Abstract
Macromolecular protein crystallisation was one of the potential tools to accelerate the biomanufacturing of biopharmaceuticals. In this work, it was the first time to investigate the roles of biotemplates, Saccharomyces cerevisiae live cells, in the crystallisation processes of lysozyme, with different concentrations from 20 to 2.5 mg/mL lysozyme and different concentrations from 0 to 5.0 × 107 (cfu/mL) Saccharomyces cerevisiae cells, during a period of 96 h. During the crystallisation period, the nucleation possibility in droplets, crystal numbers, and cell growth and cell density were observed and analysed. The results indicated the strong interaction between the lysozyme molecules and the cell wall of the S. cerevisiae, proved by the crystallization of lysozyme with fluorescent labels. The biotemplates demonstrated positive influence or negative influence on the nucleation, i.e. shorter or longer induction time, dependent on the concentrations of the lysozyme and the S. cerevisiae cells, and ratios between them. In the biomanufacturing process, target proteins were various cells were commonly mixed with various cells, and this work provides novel insights of new design and application of live cells as biotemplates for purification of macromolecules.
Highlights
Protective effect on cells, and makes bacteria dissolve and die[34]
It is obvious that plenty of S. cerevisiae cells were observed in the droplets with biotemplates at CSC[High], much fewer S. cerevisiae was observed in the droplets with biotemplates at C SC[Low]
The observation of the crystallisation process, the cell culture process (Fig. 7), the cell density (Table 1) and the SEM images (Fig. 8) supported that the lysozyme did not lead to the lysis of the S. cerevisiae cells, but obviously hindered the growth and proliferation of the cells, indicating strong interactions between the cell wall and the lysozyme molecules
Summary
Lysozyme has limited impact on the cell wall lysis of the S. cerevisiae, due to the lack of lysozyme sites in its cell w all[35,36]. To the best knowledge of the authors, the current study is the first to apply the biotemplates, live-cell templates, on protein crystallisation, this research will fill the technology gap by demonstrating the feasibility of yeast cells, S. cerevisiae, as a heterogeneous nucleant to promote the crystallisation of a model protein, lysozyme. The work demonstrated the crystallisation technology has the ability to isolate protein in the complex solution environment with various concentrations of the live cells. The knowledge provided in this study can be transformed into future applications of biotemplate design for bioseparation and purification of therapeutic proteins or other macromolecules
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