 375 nm, and emission was monitored between 400 and 600 nm using the PerkinElmer LS55 fluorescence spectrometer²².

2.7. Solid-phase binding assays

The plastic surfaces of MaxiSorp™ 96-well microplates (Nunc) were coated with 100 µL of 50 mg/ml collagen I solutions overnight at 4°C. Wells were blocked with 200 µL of 3% BSA in PBS for 1 hour. This step and other remaining steps were carried out at room temperature with a buffer comprising 20 mM Tris, 0.15 M NaCl, 2 mM MgCl₂, 20 µM CaCl₂, and 0.05% Tween-20, pH 7.8. The samples incubated by α11-I domains were diluted in a buffer with 30 μg/mL BSA. After thorough washing, the binding ligands were identified by adding 100 µL of anti-His antibody coupled with horseradish peroxidase (1:1000 dilution) and incubating for 1 hour at room temperature. Following four further washes, 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well and incubated for 10-15 minutes at room temperature in the dark. After adding 100 µL of stop solution, absorbance was measured at 450 nm. Three parallel samples were evaluated. Estimates for the dissociation constant (K_D) were obtained using the Scatchard plot and the one-binding class models using the GraphPad PrismTM software (GraphPad Software Inc., San Diego, CA) ^23,24.

3.1. Cloning and overexpression of integrin α11-I domain in E. coli C41

The DNA sequence of the integrin α11-I domain was inserted downstream of the bacteriophage T7 promoter in the pET21a expression vector. After verifying the sequence through sequencing, the construct was successfully transformed into E. coli C41 (DE3). To optimize the soluble expression conditions of the I domain, we tested various IPTG concentrations (0.1, 0.5, and 1 mM) and temperatures (18, 25, and 37 °C), as shown in Figure 1. The harvested cells were then lysed in 50 mM Tris-HCl and 100 mM NaCl at pH 7.8. The results indicated that under the different conditions, the target proteins were primarily expressed as inclusion bodies. Furthermore, the various expression conditions did not significantly differ in the protein expression amount (Figure 1).

Different lysis buffers with various additives were used to obtain the soluble form of the protein, as shown in Table 1, after expressing the protein by 0.5 mM IPTG at 25 ˚C for 14 hours.

After sonication in different buffers, the SDS–PAGE results demonstrated that buffers with varying sarkosyl concentrations can render proteins soluble (Figure 2). The study identified buffers containing sarkosyl as the most effective lysis buffers, and increasing sarkosyl concentration leads to a higher amount of soluble protein. To simplify the purification process and remove sarkosyl in the subsequent steps, we carefully selected buffer B2 containing 0.6% sarkosyl.

3.2. Purification and determining best condition storage

The target polypeptide was purified using Ni²⁺ affinity chromatography. Histidine labeled in the C-terminal domain of integrin α11-I was bound to Ni²⁺ chelate beads. A tris-HCl buffer with a range of imidazole concentrations was used to remove impurities, and the α11-I integrin domains were eluted with a buffer containing 300 mM imidazole. The purification efficiency increased after removing sarkosyl from the supernatant through dialysis. The target protein was easily eluted at 300 mM imidazole (Figure 3).

To confirm the stability of the purified recombinant protein during storage, an equal volume of the sample was stored at -80^°C and subjected to multiple freezing and thawing cycles. The results indicated that the recombinant proteins aggregated after 48 hours. To improve protein stability during storage, we added various stabilizers to the purified samples through dialysis (refer to Table 2) and stored them at -80°C for 48 and 96 hours^25–29. Subsequently, we centrifuged each sample and analyzed the supernatant solutions, all of which were of equal volume, using SDS-PAGE (Figure 4). Additionally, we used the Bradford assay to determine the soluble protein concentration in each sample (Figure 5). The results demonstrated that the recombinant protein exhibited good stability in a buffer containing 40 mM mannitol. As a result, we incorporated 40 mM mannitol into all dialysis buffers and stored samples at -80°C.

