Anti-degradation of a recombinant complex protein by incoporation in small molecular hydrogels

Article abstract of DOI:10.1039/c0cc04249h

A phenomenon of anti-degradation of a recombinant complex protein (MPP6 complex protein) in a small molecular hydrogel was reported in this study.

Full text of DOI:10.1039/c0cc04249h

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Anti-degradation of a recombinant complex protein by incoporation in
small molecular hydrogelsw
Huaimin Wang,ab Zheng Wang,bc Xiaoyi Yi,d Jiafu Long,bc Jianfeng Liuae and
Zhimou Yang*ab
Received 6th October 2010, Accepted 25th October 2010
DOI: 10.1039/c0cc04249h
A phenomenon of anti-degradation of a recombinant complex
protein (MPP6 complex protein) in a small molecular hydrogel
was reported in this study.
SM hydrogelators including derivatives of amino acids,9
sugars,10 peptides,6 and drug molecules.11 In this study, a
short peptide-based SM hydrogel of Nap-GFF (1 in Fig. 1)
developed by our group was chosen due to its versatile
synthetic pathway and its excellent gelation ability.12
Recombinant proteins, even for those being purified by size-
exclusion high performance liquid chromatography (SE-HPLC),
frequently suffer from degradation by protease, which causes
serious problems for their storage, crystal growth, etc. The
strategy of the addition of protease inhibitors during the
purification can greatly improve the stability of the recombinant
proteins.1 However, it is hard to totally inhibit the activity of
the whole family of protease by a single inhibitor. What’s
more, the purified proteins obtained by SE-HPLC usually
need to be stored in cold places (e.g. in the fridge at low
temperatures), which needs a lot of energy and is not
convenient during transportation. Therefore, a simple and
convenient method that can greatly improve the stability of
recombinant proteins at room temperature is highly desirable.
Small molecular (SM) hydrogels,2 formed by the self-assembly
of small molecules,3 might be an ideal candidate because
the proteins can be fixed in their three dimensional matrix,
thus reducing the possibility of recognition and degradation
of proteins by the protease. In this study, we demonstrated
in principle that the stability of a recombinant complex
protein—membrane-associated guanylate kinases p55 subfamily
member 6 (MPP6), residues 1-106AA complexed with Mals3—
could be greatly improved by simple incorporation into a SM
hydrogel.
After the successful synthesis of Nap-GFF by solid phase
peptide synthesis (SPPS) and purification by reverse phase
HPLC, the gelation property of 1 was tested by the invert-tube
method. The minimum gelation concentration of 1 was 0.2 wt%
in the phosphate buffered saline (pbs) buffer solution (pH = 7.4),
which meant that 1 molecule of 1 could gel about 14 950
molecules of H2O in pbs solutions. This result correlated well
with the fact that derivatives of diphenylalanine were efficient
gelators for organic solvents and aqueous solutions.13
The gel with 1.0 wt% of 1 in pbs buffer (gel I) was then used
for the encapsulation of the MPP6 complex protein purified by
SE-HPLC. Since gel I was a thixotropic hydrogel, it allowed
the encapsulation of the protein by a simple vortex. The gel
with the MPP6 complex protein (gel II) would re-form after
being kept at room temperature (22–25 1C) for about 2 h. The
MPP6 complex protein in the pbs buffer and in gel II was then
tested by the SDS-PAGE gel electrophoresis. The results
shown in Fig. 2 indicated that about 10% of the MPP6
complex protein degraded after 24 h (lane 3) and the protein
was totally degraded after 48 h (lane 4) in the pbs buffer
solution. However, there was no obvious degradation of the
complex protein being observed in gel II at room temperature
after 48 h and about 58% of the protein remained in the gel II
after 7 days, which indicated that the stability of the MPP6
complex protein was improved by a simple encapsulation in
SM gels. To understand the mechanism of stabilization of
MPP6 protein in hydrogels, we used a model protein of bovine
serum albumin (BSA) to be mixed with MPP6 protein solutions
in PBS buffer solutions and in gels. As shown in the supporting
informationw, the BSA itself was stable in PBS solutions for at
least 3 days. However, it would undergo degradation by
mixing it with the MPP6 protein solutions in the buffer
solutions. Similarly to MPP6 protein, its stability could be
It has been proved that SM hydrogels could provide suitable
environments for bioactive individuals such as cells,4 sensors,5
proteins,6,7 and drugs.8 Lots of small molecules based on
naturally occuring components have been found to be efficient
a Key Laboratory of Bioactive Materials, Ministry of Education,
College of Life Sciences, Nankai University, Tianjin 300071, China.
