Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

The Study of Stress Conditions on Growth and Proteome of Raoultella Planticola: A New Emerging Pathogen

All bacteria can survive and adapt to different stresses such as fluctuations in temperature, pH oxidative, and osmotic pressure occurring in their surrounding environments. This study aims to evaluate the effects of a variety of stress conditions on the growth, and proteome of Raoultella planticola PTCC 1598. R. planticola cells were exposed to different values of temperatures, sodium chloride, pH, and hydrogen peroxide stresses. Amongst the stress conditions, oxidative stress upon exposure to hydrogen peroxide (H2O2) at 4000 ppm concentration was selected for proteomics analysis in detail. Approximately 1400 spots were identified in two-dimensional gel electrophoresis (2-DE). Among the identified spots, 85 spots were repeatable using 2D-Platinum software and eye confirmation and, nine protein spots were differentially expressed. Among nine proteins, six proteins identified successfully with a MASCOT score greater than 40 (p<0.05) were 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (oxidoreductase family), D-galactose-binding periplasmic protein, uridine phosphorylase (glycosyltransferases), uridine phosphorylase, a single peptide match to cysteine-binding periplasmic protein, and NADP(H) nitroreductase. All identified proteins showed decreased level expression. Based on the obtained results, we concluded that hydrogen peroxide as an antiseptic compound could affect cell growth and proteomics of R.planticola. So, we recommend using an antiseptic solution containing H2O2 to prevent the spread of R.planticola as a new emerging pathogen.

A Novel Salmonella Periplasmic Protein Controlling Cell Wall Homeostasis and Virulence

Horizontal gene transfer has shaped the evolution of Salmonella enterica as pathogen. Some functions acquired by this mechanism include enzymes involved in peptidoglycan (PG) synthesis and remodeling. Here, we report a novel serovar Typhimurium protein that is absent in non-pathogenic bacteria and bears a LprI functional domain, first reported in a Mycobacterium tuberculosis lipoprotein conferring lysozyme resistance. Based on the presence of such domain, we hypothesized a role of this S. Typhimurium protein in PG metabolism. This protein, which we named ScwA for Salmonellacell wall-related regulator-A, controls positively the levels of the murein lytic transglycosylase MltD. In addition, the levels of other enzymes that cleave bonds in the PG lattice were affected in a mutant lacking ScwA, including a soluble lytic tranglycosylase (Slt), the amidase AmiC, and a few endo- and carboxypeptidases (NlpC, PBP4, and AmpH). The scwA gene has lower G+C content than the genomic average (43.1 vs. 52.2%), supporting acquisition by horizontal transfer. ScwA is located in the periplasm, stabilized by two disulfide bridges, produced preferentially in stationary phase and down-regulated following entry of the pathogen into eukaryotic cells. ScwA deficiency, however, results in a hypervirulent phenotype in the murine typhoid model. Based on these findings, we conclude that ScwA may be exploited by S. Typhimurium to ensure cell envelope homeostasis along the infection and to prevent host overt damage. This role could be accomplished by controlling the production or stability of a reduced number of peptidoglycan hydrolases whose activities result in the release of PG fragments.

Selective Release of Recombinant Periplasmic Protein From E. coli Using Continuous Pulsed Electric Field Treatment

To date, high-pressure homogenization is the standard method for cell disintegration before the extraction of cytosolic and periplasmic protein from E. coli. Its main drawback, however, is low selectivity and a resulting high load of host cell impurities. Pulsed electric field (PEF) treatment may be used for selective permeabilization of the outer membrane. PEF is a process which is able to generate pores within cell membranes, the so-called electroporation. It can be readily applied to the culture broth in continuous mode, no additional chemicals are needed, heat generation is relatively low, and it is already implemented at industrial scale in the food sector. Yet, studies about PEF-assisted extraction of recombinant protein from bacteria are scarce. In the present study, continuous electroporation was employed to selectively extract recombinant Protein A from the periplasm of E. coli. For this purpose, a specifically designed flow-through PEF treatment chamber was deployed, operated at 1.5 kg/h, using rectangular pulses of 3 μs at specific energy input levels between 10.3 and 241.9 kJ/kg. Energy input was controlled by variation of the electric field strength (28.4–44.8 kV/cm) and pulse repetition frequency (50–1,000 Hz). The effects of the process parameters on cell viability, product release, and host cell protein (HCP), DNA, as well as endotoxin (ET) loads were investigated. It was found that a maximum product release of 89% was achieved with increasing energy input levels. Cell death also gradually increased, with a maximum inactivation of -0.9 log at 241.9 kJ/kg. The conditions resulting in high release efficiencies while keeping impurities low were electric field strengths ≤ 30 kV/cm and frequencies ≥ 825 Hz. In comparison with high-pressure homogenization, PEF treatment resulted in 40% less HCP load, 96% less DNA load, and 43% less ET load. Therefore, PEF treatment can be an efficient alternative to the cell disintegration processes commonly used in downstream processing.