3.3. Investigation of protein structure by fluorescence spectroscopy

The I domain's hydrophobic surface areas were investigated in the presence of ANS, a hydrophobic fluorophore dye. By binding to exposed hydrophobic patches, ANS played a crucial role in exploring the protein's structure²². The increase in fluorescence emission confirmed the presence of hydrophobic patches on the protein's surface and its high surface hydrophobicity (Figure 6).

3.4. Bioactivity assay

The integrin α11-I domain's binding and bioactivity were assessed using solid-phase binding assays. Collagen I was used as the solid ligand. The interaction was detected using a His-tag antibody due to the histidine tag at the c-terminal of the integrin α11-I domain. The dissociation constant (K_D) of the I domain and collagen type I interaction was found to be 0.4960 µM, indicating the suitable binding of the expressed protein (Figure 7).

Recent research indicates that integrin α11 is overexpressed in the stroma of desmoplastic malignancies, including lung, breast, and Pancreatic carcinoma, as well as fibrotic tissues ^5,9,30,31. The study previously expressed the functional domain of integrin alpha 11 (domain I) in High-Five insect cells, which is responsible for binding to collagen ³². Prokaryotic expression, such as in E. coli cells, offers advantages over other expression techniques, including faster production periods, more straightforward transformation, and more effective fermentation technology ^16,32,33. Therefore, the study investigated integrin alpha 11 expression in E. coli cells. According to a study, a recombinant integrin β1 ectodomain was mainly produced as inclusion bodies in E. coli BL21 (DE3) cells ³⁴. It is important to note that prokaryotic systems often produce many proteomes, notably membrane proteins, as inclusion bodies. This can lead to meager yields of appropriately folded proteins, which may negatively affect protein function. Additionally, it can pose challenges and require extended timeframes for the refolding processes following the expression pathway ³⁵.

The pET expression system was chosen to overexpress the exogenous integrin α11-I domain. Despite significantly adjusting the expression conditions, we could not express soluble proteins, and the target proteins were primarily expressed as inclusion bodies (Figure 1). As a result, we tested and compared the effects of several detergents on obtaining active and soluble protein. Recombinant proteins, particularly those of eukaryotic origin, can tend to congregate or become packed into inclusion bodies (IB) in E. coli ³⁶. Biochemical evidence suggests that the insolubility of recombinant actin may be caused by coaggregation with bacterial outer membrane components during lysis. Several theories explain insolubility, such as the build-up of partially folded forms that will aggregate due to hydrophobic interactions and the triggering of the heat shock response, which could lead to an abnormal environment for protein folding^37–39. Several studies have demonstrated that the outer membrane lipopolysaccharide and protein have strong hydrophobic interactions and tend to assemble. As part of the lysis process, these components break apart, revealing hydrophobic and anionic surfaces that can trap the exposed hydrophobic or cationic surfaces of produced proteins³⁹.

Inclusion bodies, also known as protein aggregation, occur when a protein loses its original structure and takes on a different shape, leading to aggregation³⁸. These aggregates come in various forms, such as fibrils, amorphous aggregates, and non-classical inclusion bodies. Non-classical inclusion bodies contain large amounts of proteins that are likely misfolded but have higher solubility. As a result, they can be extracted using mild, non-denaturing solutions ^40,41.

Detergents are versatile compounds with unique properties that allow them to manipulate hydrophobic-hydrophilic interactions in biological samples. They are commonly used in lysis buffers to break down cell membranes, releasing soluble proteins and lipids. Additionally, detergents help prevent non-specific binding during various processes^39,42. It is important to note that the type and concentration of detergent used can significantly impact cell lysis efficiency and the recovered proteins' stability and activity⁴². Triton X-100 is a non-ionic mild detergent, 3-[(3-Cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS or CHAPSO) is a zwitterionic detergent with nondenaturing characteristics, and urea is a chaotropic chemical used to increase protein solubility ^16,18. Non-classical inclusion bodies can be solubilized using low concentrations of organic solvents, such as 5% n-propanol and DMSO, as well as detergents like 0.2% N-lauroyl sarcosine ⁴⁰. When investigating the His6-tagged fusion protein glutathione S-transferase (GST), harvested cells were incubated in lysis buffer with 2-5% (up to 10%) sarkosyl for 12-24 hours at room temperature with shaking. This process ensures complete cell lysis and enhances protein solubility ¹⁸.

