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FEATURED CASE STUDY
Process Optimization to Express 10 Different Novel Proteins
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Background:

Recombinant protein expression in E. coli depends on several parameters which influence the expression pattern individually or relative to each other. Optimization of protein expression conditions can significantly improve the resulting parameters which, in turn decreases the overall costs and increases the throughput for the development of novel biomolecules.

Problem/Rationale:

Due to lack of available literature, individual and combinatorial effects of the experimental conditions (such as selection of host strain, media compositions, inducer concentration and induction temperature) on expression of client’s proteins was not available. The initial screening procedures for these pre-clinical targets was a challenge for the client with respect to time and money.

Solution/Approach:

To solve this problem, we thoroughly studied the behaviour of similar kinds of proteins in literature and found that these recombinant proteins could be inherently toxic to the cells. Since there are various parameters that can influence the toxic effect of recombinant proteins, we applied the matrix-based approach. This approach captures approximately 1200 data points within a short period of 8 weeks and screened optimum expression conditions for novel therapeutic proteins. In this approach, we designed the optimization process, which was applied to various steps within the expression study, which included eight different E. coli strains, transformation combinations, optimum incubation temperatures, induction temperature, and inducer conditions in different growth media compositions. (Table 1).

Table 1: Experimental design for the expression optimization


We propose that the toxicity of these recombinant proteins may be due to their leaky expression before or after induction. One of the reasons for leaky expression can be insufficient transcription repression by high IPTG concentrations. IPTG is not only an innocuous inducer; instead, it exacerbates the toxicity caused by recombinant proteins and may cause appreciable damage to E. coli. Similarly, protein toxicity may be reduced or eliminated at a lower temperature. Lower temperature also results in increased protein solubility. Therefore, we optimized the expression of the desired proteins at three different IPTG concentrations at four different temperatures in all eight strains. Our results showed that induction at correct temperature and ITPG concentration in three strains of E. coli mitigated the toxic effect and led to the desired protein in a certain quantity, which we could visualize and estimate during analysis (Figure 1). Out of ten, four proteins displayed appropriate migration and expression at appropriate protein sizes (at a certain set of optimal conditions) and can be taken further for experimentation, production, purification, and supply. However, other proteins showed smaller size, which could be due to the degradation of these proteins by cells for their survival.

Figure 1: SDS-PAGE confirmation of four novel proteins


Measurable Impact:

This DoE matrix can be employed to novel proteins making the process fast, efficient, and cost-effective.


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