Single Protein Production System Research Articles

Improvement of stability and in vivo antioxidant effect of human glutathione peroxidase mutant by PEGylation

Human glutathione peroxidase (GPx), as an important kind of antioxidant enzyme, is often used for the removal of reactive oxygen species. Unfortunately, the application has been hindered by its limited source and poor stability. To solve these problems, human glutathione peroxidase mutant (GPxM) with high activity and yield was obtained using Escherichia coli BL21(DE3) cys auxotrophic strain and the single-protein production system in our previous work. However, the antioxidant effect of this novel recombinant protein drug in animals has not been demonstrated, and its immunogenicity and short biological half-life as a biological macromolecule may have seriously hindered its clinical application. Therefore, it is important to find an effective strategy to address the above issues. In this work, PEGylated GPxM was prepared by conjugating the corresponding mutant with monomethoxy polyethylene glycol succinimidyl succinate (SS-mPEG). We researched the structure, stability, pharmacokinetic properties, antioxidant effect in vivo and protective mechanism against adriamycin (ADR)-mediated cardiotoxicity of modified products, and compared with the above properties of GPxM. The results revealed that GPxM had an excellent antioxidant effect in vivo, and PEGylation can enhance the stability, half-life and antioxidant effect of GPxM while reducing immunogenicity. In addition, the above improvement of PEGylated GPx1M was stronger than that of monoPEGylated GPx4M. Hence, PEGylation might be an effective method to broaden the applications of GPxM as the important antioxidant drug, especially the PEGylated GPx1M with high antioxidant effect.

Cold-Shock Expression System in E. coli for Protein NMR Studies.

The cold-shock system using the pCold vector is one of the most effective Escherichia coli heterologous protein expression systems. It allows the improvement of the expression level of the protein of interest in a soluble fraction. In this chapter, we describe practical procedures for the overexpression of heterologous protein of interest by using the pCold vector or the single-protein production system. The latter is one of the most advanced pCold technologies for isotope labeling of the target protein and its NMR studies.

Suppression of the toxicity of Bac7 (1-35), a bovine peptide antibiotic, and its production in E. coli.

Bac7 (1–35) is an Arg- and Pro-rich peptide antibiotic, produced in bovine cells to protect them from microbial infection. It has been demonstrated to inhibit the protein synthesis in E. coli, leading to cell death. Because of its toxicity, no cost effective methods have been developed for Bac7 production in Escherichia coli for its potential clinical use. Here, we found a method to suppress Bac7 (1–35) toxicity in E. coli to establish its high expression system, in which Bac7 (1–35) was fused to the C-terminal end of protein S, a major spore-coat protein from Myxococcus xanthus, using a linker containing a Factor Xa cleavage site. The resulting His6-PrS2-Bac7 (1–35) (PrS2 is consisted of two N-terminal half domains of protein S connected in tandem) was well expressed using the Single-Protein Production (SPP) system at low temperature and subsequently purified in a single step by using a Ni column. The combination of protein S fusion and its expression in the SPP system at low temperature appeared to suppress Bac7 (1–35) toxicity. Both the purified His6-PrS2-Bac7 (1–35) and His6-PrS2-Bac7 (1–35) treated by Factor Xa were proven to be a potent inhibitor for cell-free protein synthesis.

Characterization of recombinant human gastrointestinal glutathione peroxidase mutant produced in Escherichia coli

Gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenium-dependent enzyme and regarded as the first line of defense against oxidative stress caused by ingested pro-oxidants or gut microbes. As the essential part of the catalytic site of GPx2, selenocysteine (Sec) is encoded by an in-frame UGA stop codon, which makes the expression of human GPx2 (hGPx2) using traditional recombinant DNA technology difficult. In order to produce bioactive recombinant hGPx2, the gene of hGPx2 was designed with the conversion of the codons for four cysteine (Cys) residues to the codons for serine (Ser) residues and the codon for Sec-40 was changed to the codon for Cys. This recombinant seleno-hGPx2 mutant was obtained using a single protein production system in a cysteine (Cys) auxotrophic strain, in which Sec was introduced into the protein via tRNACys misleading. The activity of this mutant was in the same order of magnitude as that of hGPx4, but about one order of magnitude lower than that of hGPx1 and hGPx3. Further study showed that the mutant exhibited pH and temperature optima of 7.4 and 25°C, respectively. The results obtained from the kinetic analysis demonstrated that it followed a typical ping-pong mechanism similar to native GPx. As there was no report on the activity of purified GPx2, this research was valuable in recognizing native GPx2. In addition, a three-dimensional structure of seleno-hGPx2 mutant was constructed, which could facilitate further analysis of the role and the catalytic mechanism of native GPx2.

