A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

Abstract

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them ‘difficult‐to‐express’. This study aimed to define the molecular features that restrict the production of a model ‘difficult‐to‐express’ recombinant protein, Tissue Inhibitor Metalloproteinase‐3 (TIMP‐3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass ‘secretory’ bottlenecks and result in efficient recombinant protein production.

A comparative analysis of recombinant Fab and full‐length antibody production in Chinese hamster ovary cells

“…Proteins were resolved via SDS‐PAGE and transferred onto nitrocellulose membrane as detailed previously (Hussain et al, 2018). Membranes were blocked in 5% (w/v) milk in phosphate‐buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , and pH 7.4) with 0.1% (v/v) Tween‐20 (5% mPBS‐T) for 1 h at room temperature before incubation with primary antibodies in 5% mPBS‐T solution for 1 h at room temperature.…”

“…Proteins were resolved via SDS-PAGE and transferred onto nitrocellulose membrane as detailed previously (Hussain et al, 2018).…”

Predicting protein stability and solubility changes upon mutations: data perspective

“…[4] Improving properties of valuable but difficult-to-work-with proteins that have borderline stability or are poorly soluble outside living cells presents another major challenge in biotechnology. [5,6] Moreover, human neurodegenerative disorders, metabolic diseases, and cancer are often linked to mutations leading to protein misfolding, aggregation, [7][8][9] or decreased solubility, [10,11] and unstable or insoluble proteins may lead to precipitates triggering an unwanted immune response in patients. [12] Given the astoundingly vast protein sequence space to explore in the pursuit of improved stability and solubility, computational tools are used increasingly to narrow down the search to ideally only a few promising mutations to be tested experimentally.…”

“…Biocatalyst production suffers losses from time and resources wasted on poorly soluble and unstable protein mutants, [3] and in industrial applications, improved stability against harsh environments often becomes critical [4] . Improving properties of valuable but difficult‐to‐work‐with proteins that have borderline stability or are poorly soluble outside living cells presents another major challenge in biotechnology [5,6] . Moreover, human neurodegenerative disorders, metabolic diseases, and cancer are often linked to mutations leading to protein misfolding, aggregation, [7–9] or decreased solubility, [10,11] and unstable or insoluble proteins may lead to precipitates triggering an unwanted immune response in patients [12] …”

Surface patches on recombinant erythropoietin predict protein solubility: engineering proteins to minimise aggregation

“…Here we report a novel experimental approach targeted at improvement of rHuEPO solubility for expression in E. coli, following the observation that soluble expression of proteins is inversely correlated with the size of the largest positively-charged patch on the protein surface [29,32]. This result is based on data from cell-free expression [33], and here we test the hypothesis by mapping surface charge of rHuEPO, focusing on modulation of positively-charged patches through mutagenesis.…”