A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli

In prokaryotes, acyl carrier protein (ACP) is a cofactor central to a myriad of syntheses, including fatty acid and phospholipid synthesis. To fulfill its function, ACP must therefore interact with a multitude of different enzymes, which includes the thioesterase YbgC. We found a specific interaction between ACP and YbgC whose thioesterase activity has been demonstrated in vitro on acyl-CoA derivatives, but whose physiological function in bacteria remains unknown. Therefore, YbgC could be a thioesterase active on some specific acyl-ACPs. We then assigned a function to the ACP/YbgC pair by employing a proteomic approach derived from tandem affinity purification, the split tag method. This technique allowed us to purify proteins interacting with ACP and YbgC proteins at the same time. Interactions with PlsB, a sn-glycerol-3-phosphate acyltransferase and PssA, a phosphatidylserine synthase, were identified and validated, showing that YbgC is involved in phospholipid metabolism. Furthermore, using an in vivo bacterial two-hybrid interaction analysis, we showed for the first time that enzymes of the phospholipid synthesis pathway form a complex in the inner membrane. Taken together, these results describe an integrated protein network that could be involved in the coordination of phospholipid metabolism.     

Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase

Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several alpha-keto acids. In its functional form, it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg(-1), indicating a turnover number of approximately 0.2 s(-1), and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2+/-4.4 and 13.2+/-2.9 microM, respectively.     

Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis.

In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2). Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene. Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen. An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy. Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically. An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media. These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene. Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.     

Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli

Abstract

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85?g/L in the shake flask and 7.7?g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.     

A novel amino acid supplementation strategy based on a stoichiometric model to enhance human IL-2 (interleukin-2) expression in high-cell-density Escherichia coli cultures

A novel amino acid supplementation strategy was developed for enhancing the production of IL-2 (interleukin-2; as a model protein) by recombinant Escherichia coli BL21 (pET21a-hil2) in fed-batch high-cell-density cultures. The amino acids most needed and their amounts were determined using a stoichiometric model, and full factorial design experiments were conducted to determine the effects of single amino acids and amino acid mixtures on production. One of the most effective amino acid mixtures was found to be leucine, aspartic acid and glycine. This amino acid mixture was utilized for the production of IL-2 in batch and fed-batch fermentations. The amount of IL-2 produced increased from 403 to 722 mg/l and from 5.15 × 103 to 8.08 × 103 mg/l in batch and fed-batch cultures respectively. The results also revealed that the above amino acid mixture specifically increases IL-2 concentration in the cells.     

Gene expression and characteristics of a novel fibrinolytic enzyme (subtilisin DFE) in Escherichia coli

AIMS: The objective of this study is to actively express a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme), in Escherichia coli. METHODS AND RESULTS: The DNA fragments encoding pro-subtilisin DFE was amplified and cloned into the vector pET32a to obtain N-terminal Trx fusion expression plasmid. The recombinant subtilisin DFE was successfully expressed and processed in the soluble fraction of E. coli BL21(DE3) in a similar fashion as the endogenous one of Bacillus amyloliquefaciens DC-4, resulting in an active enzyme. Moreover, active enzyme can also be refolded from inclusion body. CONCLUSIONS: Active subtilisin DFE can be expressed and processed in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidences that subtilisin DFE can be actively expressed in E. coli and the pro-peptide is essential for guiding the proper folding into the active conformation. As such, large quantities of recombinant subtilisin DFE can be produced for pharmacological and clinical research.     

Redox regulation of PTEN and protein tyrosine phosphatases in H(2)O(2) mediated cell signaling

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H₂O₂ are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disulfide bond between two cysteines, one in the active site and the other nearby, during oxidation by H₂O₂. The disulfide bond likely confers efficiency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disulfide bond formation in the PTPs.     

Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2[4Fe-4S] ferredoxin

Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin (Fd) in Escherichia coli. The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers. The incorporation of the [4Fe-4S] clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C. pasteurianum apoFd. In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters. The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 2[4Fe-4S] Fd in E. coli, irrespective of the expression system used. These features are characteristic of 2[4Fe-4S] Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields. The reasons for the high turnover of 2[4Fe-4S] Fds are discussed.     

大肠杆菌重组乳链菌肽抗性蛋白(NSR)的表达纯化及其功能分析

乳链菌肽(Nisin)是由某些乳酸菌产生的一种阳离子抗菌肽, 而Nisin抗性蛋白(Nisin resistance protein, NSR)的表达则使一些非 Nisin产生菌获得Nisin抗性。为深入探索NSR的作用机制, 本研究在大肠杆菌中表达了去除N末端38个氨基酸残基的NSR(NSRΔ38)与GST的融合蛋白GST-NSRΔ38。通过谷胱甘肽(GSH)亲和层析和GST标签的切除后, 得到纯化的NSRΔ38, 并测定了该蛋白可能的nisin降解活性。反应产物的抑菌活性测定结果表明被NSRΔ38作用     

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand–HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.     

Purification and refolding of human alpha5-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli

The 20S proteasome is the central enzyme of nonlysosomal protein degradation in both the cytosol and nucleus. It is composed of 28 protein subunits which are arranged into four staggered heptameric rings. The outer rings consist of alpha-subunits which are responsible for binding of proteasome activators, inhibitors, and regulators. To better characterize human alpha5-subunit (PSMA5) of the 20S proteasome, we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the human PSMA5, which was subcloned into the vector pET-22b (+), has been expressed as inclusion bodies in E. coli BL21 (DE3). To produce the native PSMA5, straightforward protocols have been developed for refolding the human PSMA5 in the presence of surfactants using dilution refolding and size-exclusion chromatography matrix refolding methods. After refolding, recovery yields of about 20% were obtained, respectively, with purity above 95%. The human PSMA5 was detected by dynamic light scattering in refolding process, and the molecular weight of the final refolded product was measured using gel filtration chromatography, which indicates that the human PSMA5 exists mainly as tetramer.     

Soluble expression of a functionally active Plasmodium falciparum falcipain-2 fused to maltose-binding protein in Escherichia coli

Falcipain-2 (fp2) is a hemoglobinase required for supplying peptides and amino acids for the proliferation of Plasmodium falciparum in blood. The prospect of circumventing its activity thereby serves as a potential strategy for mining drugs for anti-malarial therapy. However, to date, efforts to express soluble and active fp2 in Escherichia coli have been futile. To overcome this problem, fp2 was expressed under an array of conditions including the exploitation of multiple gene constructs in eukaryotic and prokaryotic hosts. A series of experiments led to the finding that the placement of maltose-binding protein (MBP) before the fp2 mature domain was best in availing the soluble expression of the protease. The results also indicate that the prodomain impaired the bacterial expression of the protease and the amino acid residues at the N-terminal segment of mature fp2 can have a significant effect on the folding and solubility of the enzyme. The overexpressed MBP-fp2 fusion protein was purified and shown to be functionally active, providing a very useful alternative to the use of resolubilized enzyme for future study of structure and function of fp2.     

Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.