The Protective Effect of a Metallic Selenopeptide with Superoxide Dismutase and Glutathione Peroxidase Activities Against Alcohol Induced Injury in Hepatic L02 Cells

A 65-mer peptide (Se–CuZn–65P) is an enzyme mimic with antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX). The metallic selenopeptide imitate was obtained through a single protein production system and cysteine auxotrophic expression technique, and it enters cells in the assistance of the trans-activator of transcription peptide. Because Se–CuZn–65P was provided with the antioxidant activities of SOD and GPX, it can be a potential drug for the prevention of alcoholic liver diseases. Therefore, the protective effects of Se–CuZn–65P on ethanol-induced L02 cells were evaluated in vitro. Our results revealed that Se–CuZn–65P significantly protected the hepatocytes against ethanol-induced cell cytotoxicity which displayed by increase of cell viability, migration and decrease of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH), aspartate aminotransferase (AST). Furthermore, the experiments of TUNEL assay, flow cytometry assay, expression levels determination of apoptosis-related gene (Bad, Bax, Bcl-xL, Bcl-2) and protein (pro-caspase3, PARP cleavage) indicated that Se–CuZn–65P inhibited ethanol-induced L02 cell apoptosis. Mechanically, Se–CuZn–65P could attenuate the oxidative damage of mitochondria in ethanol-induced L02 cells. Se–CuZn–65P prevented ethanol-triggered ROS and MDA generation in L02 cells and inhibited the release of the AST and LDH. Se–CuZn–65P also restored the equilibrium of proapoptotic and antiapoptotic genes including Bad, Bax, Bcl-xL, and Bcl-2. And the reduction of procaspase-3 and the cleavages of PARP were attenuated by Se–CuZn–65P. Thereby, the L02 cell apoptosis were significantly prevented by treatment with Se–CuZn–65P, mainly due to attenuation of oxidative stress. In summary, Se–CuZn–65P could prevent ethanol-induced liver injury through enhancing hepatocyte antioxidant abilities in intracellular.

High level soluble expression, purification, and characterization of human ciliary neuronotrophic factor in Escherichia coli by single protein production system

Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (rhCNTF) in heterologous expression system is demanded. Many attempts had been undertaken to product rhCNTF in Escherichia coli (E. coli), however, the expression level of rhCNTF was low and most of which formed insoluble inclusion bodies. In this study, we described a new and efficient method to express rhCNTF. The human CNTF gene was codon optimized and then expressed by the single protein production (SPP) expression system in E. coli. The results showed that rhCNTF was expressed as a soluble biologically active protein, and upon purification, the final yield was about 250mg/L in shake flask with a specific neuroprotective activity in Aβ-induced SH-SY5Y cell injury model. Our study might open up a new strategy for large-scale production of functional rhCNTF for clinical applications as well as basic research.

Can Recombinant Human Glutathione Peroxidase 1 with High Activity Be Efficiently Produced inEscherichia coli?

Cellular glutathione peroxidase 1 (GPx1) is an important antioxidant selenoenzyme. Due to the presence of selenocysteine (Sec) encoded by UGA, normally recognized as a stop codon, the expression of mammalian GPx with traditional recombinant DNA technology is extremely difficult. In this study, a series of human GPx1 (hGPx1) mutants with significantly high catalytic activities were produced for the first time in an Escherichia coli BL21(DE3)cys auxotrophic strain using the single-protein production system. Cys residues in hGPx1 were mutated to Ser in turn because untargeted substitution of Sec in place of Cys resulted in the decline of recombinant selenoenzyme activity. The results of this work showed that the catalytic activities of the mutants increased progressively with decreasing number of noncatalytic Sec residues. Seleno-hGPx1-C2/78/115/156/202S with all Cys residues changed to Ser showed the highest activity (21,268 U/μmol), which was more than 10-fold higher than bovine liver GPx. This increase could be explained by structural analysis of hGPx1 mutants based on homology modeling and binding site analysis. These results lead to the hypothesis that the conversion of noncatalytic Sec residues to Ser may optimize the structure of seleno-GPx in this expression system and consequently increase the catalytic efficiency.

Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

Generation of selenoprotein with glutathione peroxidase activity by chemical modification of the single-chain variable fragment expressed in a single-protein production system and its antioxidant ability

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. Single-chain variable fragment (scFv) can be converted into seleniumcontaining single-chain variable fragment (Se-scFv) by chemical modification of the hydroxyl groups in scFv, thus Se-scFv possesses GPX activity and becomes a prodrug. To improve the expression of scFv and simplify its purification steps, Single-protein production (SPP) system was used to express scFv and chemical modification was used to synthesize Se-scFv. Therefore, we must construct a new scFv-WCD1-lessACA gene, which can express its mRNA not containing any ACA sequences and express its amino acid sequence of target protein (scFv) being same to scFv-WCD1. In this way, the scFv-WCD1-lessACA can be only expressed in SPP system and no other background proteins in the cells could be expressed. The expression results showed that high level of scFv-WCD1-lessACA synthesis was at least sustained for 96 h in the virtual absence of background protein synthesis. Then, selenocysteine (Sec) was incorporated into the scFv-WCD1-lessACA by chemical modification and resulted in Se-scFv-WCD1-lessACA. The enzymatic characteristics of Se-scFv-WCD1-lessACA were determined. GPX activity was 2,563 U/μmol, its binding constant for GSH was 0.687 ×105/mol. Moreover, Se-scFv-WCD1-lessACA was confirmed to have a strong antioxidant ability to protect mitochondria against oxidative damage induced by Vc/Fe2+ (mitochondrial damage model), suggesting that Se-scFv-WCD1-lessACA has potential application for protection of mitochondrial damage induced by reactive oxygen species (ROS).

An Assay Suitable for High Throughput Screening of Anti-Influenza Drugs

We developed a novel drug screening system for anti-influenza A virus by targeting the M2 proton channel. In the SPP (Single Protein Production) system, E. coli cell growth occurs only in the presence of effective M2 channel inhibitors, and thus simple measurement of cell growth was used as readouts for drug screening. Two potential inhibitors for M2 (V27A) mutant were verified using this method, which inhibit both the mutant and wild-type M2 channels.

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that (13)C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that (13)C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

Secretion of human superoxide dismutase in Escherichia coli using the condensed single‐protein‐production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ∼20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins, OmpA and OmpX in E. coli

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing ¹⁵N-NH₄Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [¹H-¹⁵N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment without the need of purification and reconstitution.

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-beta-d-thiogalactopyranoside]), the mutated MazF [MazF(DeltaW)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(DeltaH)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of (13)C, (15)N, and (2)H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.

Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.

In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.

The E. coli single protein production system for production and structural analysis of membrane proteins

At present, only 0.9% of PDB-deposited structures are of membrane proteins in spite of the fact that membrane proteins constitute approximately 30% of total proteins in most genomes from bacteria to humans. Here we address some of the major bottlenecks in the structural studies of membrane proteins and discuss the ability of the new technology, the Single-Protein Production system, to help solve these bottlenecks.

Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment

The ability to produce isotope-enriched proteins is fundamental to the success of modern protein NMR, and is particularly essential for NMR activities in structural genomics projects. Conventional methods of protein production often prove to be cost prohibitive for obtaining samples, particularly perdeuterated and site-specifically labeled proteins. The condensed single protein production system (cSPP), providing protein expression following condensation of cells 10-40 fold, allows for the production of such samples at a fraction of the cost. The previously described cSPP system is a two plasmid system where both the MazF toxin and ACA-less target gene are coinduced with IPTG. Coinduction results in 10-20% of the target protein produced without isotopic enrichment. Though the unlabeled protein is generally not visible in isotope-filtered NMR experiments, it results in an effective reduction in yield of the observable sample. By altering the cSPP system and separating the induction of the MazF toxin, required to convert cells into a semiquiescent state prior to condensation, from the expression of the target gene, we are now able to eliminate the unlabeled protein fraction and improve the isotope incorporation. Here we describe a series of pCold(tet) vectors with various features that can be used in the dual inducible cSPP(tet) system to obtain high-quality isotopically enriched protein at as little as 2.5% the cost of traditional methods.

Single protein production (SPP) system in Escherichia coli

Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 degrees C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20-30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including high-yield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.

Finding one of a kind: advances in single-protein production

An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies.