Rhodopseudomonas Palustris Collagen-Like Recombinant Protein Purification Using an Aqueous Two-Phase System
Keywords:
Recombinant collagen-like protein, R. palustris, Recombinant collaqueous two-phase system, purification, chromatographyAbstract
The potential use of recombinant collagen-like protein (recCLP) extracted from bacteria as disease-free collagen has been studied over the past decade. However, the complexity of the downstream processing generates high demand for an efficient and low-cost purification method. Aqueous two-phase system (ATPS) was adopted as a new approach to the recovery of biomolecules due to its simple, benign, and straightforward process. This study aimed to purify recombinant collagen-like protein from Rhodopseudomonas palustris using ATPS formed by a polymer/salt system. Recombinant collagen-like protein from R. palustris was partitioned in ATPS composed of polyethylene glycol (PEG) and potassium phosphate and several factors that influence the protein partitioning such as volume ratio, system pH, the concentration of polymer and salt were studied. Then, optimization of the selected ATPS conditions (PEG and salt concentration) were performed using response surface methodology (RSM). Results showed that the optimum conditions were found in ATPS with 24.80% (w/w) PEG 2000 and 29.23% (w/w) potassium phosphate with recCLP concentration of 3.23 ± 0.12 mg/mL with purification factor 7.48 ± 0.3. In comparison with the affinity chromatography method, ATPS was found to be low-cost, and time-saving with a higher protein recovery. Hence, this study demonstrated the potential application of ATPS in the recovery of recombinant CLPs for large-scale downstream processing.
ABSTRAK: Potensi penggunaan protein seperti kolagen rekombinan (recCLP) yang diekstrak daripada bakteria sebagai kolagen bebas penyakit telah dikaji sejak sedekad yang lalu. Walau bagaimanapun, kerumitan pemprosesan hiliran menjana permintaan yang tinggi untuk kaedah penulenan yang cekap dan berpatutan. Sistem akueus dua fasa (ATPS) telah diterima pakai sebagai pendekatan baharu dalam pemulihan biomolekul kerana prosesnya yang mudah. Tujuan utama kajian ini adalah untuk menyaring protein seperti kolagen rekombinan daripada Rhodopseudomonas palustris menggunakan ATPS yang dibentuk oleh sistem polimer/garam. Protein seperti kolagen rekombinan daripada R. palustris telah dibahagikan dalam ATPS yang terdiri daripada polietilena glikol (PEG) dan kalium fosfat dan beberapa faktor yang mempengaruhi pembahagian protein seperti nisbah isipadu, pH sistem, kepekatan polimer dan garam telah dikaji. Kemudian, keadaan ATPS terpilih (PEG dan kepekatan garam) telah dioptimumkan menggunakan metodologi permukaan tindak balas (RSM). Keputusan menunjukkan bahawa keadaan optimum dalam ATPS adalah 24.80% (b/b) PEG 2000 dan 29.23 % (b/w) kalium fosfat dengan kepekatan recCLP 3.23 ± 0.12 mg/mL dengan faktor penulenan 7.48 ± 0.3. Berbanding dengan kaedah kromatografi afiniti, ATPS didapati menjimatkan kos dan masa dengan pemulihan protein yang lebih tinggi. Oleh itu, kajian ini menunjukkan potensi aplikasi ATPS dalam pemulihan CLP rekombinan untuk pemprosesan hiliran berskala besar.
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Ministry of Higher Education, Malaysia
Grant numbers TRGS/1/2018/UIAM/01/1/1),
