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FEATURED CASE STUDY
Expression of Highly Active Protein in E. coli for Diagnostics Application
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Introduction:

The production of recombinant enzymes has led to the development of many diagnostics strategies in various medical fields. The increasing demand for recombinant proteins has created the need to develop efficient protein expression techniques to increase the productivity of the product.

Problem/Rationale:

Protein X has a hydrophobicity index of 10.65%, has ten cysteine residues with five disulfide bonds, and a pI of 10, which negatively influences the purification of the protein.

Solution/Approach:

We have developed a robust process to express cysteine-rich and functionally active small proteins from two different species (human and mouse) using a suitable host strain of E. coli, expression optimization, and purification techniques.

Protein was successfully expressed in BL21 and Origami-2 strains of E. coli, recovered from inclusion bodies and confirmed by immunoblot (Figure 1). For one-liter scale-up expression studies, we have next optimized various parameters such as the choice of host strain, induction temperature, and media formulations with various carbon sources for high expression of functionally active protein. Single-step ion-exchange chromatography (rapid and simple) resulted in a purified protein product, the activity of which was determined by functional ELISA. The developed ELISA was sensitive to the tertiary structure of the protein, and hence, any misfolding of the protein will result in low or no binding at all. ELISA activity assay demonstrated a linear graph, confirming an increase in protein-binding activity with increased protein concentration. Results of ELISA demonstrated that protein expressed at Premas was comparative to the gold standard (Figure 2). Mass spectrometry analysis confirmed the calculated mass of the protein to be the same as their actual molecular weight (Figure 3).

The represented data highlights a very efficient protein expression system, well suited for rapid and high yield production of active proteins with an appropriate folding and 3D structure in Escherichia coli host.

Fig 1: Confirmation of protein by immunoblot


Fig 2: ELISA demonstrating activity of proteins 1) Gold standard 2) Human 3) Mouse


Fig 3: Confirmation of the protein size by Mass spectrometry. A) Human protein B) Mouse protein



Measurable Impact:

1) Expression of functionally active protein having 10 cysteine residues with an appropriate folding.
2) Testing of functional activity of the protein by ELISA demonstrated to be comparatively equal to the standard.
3) Have supplied protein with >85% purity for preclinical studies.



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