Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

Abstract

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

Keywords: E. coli; industrial applications; protein folding; protein inclusion bodies; recombinant proteins.

FIGURE 1

The protein homeostasis network in E. coli cells. Protein homeostasis refers to the control of concentration, conformation, binding interactions, and localization of individual proteins making up the proteome by readapting the innate biology of the cell. It involves multiple pieces of cellular machinery for transcription, translation, protein folding, and protein degradation. Protein IBs formation in E. coli cells result from an unbalanced equilibrium among protein’s proper folding, aggregation and/or degradation. This figure was generated based on Balch et al. (2008) and Morimoto et al. (2019) with modifications.

FIGURE 2

New developments in E. coli strain’s engineering to tackle the issue of IBs formation in recombinant protein expression. Amongst the issues is its inability to catalyze di-sulfide bond formation, glycosylation, and the high strength of expression. (A) SHuffle strains enable disulfide bond formation by oxidized thioredoxins and mutant AhpC* transfers electrons to GSH/glutaredoxin pathway allowing for the reduction of oxidized ribonucleotide reductase which is essential for growth. (B) N- or O-glycosylation in glycoengineered E. coli is conducted by the protein glycosylation (pgl) locus which is responsible for the biosynthesis of the glycans. Glycoengineered strains like E. coli CLM24 could potentially be engineered to be “leaky” facilitating secretion of the glycoprotein to culture media. As proof of concept, E. coli CLM37 was engineered to be “leaky” by deleting the Braun’s lipoprotein (lpp gene) which connects the outer membrane to the peptidoglycan layer. (C) Various E. coli strains have been engineered (1, 2, 3) to modulate transcription or (4, 5) inhibition of either orthogonal T7 RNA polymerases (RNAP). There are also those strains engineered in (6) decoupling of host cell growth from recombinant protein production via inhibition of E. coli RNAPs.

FIGURE 3

Design of a fusion expression vector to aid in solubilizing the expressed recombinant protein. Expression vectors containing a fusion protein or peptide tags may help increase the solubility of the target protein by promoting proper folding and stabilization. These fusions may be attached to either the N- or C-terminus. Various protein fusions and peptide tags are available for selection, however, they must be carefully chosen to be compatible with the target protein. Examples of protein fusion tags include glutathione-S-transferase (GST), mannose binding protein (MBP), while peptide tags include commonly utilized histidine (His). Plasmid display system technology makes use of fusion partners to attach the fusion and target protein to the plasmid to improve stability of the expressed protein. If selecting a fusion partner to use in a plasmid display system, consideration must be given to an appropriate and soluble fusion partner. For example, in the following figure the transcription factor Oct-1 is utilized which possesses a DBD that recognizes and attaches to the recognition sequence.