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).