E-mail: yangzm@nankai.edu.cn
b Department of Biochemistry and Molecular Biology,
College of Life Sciences, Nankai University, Tianjin 300071, China
c Tianjin Key Laboratory of Protein Science, College of Life Sciences,
Nankai University, Tianjin 300071, China
d School of Chemistry and Chemical Engineering,
Central South University, Changsha 410083, China
e Tianjin Key Laboratory of Molecular Nuclear Medicine,
Institute of Radiation Medicine, Chinese Academy of Medical
Sciences & Peking Union Medical College, Tianjin 300192, China
w Electronic supplementary information (ESI) available: Synthesis
of 1, expression and purification of the MPP6 complex protein,
protocol of the protein encapsulation, determination of phosphatase
activity, optical image of gels, and emission spectra. CCDC 784334.
For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c0cc04249h
Fig. 1 The chemical structure of Nap-GFF and an optical image of
gel I with 1 wt% of 1 in PBS buffer (pH = 7.4).
c
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Fig. 3 (A) Dynamic frequency of gel I with 1.0 wt% of 1 and gel II
with 1.0 wt% of 1 and 0.005 wt% of MPP6 complex protein in pbs
solutions at the strain of 2% (both gels were formed by the heating-
cooling cycle, gel I: circles, gel II: triangles, G0: filled symbols, and G0 0:
open symbols) and (B) the recovery property of gel I with 1.0 wt% of 1
(gel I was firstly subjected to a large strain of 50% for 10 min and then
its recovery was probed at the strain of 2% and frequency of 2 rad s-1).
Fig. 2 The scanning image of SDS-PAGE gel electrophoresis of
recombinant MPP6 complex protein in the PBS buffer solution
(pH = 7.4) and in gel II at day 0–3.
value of G0 was observed after 10 min under the external large
stress (strain = 50%). After the removal of the external large
stress, the speed of recovery of the strength of gel I was quite
slow—only 24% of the original value of G0 was achieved after
1 h. These results implied that gels formed by 1 were weak and
thixotropic hydrogels that possessed a shear thinning and slow
recovery property.
improved by incorporation of it in the SM hydrogels. These
results indicated the presence of the digestion enzyme/enzymes
in MPP6 protein solutions, which could not be separated from
the MPP6 complex protein by SE-HPLC. These results also
indicated that such kind of great improvement of the stability
of the proteins was due to the fixation of the protein and the
digestion enzymes in the fiber networks, which dramatically
reduced the diffusion rate of the enzymes and the proteins and
decreased the possibility of the proteins being recognized by
the digestion enzymes.
Atomic force microscopy (AFM) was used to characterize
the nanostructures within the gels (Fig. 4). Small fibrils with a
diameter of about 20 nm and ribbons with width of about
60 nm were observed in gels I (Fig. 4A). Both the fibrils and
ribbons in gel I were longer than 2 mm and they entangled with
each other to form a three dimensional network for gel I. To
understand what happened for the gel I before and after
vortex, we also obtained the AFM image of gel I after vortex.
