Purification of recombinant collagen-like protein from Rhodopseudomonas palustris expressed in Escherichia coli using aqueous two-phase system

Abstract

Recombinant collagen-like protein (recCLP) is a collagen-like molecule extracted from microorganisms and expressed in Escherichia coli host. The surge in demand for highquality collagen is due to the multitude of applications in the end-user industries such as pharmaceuticals, food, nutraceuticals and cosmetics. The awareness of the halal status of collagen among Muslim consumers encouraged the discovery of protein from non-mammalian sources, especially from microorganisms. The collagens from microorganisms have great industrial potential because they are free from any zoonotic diseases, contamination and side effect issues. Moreover, the difficulties of the present downstream processing, especially at the purification step, encourage the application of an aqueous two-phase system (ATPS). This study aimed to identify the optimum ATPS conditions for purification of recombinant collagen-like protein expressed in E. coli that initially extracted from Rhodopseudomonas palustris. Recombinant collagen-like protein from R. palustris was purified using the aqueous two-phase system consisting of polyethylene glycol (PEG)/ potassium phosphate. First, the five binodal curves representing five different molecular weights of PEG (1500, 2000, 4000, 6000 and 8000 g/mol) were constructed using the node determination method. Binodal curve that divides the region of two aqueous phases from one phase is important so that a systematic choice of system can be used for portioning experiments. Then, several factors involved in the partitioning behaviour of recCLP such as volume ratio, system pH, the concentration of polymer and salt were studied. The selected ATPS conditions (PEG and salt concentration) were optimised using the response surface methodology (RSM) method. Purification by affinity chromatography was carried out and further compared with ATPS in terms of efficiency and economic aspects to evaluate its potential application as a purification method for recCLP. The binodal curves obtained proved that a high molecular weight of PEG required a low concentration of potassium phosphate to form a two-phase system. As PEG molecular weight increased, the curved was distorted towards the origin. The highest partition coefficient (KE) was found in the system with 26 % (w/w) PEG 2000 and 26 % (w/w) potassium phosphate, making it the best ATPS combination for the OFAT analysis and optimisation process. The range of volume ratio, pH and concentration of PEG and potassium phosphate on the partitioning of recombinant collagen-like protein by ATPS were successfully obtained from OFAT method prior to the optimisation study. Optimisation of ATPS conditions using face-centered central composite design (FCCCD) in Response Surface Methodology (RSM) with 11 runs showed the optimum conditions of ATPS with 24.80 % (w/w) PEG 2000 and 29.20 % (w/w) potassium phosphate with recCLP concentration of 3.23 ± 0.12 mg/mL. Analysis of variance showed the coefficient of determination (R2) were 0.8823, 0.8823, and 0.8193 for fluorescence intensity, the concentration of collagen-like protein and purification factor, respectively. Lastly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the molecular weight of the recCLP, which is 36 kDa. In addition, results showed that ATPS is a low cost, time-saving with a high recovery method that may raise the consideration for substitution of chromatography method. In conclusion, the purification method through ATPS to purify recCLP has high potential, cost-effective, can replace the tedious and expensive downstream processing. Furthermore, this study can serve as a reference and deliver new information for future work.