Abstract
Solubility is the prime criterion for determining the quality of recombinant proteins, yet it often fails to represent functional activity due to the involvement of non-functional, misfolded, soluble aggregates, which compromise the quality of recombinant proteins. However, guidelines for the quality assessment of soluble proteins have neither been proposed nor rigorously validated experimentally. Using the aggregation-prone enhanced green-fluorescent protein (EGFP) folding reporter system, we evaluated the folding status of recombinant proteins by employing the commonly used sonication and mild lysis of recombinant host cells. We showed that the differential screening of solubility and folding competence is crucial for improving the quality of recombinant proteins without sacrificing their yield. These results highlight the importance of screening out incorrectly folded soluble aggregates at the initial purification step to ensure the functional quality of recombinant proteins.
Highlights
Bacterial hosts, in particular Escherichia coli, have been favored for the recombinant expression of heterologous proteins as the procedure is inexpensive, fast, simple, and easy to scale-up
We fused hRID(2m) to the N-terminal of enhanced green-fluorescent protein (EGFP) to induce the formation of soluble aggregates. Using this model protein prone to soluble aggregation, and employing commonly used cell extraction methods from the E. coli recombinant host, we showed that the differential screening of solubility and folding competence is a prerequisite for improving the quality of recombinant proteins in subsequent purification processes
Since EGFP is expressed predominantly in its insoluble form in E. coli under harsh conditions, hRID(2m)-fusion was used to regulate the formation of soluble aggregates
Summary
In particular Escherichia coli, have been favored for the recombinant expression of heterologous proteins as the procedure is inexpensive, fast, simple, and easy to scale-up. The cytoplasm of E. coli often fails to provide the optimal environment for the folding of recombinant proteins; various approaches have been used to overcome this inherent problem [1] These approaches include: (1) co-expression with molecular chaperones or specific binding partners that may help to fold target proteins [2,3,4,5], and (2) fusion with highly soluble proteins, such as Glutathione S-transferase(GST), Maltose-binding protein(MBP), N utilization substance protein A(NusA) [6], DnaK [7,8], Small ubiquitin-like modifier(SUMO) [9], or Lysyl tRNA synthetase(LysRS) [10], to enhance the solubility of target proteins [11]. Fusion with highly soluble partners or co-expression with molecular chaperones may greatly enhance their overall solubility, it has long been observed that these proteins often remain biologically inactive Further examination of their physico-chemical properties frequently reveals that these proteins are present predominantly as aggregates rather than in their mono-dispersed forms [18]. Such negative results are regularly observed by researchers, but are not usually shared in the public domain
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