As shown in the supporting information (Fig. S-7w), gel I after
vortex exhibited a relatively higher amount of small fibrils with
a size of about 20 nm than gel I before vortex. This result
suggested that the ribbons or fibers with larger sizes were
formed by the bundles of the small fibrils and the vortex would
weaken the interaction between these small fibrils, thus leading
to mechanically weaker hydrogels. Compared to gel I before
vortex, gel II showed much smaller and uniform fibrils with a
diameter of about 15 nm and there were no ribbons being
observed (Fig. 4B). These observations correlated well with the
result of a much smaller value of G0 of gel II than that of
gel I. These results further suggested that the existence of a
tiny amount of protein might have strong influences on the
microscopic (morphology and molecular arrangement) and
macroscopic (mechanical property) properties of the gels.
In order to test whether the activity of the encapsulated
protein had been changed or not, the protein of an enzyme
(phosphatase) was incorporated into the gel and its activity
was measured at different time points. The results showed that
the activity of the phosphatase in the gel after 48 h was almost
the same to that of free phosphatase in pbs solution at day 0
(98% of the activity remained),14 which meant that the self-
assembled fiber network had no obvious effects on the activity
of the encapsulated enzyme in the short time.
We then went back to characterize both kinds of the
hydrogels (gel I and gel II). The mode of dynamic frequency
sweep was firstly used to characterize the mechanical property
of both gels. As shown in Fig. 3A, the values of G0 of gel I and
gel II were higher than those of G00 of gel I and gel II
respectively at the low frequency regions, indicating that both
gels were solid-like samples. The values of G0 and G0 0 of both
gels showed strong dependencies on the frequency—both
values of G0 and G00 increased with enhancement of the
frequency and the values of G00 of gel I and gel II were almost
the same as those of G0 of both gels respectively at the high
frequency regions. Comparing the values of G0 of both gels, it
was found that the value of G0 decreased from 120 Pa in gel I
to 6 Pa in gel II formed by the heating-cooling cycle after the
addition of a small amount of the protein (0.005 wt%). This
result suggested that the protein might interact with the small
molecules, thus inhibiting the formation of large bundles of
the self-assembled structures and weakening the mechanical
property of the gels. Since gel I was a thixotropic hydrogel,
the mode of dynamic time sweep was used to test its
recovery property. As shown in Fig. 3B, both values of
G0 and G0 0 dropped rapidly and only 18% of the original
Fig. 4 AFM images of (A) gel I and (B) gel II.
c
956 Chem. Commun., 2011, 47, 955–957
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from College of Life Sciences, Nankai University (B08011)
for their financial support.
Notes and references
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Fig. 5 (A) The crystal structure of Nap-GFF obtained from ethanol
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Unlike the Nap-FF, in which the naphthalene group inter-
acted with the phenyl ring of its own phenylalanine (F) and
two Nap-FF molecules formed a dimer with a ‘X’ shape,15 the
naphthalene group on Nap-GFF was far away from the phenyl
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of the dimer with a ‘//’ shape. This observation helped us to
understand the super-gelation ability of the derivatives of
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molecules between two dimers helped the formation of exten-
sive hydrogen bonds between Nap-GFF and both the hydrogen
bonds and aromatic interactions helped to efficiently extend
the supramolecular chains to form superstructures.
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In summary, we demonstrated the first example of the
application of SM hydrogels in anti-degradation of complex
proteins by a simple encapsulation strategy. Such a strategy
would not dramatically decrease the activity of the encapsulated
enzyme in the SM hydrogels. Though only 58% of the
encapsulated enzyme remained in the gel after storage at room
temperature for 7 days, the stability of the encapsulated
protein at room temperature might be further improved by
using SM gels with higher mechanical property. The results
indicated that SM hydrogels not only had potential to be
developed into promising biomaterials for long term storage of
recombinant proteins at room temperature, but also would be
useful for the delivery of proteins to treat different diseases. It
was also quite unusual that the addition of a tiny amount of
protein caused such dramatic effects on the microscopic and
macroscopic properties of the hydrogels, which will be further
studied and reported in due course.
The authors acknowledge NSFC (20974054 and 30700178),
the TBR program (08JCZDJC20500), and the 111 project
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 955–957 957