Structure- and Design-Based Difficulties in Recombinant Protein Purification in Bacterial Expression

Abstract

Protein purification is not a simple task. Yet, overexpression at bacterial systems with recombinant modifications brings further difficulties. Adding a tag, an affinity label, and expressing particular domains of the whole protein, especially hydrophobic sections, make purification a challenging process. Protein folding pattern may perturb N- or C-terminal tag and this terminal preference may lead to poor purification yield. Codon optimization, solvent content and type, ionic conditions, resin types, and self-cleavage of recombinant proteins bring further difficulties to protein expression and purification steps. The chapter overviews problems of protein purification through a small peptide overexpression in bacteria (Recombinant anti-SARS Coronavirus 2 (SARS-Cov-2) Spike protein Receptor Binding Domain (RBD) antibody (Clone Sb#14). The chapter also covers troubleshooting at distinct steps and highlights essential points to solve crucial issues of protein purification.

Investigation of the effect of different culture conditions on recombinant protein production

“…In addition, a carbon-energy source (glucose, glycerol, or other sugars) and a nitrogen source are needed for medium formulations. Therefore, Luria Bertani (LB) and Terrific Broth (TB) are chosen as the most suitable media for recombinant protein production (Coskun et al 2022, Thongbhubate et al 2021. Another important parameter is the identification of the inducer.…”

“…In addition to high production, high recovery of recombinant proteins is also important in the efficiency of production. Therefore, different parameters such as resin type, solvent, pH, buffer system, protein structure and ionic strength should be considered in the purification optimization of recombinant proteins (Coskun et al 2022).…”