E.coli problems Flashcards
Intro
Escherichia coli (E. coli) is widely used for the expression of recombinant proteins due to its low cost, fast growth, and well-characterized genetic system. However (problems in qs) can appear. These challenges arise due to the inherent differences between E. coli and eukaryotic systems, such as protein folding, post-translational modifications, and cellular stress responses. Key factors attributing to these issues and their solutions are outlined below.
First factor - protein aggregation
One of the primary challenges when expressing human proteins in E. coli is the formation of insoluble aggregates known as inclusion bodies. Human proteins often require specific post-translational modifications (PTMs) like glycosylation, disulfide bond formation, or phosphorylation, which E. coli is unable to perform. As a result, many recombinant human proteins do not fold correctly and aggregate into inclusion bodies (Baneyx, 1999). The accumulation of inclusion bodies often leads to low yields of active protein and necessitates costly and complex refolding procedures.
Second factor - codons
E. coli and humans differ in their codon usage, with E. coli often having a preference for certain codons that may not be optimal for human gene expression. This discrepancy can result in inefficient translation and premature termination, which can compromise protein yield and quality (Terpe, 2006). Additionally, the slower translation of rare codons may affect the overall speed and efficiency of recombinant protein synthesis.
Third factor - cellular stress
High-level expression of recombinant proteins can overwhelm the protein-folding machinery in E. coli, leading to cellular stress responses such as the heat shock response and the Unfolded Protein Response (UPR). These responses can further inhibit protein production and result in the accumulation of misfolded proteins, which decreases the overall yield of functional recombinant protein (Chung et al., 2014)
First solution - molecular chaperones
One effective approach to addressing solubility issues is the co-expression of molecular chaperones or foldases. These proteins assist in the proper folding of recombinant proteins and can prevent the formation of inclusion bodies. Commonly used chaperones in E. coli include GroEL/GroES, DnaK/DnaJ, and trigger factor, which help stabilize folding intermediates and promote the correct folding of proteins (Chung et al., 2014). By enhancing the folding capacity of the host cell, co-expression of chaperones can improve both the solubility and yield of recombinant proteins.
Second solution - fusion tags
Fusion tags, such as His-tags, glutathione S-transferase (GST), or maltose-binding protein (MBP), can be added to the recombinant protein to improve its solubility and facilitate purification. These tags often enhance the stability of the protein and can also assist in the folding process (Baneyx, 1999). After expression, the tag can be cleaved to yield the recombinant protein in its native form. This approach is particularly useful when dealing with proteins that are prone to aggregation.Thor
CONC
While E. coli is an invaluable tool for recombinant protein production, issues such as solubility, yield, and functionality can arise, particularly when expressing human proteins. These challenges can be mitigated through strategies such as co-expression of chaperones, codon optimization, altering expression conditions, using fusion tags, and managing cellular stress. With continued advancements in synthetic biology and expression systems, it is likely that more effective solutions will emerge to improve the expression of human proteins in E. coli.
