Simple Protein Purification Through Affinity Adsorption On Regenerated Amorphous

Cellulose Followed By Intein Self-Cleavage

A simple, low-cost, and scalable protein purification method was developed by using
a biodegradable regenerated amorphous cellulose (RAC) with a binding capacity of up to
365mgprotein per gram of RAC. The recombinant protein with a cellulose-binding module
(CBM) tag can be specifically adsorbed by RAC. In order to avoid using costly protease and
simplify purification process, a self-cleavage intein was introduced between CBM and target
protein. The cleaved target protein can be liberated from the surface of RAC by intein self-
cleavage occurring through a pH change from 8.0 to 6.5. Four recombinant proteins (green
fluorescence protein, phosphoglucomutase, cellobiose phosphorylase, and glucan
phosphorylase) have been purified successfully.

Developing simple, low-cost, and environmentally friendly methods for recombinant

protein purification on a large scale remains challenging [1,2]. Affinity chromatography by
using var- ious affinity tags on various resins is popular in laboratories and biotechnology
companies [1,3,4], but it cannot be applied to low- selling-price non-therapeutic proteins,
such as industrial enzymes [5]. A low-cost and scalable method for large-scale protein purifi-
cation is prerequisite for commercialization of industrial scale
biocommodityproductionmediatedbyenzymesorsyntheticenzy- matic pathways [6–8].

Materials and methods:

1. Chemicals and strains

2. Regenerated amorphous cellulose preparation
3. Recombinant protein expression plasmids
4. Recombinant protein expression
5. Self-cleavage efficiency of intein at different temperature and Ph
6. Protein purification
7. Enzyme assays and protein analysis

Results: A simple protein separation method was proposed based on high-affinity

adsorption of the CBM-tag protein on the surface of RAC (Fig. 1). RAC has a much higher
binding capacity (e.g., 365mg of CBM-GFP per gram of RAC) than that of crystalline
cellulose (14.8mgofCGpergramofAvicel)andthanthoseofanycommercial protein purity resins
(e.g., 10–40mg of protein per gram of resin). The dissociation constant based on RAC is
168L/g RAC, indicating high affinity adsorption of the family three CBM on RAC. The bind-
ing capacity based on the RAC volume is approximately 15mg of protein per mL of bed
volume. The crude protein solution contain- ing the fusion proteins of CBM-intein-target
protein was mixed well with the RAC absorbent. After centrifugation, the supernatant con-
taining most of impure protein swas decanted. The impure proteins that stayed in the porous
RAC were washed away in a buffer once or several times. The cleaved soluble target protein
can be obtained through intein self-cleavage in the cleaving buffer (low pH and high salt
concentration). After centrifugation, the purified cleaved target protein can be obtained in the
supernatant. The above pro- tein purification processes involving only solid/liquid separation
(centrifugation or filtration) can be easily scaled up.

The protein purification process for the GFP-intein-CBM fusion protein was
optimized. One gram of RAC has a maximum bind- ing capacity of 365±20mg of CIG,
determined by the Langmuir isotherm as described elsewhere [15,28]. In order to bind ∼95%
of the target protein, a 1.7-fold overload of RAC was needed (i.e., 215mg of adsorbed
protein/g of RAC). After centrifugation, the impure protein in the supernatant was decanted.
The RAC pellet with the adsorbed CIG was suspended in the buffer once or twice to remove
the impure protein remaining in the adsorbent matrix.
Pemeliharaan Protein Sederhana Melalui Adsorpsi Afinitas Pada Selulosa Amorf yang Diatur
Dilanjutkan Dengan Pembelahan Diri Intein