These amino acids were then analyzed using the UCSF Chimera tool, which revealed hydrophobic patches in the α11-I domain protein (see Figure S2). The Kyte & Doolittle hydropathy index of amino acids was also used to represent the hydrophobic patch in the integrin α11-I domain (see Figure S3) confirmed by the fluorescence spectrum in the presence of ANS (Figure 6)

The integrin α11-I domain has a His6-tag at its C-terminal, which allows for easy purification of recombinant proteins using Ni-NTA affinity chromatography. The purification yield is low when sarkosyl is present (see Figure 3a). The critical micelle concentration (CMC) of sarkosyl is 14 mM. Therefore, it is possible that dialysis can remove sarkosyl in subsequent purification steps ^18,43. Following the reduction of sarkosyl through dialysis, the purification yield significantly increases (Figure 3b). On the other hand, removing sarkosyl causes protein aggregation. Numerous studies indicate that various osmolytes can enhance protein stability, including trehalose, glycine betaine, mannitol, L-arginine, CuCl₂, proline, xylitol, CTAB, and others ^44–48. Osmolytes play a significant role in restoring the folded structure and mobility of denatured proteins by changing the characteristics of the solvent ^49,50. To increase protein stability during storage, we used different additives (Table 2), and the results showed that 40 mM mannitol can increase protein stability at -20 °C (Figures 4 and 5).

Integrin α11β1 is a collagen receptor that can recognize the GFOGER sequence in collagens. The I domain plays a crucial role in collagen binding ¹². Solid phase tests can measure the ligand binding of integrin α11β1 ²⁴. The interaction of the α11-I domain with collagen type I demonstrated that the recombinant integrin α11-I domain is biologically active (see Figure 7).

This study outlines a method for expressing, purifying, and enhancing the stability of the soluble recombinant I domain of integrin α11 in E. coli. We examined the impact of adding various additives at different concentrations to the lysis buffer to obtain the recombinant protein in a soluble form. Our observations revealed that the optimal additive was 0.6% sarkosyl, and the most effective additive for enhancing protein stability after purification and removing imidazole was 40 mM of mannitol. The recombinant protein exhibited the expected activity, indicating that the I domain of integrin α11 is suitable for further characterization of integrin α11. Notably, the methodology outlined in this study has broader applications and could be utilized to optimize buffer conditions for similar proteins, thereby advancing research in this field.

Acknowledgment

The authors express their gratitude to the research council of Tarbiat Modares University for funding this project.

Author contributions

Elaheh Tavili: Writing – original draft, Visualization, Validation, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Bahareh Dabirmanesh: Writing–review & editing. Massoud Amanlou: editing. Khosro Khajeh: Project administration,Writing – review & editing.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

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Table 1- The lysis buffer formulations

Buffer	Component
B1	50 mM Tris-HCl pH 7.8, 0.3% Sarkosyl
B2	50 mM Tris-HCl pH 7.8, 0.3% Sarkosyl, 2 M Urea
B3	50 mM Tris-HCl pH 7.8, 0.5% (w/v) SDS
B4	50 mM Tris-HCl pH 7.8, 1% Sarkosyl
B5	50 mM Tris-HCl pH 7.8, 0.6% Sarkosyl
B6	50 mM Tris-HCl pH 7.8, 2 M Urea
B7	2% Triton X-100, 0.01 % Tween 20
B8	Radio-immunoprecipitation assay buffer (RIPA buffer) pH 7.8
B9	50 mM Tris-HCl pH 7.8, 20% Glycerol

Table 2. Additive’s composition of storage buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.8)

Additive	Final concentration
Mannitol	40 mM
Trehalose	1.75 mM
L-Arginine	0.065 mg/ml
Sucrose	40 mg/ml
Glycerol	40 % v/v
No additive	-

No competing interests reported.

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Characterization of the overexpressed recombinant human α11 integrin I domain in Escherichia coli for functional analysis

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