1. Introduction
The production of proteins is fundamental to the fields of bio-pharmaceutical production, functional foods, and biosensors, and demand continues to increase (Menzella, Gramajo, & Ceccarelli, 2002; Rasala & Mayfield, 2015; Romanov, Kostromina, Miroshnikov, & Feofanov, 2016; Wells & Robinson, 2017). Proteins can be fabricated using a range of hosts, including Escherichia coli , yeast, mammalian cells, and green algae. Among these, E. coli , which is inexpensive and has a high proliferation rate, is a valuable resource for mass culture for protein production. However, E. coli often produce insoluble protein aggregates, which do not have active three-dimensional structures, and sometimes produce toxic proteins or self-digestive proteases. To recover protein activity, the aggregate must be solubilized using denaturants such as urea or guanidine hydrochloride (GdnHCl), and then reconstructed to produce the original active structure; this process is called “refolding.” A recent steep increase in the demand for highly-functional proteins requires technologies for rapid refolding. From an industrial perspective, the size of facilities required for this refactoring should be minimized, requiring efficient production of high concentrations of proteins (Clark, 2001; Eiberle & Jungbauer, 2010; Zhao et al., 2014).
Current refolding techniques can be generally classified into one of three strategies: dilution; dialysis; or solid-phase treatment (Yamaguchi, Yamamoto, Mannen, & Nagamune, 2013). The dilution method involves refolding proteins by diluting denatured proteins by adding 10 to 1000-fold concentrations of buffer to lower the denaturant concentration to allow protein refolding. While the procedure is simple, the concentration of proteins produced after dilution is generally low, so stirring and storing tanks must be quite large. (Clark, 2001; Eiberle & Jungbauer, 2010) In the dialysis method, proteins denatured using a denaturant are placed in a dialysis membrane bag. As only the denaturant can permeate through the membrane, the protein concentration within the dialysis membrane can be maintained at high levels during the refolding process. The downside of this method is that the process of refolding can take days, lowering productivity and inducing the aggregation of high concentrations of intermediate proteins (Yamaguchi, Miyazaki, Briones-Nagata, & Maeda, 2010). The third approach is the solid-state method. In this method the denaturant is removed using chromatography, solid particles, or gels (Batas & Chaudhuri, 1996; Lanckriet & Middelberg, 2004; Li et al., 2009). Column chromatography can be used for protein purification and is easily automated. As the denaturant is rapidly removed from the protein, aggregation generally occurs in the top section on the column (Yamaguchi et al., 2013).
For industrial scale refolding, the protein concentration after refolding should be high enough to reduce the amount of processing required. There has been some research into high-concentration refolding (Batas & Chaudhuri, 1996; Li et al., 2009; West, Chaudhuri, & Howell, 1998; Zhao et al., 2014). While protein concentrations are lower during refolding using the dilution or chromatography methods, the dialysis method can produce high levels of refolded proteins because of the reduction in protein dilution achieved by using a dialysis bag. However, this method can be time consuming, because the rate-determining step of the process is the removal of denaturant through dialysis membrane. The membrane surface area—that is, membrane area per volume of denatured protein solution—is small in the conventional method (Kohyama, Matsumoto, & Imoto, 2010; Maeda, Koga, Yamada, Ueda, & Imoto, 1995). If the surface area of the dialysis membrane can be enlarged, higher concentrations of protein could be recovered over shorter time periods.
To address this problem, in the work reported in the present study we developed a dialysis refolding method using microchannels, which can produce a large surface area to volume ratio. There have been studies on the application of microchannels to refolding, but all of them have been developed for dilution methods (Kashanian, Masoudi, Shamloo, Habibi-Rezaei, & Moosavi-Movahedi, 2018; Yamaguchi & Miyazaki, 2015; Yamaguchi et al., 2010; Yamamoto et al., 2010; Zaccai, Yunus, Matthews, Fisher, & Falconer, 2007), rather than dialysis methods. In research combining dialysis and microchannels, there have been reports on areas such as the development of bioassays (Imura, Yoshimura, & Sato, 2013), separation of single- and double-stranded DNA (Sheng & Bowser, 2014), and pH adjustment of microemulsions (Hood, Vreeland, & DeVoe, 2014). There has been no application to protein refolding.
In this study, to facilitate the preparation of microchannels for dialysis refolding, rational design involving the permeability of denaturant through dialysis membranes was used. First, the permeation coefficient of the denaturant through the dialysis membrane was determined. Then, the details of the microchannels, which can reduce the denaturant concentration in a designated time, were designed using this coefficient. Finally, using the fabricated microchannels, the reductions in denaturant concentration and the refolding of a model protein with a short residence time were investigated. As a model protein, Carbonic Anhydrase, the enzymatic reaction of which can be traced by hydrolysis of p -nitrophenyl acetate to p -nitrophenol, was used (Ikai, Tanaka, & Noda, 1978).