Roles of DegP in Prevention of Protein Misfolding in the Periplasm upon Overexpression of Penicillin Acylase in Escherichia coli

Autor: C. Perry Chou, Kao-Lu Pan, Ming-Sheng Wu, Hsu-Chou Hsiao, Chiao-Ling Weng
Jazyk: angličtina
Rok vydání: 2003
Předmět:
Popis: The well-known genetic information of Escherichia coli and successful applications of recombinant DNA technology make it possible for a variety of attempts with genetic engineering techniques to overproduce recombinant proteins. Upon performing the cultivation for recombinant protein production, there are two primary goals: high-cell-density cultivation and high-level gene expression. Culture performance can be optimized when the two goals are achieved simultaneously. High-cell-density culture can be obtained by fed-batch cultivation (48), in which concentrated medium is fed gradually into the bioreactor. The primary concern of this operation is developing an optimum feeding strategy, based on which cells can be maintained in a high-energy state for enhancing gene expression while cells are growing. On the other hand, various genetic strategies have been developed for high-level gene expression (24). The use of strong promoters (e.g., tac, trc, and T7) for regulation of gene expression has been successfully applied to enhance recombinant protein production by improving transcriptional efficiency and perhaps translational efficiency as well (35). However, it is a common problem that insoluble protein aggregates, known as inclusion bodies, tend to accumulate inside the cells upon overproduction of gene products (14, 44). While the mechanism of inclusion body formation is not completely understood, it is believed that the overexpressed gene products cannot be suitably processed by folding modulators to develop a proper protein structure (46). For proteins destined to be exported, the protein formation mechanism would be more complicated, and the efficiency of translocation, posttranslocational folding, processing, and targeting becomes important. In principle, the precursors, intermediates, or final gene products can possibly form inclusion bodies in the cytoplasm and/or periplasm upon gene overexpression (4). This raises an important issue that, in addition to improving the efficiency of each gene expression step (i.e., transcription, translation, and posttranslational steps), balanced protein synthesis flux throughout these steps should be properly maintained to avoid the accumulation of any protein species upon the overproduction of recombinant proteins. Within the past decade, the issues of protein misfolding in the bacterial periplasm and heat shock responses to extracytoplasmic stresses began to gain attentions. A specific periplasmic heat shock regulon of σE, which is similar to the σH heat shock regulon in the cytoplasm, has been identified in E. coli (9, 29, 32). Just as occurs in the cytoplasm, the heat shock responses to extracytoplasmic stresses were driven by synthesis of a variety of heat shock proteins expressing protease activity that degrades misfolded proteins and/or chaperone activity that renatures misfolded proteins. From the viewpoint of application, periplasmic proteins with these protease and/or chaperone activities would be proper candidates for relieving extracytoplasmic stresses when cells are overexpressing foreign gene products. More than 10 periplasmic proteases have been identified in E. coli (25). On the other hand, several periplasmic proteins, including DegP (also known as HtrA or Do) (42), Skp (11), SurA (20), and FkpA (2), have been identified as having chaperone activity and could play a role in the folding or targeting of extracytoplasmic proteins. Among these periplasmic proteases and chaperones, DegP and FkpA are two gene products induced in response to extracytoplasmic stresses, such as heat shock or the presence of misfolded proteins, via the σE heat shock regulon and Cpx two-component systems (32). DegP is perhaps the only periplasmic heat shock protein with both protease and chaperone activities. It has an inducible serine protease activity for breakdown of aberrant periplasmic proteins arising upon extracytoplasmic stresses (17, 45). Another uncommon feature of DegP is that this protein can exhibit either protease or chaperone activity with temperature as the regulatory switch (42). With these multiple functions, DegP is known to be involved in relieving extracytoplasmic stresses upon overexpression of several gene products, including alkaline phosphatase (PhoA) (13), DsbA′-PhoA (10), MalS (42), and maltose-binding protein (MalE) variants (3, 28). We previously demonstrated that the presence of exogenous DegP could enhance the production of recombinant penicillin acylase (PAC), an important industrial enzyme for the production of many β-lactam antibiotics (37), in E. coli (21). The formation of mature PAC in the periplasm involves a series of posttranslational steps, including translocation and periplasmic processing/folding steps, which are unusual for prokaryotic proteins (Fig. ​(Fig.1)1) (38). The periplasmic processing mechanism is known to consist of various proteolytic steps via intramolecular autoproteolysis (15, 38). The formation of inclusion bodies, which are composed primarily of PAC precursors in the periplasm, was recently identified as an important obstacle to the overproduction of PAC in E. coli (8, 36, 43). Hence, efforts have been directed to reducing the number of periplasmic PAC inclusion bodies. FIG. 1. Synthesis and maturation of PAC in E. coli. The structural pac gene from E. coli ATCC 11105 encodes a 95-kDa polypeptide precursor (preproPAC) composed of, from the N terminus to the C terminus, a signal peptide (S), α subunit (α), connecting ... In this study, we provide evidence that DegP functions primarily as a protease to improve cell physiology by preventing the misfolding and aggregation of periplasmic PAC precursors when pac is overexpressed. Cultivation performance for the production of recombinant PAC was significantly enhanced due to improved cell physiology as well as simultaneous increases in the pac gene expression level and culture cell density.
Databáze: OpenAIRE
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