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 folding37–39. 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 processes39,42. 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.