The hypothetical periplasmic protein PA1624 from Pseudomonas aeruginosa folds into a unique two-domain structure

The crystal structure of the 268-residue periplasmic protein PA1624 from the opportunistic pathogen Pseudomonas aeruginosa PAO1 was determined to high resolution using the Se-SAD method for initial phasing. The protein was found to be monomeric and the structure consists of two domains, domains 1 and 2, comprising residues 24–184 and 185–268, respectively. The fold of these domains could not be predicted even using state-of-the-art prediction methods, and similarity searches revealed only a very distant homology to known structures, namely to Mog1p/PsbP-like and OmpA-like proteins for the N- and C-terminal domains, respectively. Since PA1624 is only present in an important human pathogen, its unique structure and periplasmic location render it a potential drug target. Consequently, the results presented here may open new avenues for the discovery and design of antibacterial drugs.

RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae

Vibrio cholerae, the cause of cholera disease, exhibits a characteristic curved rod morphology, which promotes infectivity and motility in dense hydrogels. Periplasmic protein CrvA determines cell curvature in V. cholerae, yet the regulatory factors controlling CrvA are unknown. Here, we discover the VadR small RNA (sRNA) as a post-transcriptional inhibitor of the crvA mRNA. Mutation of vadR increases cell curvature, whereas overexpression has the inverse effect. We show that vadR transcription is activated by the VxrAB two-component system and triggered by cell-wall-targeting antibiotics. V. cholerae cells failing to repress crvA by VadR display decreased survival upon challenge with penicillin G indicating that cell shape maintenance by the sRNA is critical for antibiotic resistance. VadR also blocks the expression of various key biofilm genes and thereby inhibits biofilm formation in V. cholerae. Thus, VadR is an important regulator for synchronizing peptidoglycan integrity, cell shape, and biofilm formation in V. cholerae.

Role of LmeA, a Mycobacterial Periplasmic Protein, in Maintaining the Mannosyltransferase MptA and Its Product Lipomannan under Stress

ABSTRACT The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, a periplasm, and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan chain. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in an lmeA deletion (ΔlmeA) mutant under both stress conditions. The transcript levels of mptA in the ΔlmeA mutant were similar to or even higher than those in the wild type, indicating that the decrease of MptA protein was a posttranscriptional event. The ΔlmeA mutant was unable to maintain the cellular level of LM under stress, consistent with the decrease in MptA. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase, particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity. IMPORTANCE Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria.

The role of LmeA, a mycobacterial periplasmic protein, in stabilizing the mannosyltransferase MptA and its product lipomannan under stress

ABSTRACTThe mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, periplasm and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan backbones. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis. Here, we examined the role of LmeA under stress conditions. We found that the lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in lmeA deletion mutant (ΔlmeA) in both stress conditions. The transcript levels of mptA in ΔlmeA were similar to or even higher than those in the wildtype, indicating that the decrease of MptA protein was a post-transcriptional event. Consistent with the decrease in MptA, ΔlmeA was unable to maintain the cellular level of LM under stress. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.

Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica

The characterization of Zn(ii) and Cu(ii) complexes with periplasmic protein ZinT suggests that the N-terminal histidine-rich loop plays a role as a primary metal scavenger and that ZinT can possibly transfer a Zn(ii) ion to ZnuA.