Metode pengrusakan protein sederhana, murah, dan terukur dikembangkan dengan

menggunakan selulosa amorf (RAC) teregenerasi biodegradable dengan kapasitas pengikatan
hingga 365 mg protein per gram RAC. Protein rekombinan dengan tag modul pengikat
selulosa (CBM) dapat secara khusus diadsorpsi oleh RAC. Untuk menghindari penggunaan
protease yang mahal dan menyederhanakan proses pengrusakan, intestin pembelahan diri
diperkenalkan antara CBM dan protein target. Protein target yang terbelah dapat terbebaskan
dari permukaan RAC dengan pembelahan diri intestin yang terjadi melalui perubahan pH dari
8,0 menjadi 6,5. Empat protein rekombinan (protein fluoresen hijau, fosfoglucomutase,
fosforilase selobiosa, dan glukan fosforilasa) telah berhasil dipastikan.

Mengembangkan metode sederhana, berbiaya rendah, dan ramah lingkungan untuk

pemurnian protein rekombinan dalam skala besar tetap menantang. Kromatografi afinitas
dengan menggunakan tag afinitas yang unik pada berbagai resin sangat populer di
laboratorium dan perusahaan bioteknologi, namun tidak dapat diterapkan pada protein non-
terapeutik dengan harga rendah, seperti enzim industri. Metode biaya rendah dan terukur
untuk pemurnian protein skala besar merupakan prasyarat untuk komersialisasi biokomoditas
skala industri yang diproduksi melalui jalur sterilisasi neurologis.

Bahan dan metode:

1. Bahan kimia dan strain
2. Persiapan selulosa amorf yang diregenerasi
3. Ekspresi protein rekombinan plasmid
4. Ekspresi protein rekombinan
5. Efisiensi pembelahan diri intravena pada suhu yang berbeda dan Ph
6. Paste fi kasi protein
7. Uji enzim dan analisis protein

Hasil: Metode pemisahan protein sederhana diusulkan berdasarkan adsorpsi afinitas tinggi
protein tag CBM pada permukaan RAC (Gambar 1). RAC memiliki kapasitas pengikatan
yang jauh lebih tinggi (mis., 365mg CBM-GFP per gram RAC) daripada selulosa kristal
(14,8mgCCpergramof Avicel) dan kandungan resin kemurnian protein komersil (misalnya,
10-40 mg protein per gram resin). Konstanta disosiasi berdasarkan RAC adalah 168L / g
RAC, menunjukkan adsorpsi afigitas tinggi dari keluarga tiga CBM pada RAC. Kapasitas
pengikatan berdasarkan volume RAC kira-kira 15mg protein per mL volume tempat tidur.
Larutan protein kasar yang mengandung protein fusi protein target-CBM-intang dicampur
dengan baik dengan penyerap RAC. Setelah sentrifugasi, supernatan yang menahan sebagian
besar protein yang tidak murni tertuang. Protein tidak murni yang tinggal di RAC berpori
hanyut dalam penyangga sekali atau beberapa kali. Protein terlarut protein yang dapat larut
dapat diperoleh melalui pembelahan diri intein pada buffer pembelahan (pH rendah dan
konsentrasi garam tinggi). Setelah sentrifugasi, protein target yang dipecah belah dapat
diperoleh dalam supernatan. Proses pemurnian protein di atas yang hanya melibatkan
pemisahan padat / cair (sentrifugasi atau penyaringan) dapat dengan mudah ditingkatkan.
Proses pemurnian protein untuk protein fusi GFP-intein-CBM dioptimalkan. Satu gram RAC
memiliki kapasitas pengikatan maksimum 365 ± 20mg CIG, yang ditentukan oleh isoterm
Langmuir seperti yang dijelaskan di tempat lain [15,28]. Untuk mengikat ~ 95% protein
target, diperlukan pembebanan RAC sebanyak 1,7 kali lipat (yaitu, 215mg protein teradsorpsi
/ g RAC). Setelah sentrifugasi, protein tak murni di supernatan dituang. Pelet RAC dengan
CIG yang teradsorpsi disuspensikan dalam buffer sekali atau dua kali untuk menghilangkan
protein tidak murni yang tersisa dalam matriks adsorben.