Corrigendum to "establishing a novel assay system for measuring renin concentration using cost effective recombinant ovine angiotensinogen".

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01409.].

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs.

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.

Identification of a radical SAM enzyme involved in the synthesis of archaeosine.

Archaeosine (G+), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ0)-containing tRNA (q0N-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G+-containing tRNA (G+-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to q0N-tRNA to form q0kN-tRNA, which has a preQ0 lysine adduct as a base. Through comparative genomics and biochemical experiments, we found that ArcS forms a robust complex with a radical S-adenosylmethionine (SAM) enzyme named RaSEA. The ArcS-RaSEA complex anaerobically converted q0N-tRNA to G+-tRNA in the presence of SAM and lysine via q0kN-tRNA. We propose that ArcS and RaSEA should be considered an archaeosine synthase α-subunit (lysine transferase) and ß-subunit (q0kN-tRNA lyase), respectively.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2.

Hydrogen peroxide (H2O2) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H2O2 in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H2O2 application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H2O2 but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H2O2 response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H2O2 response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Establishing a novel assay system for measuring renin concentration using cost effective recombinant ovine angiotensinogen.

BACKGROUND: Plasma renin can predict future cardiovascular events as well as the prevalence of chronic renal disease in hypertensive subjects. Ovine angiotensinogen (oANG) is a better substrate for measuring renin concentration through activity assay. Recombinant oANG expressed in Escherichia coli cells can be utilized as the substrate while measuring plasma renin. We aim to establish an immunoassay for measuring renin concentration at picomolar level using recombinant oANG. MATERIAL AND METHODS: Recombinant oANG was expressed in E. coli cells and purified to homogeneity. Various concentrations (0-1.5 pM) of recombinant human renin standard were prepared and incubated with recombinant oANG. Renin activity was determined by angiotensin-I specific enzyme-linked immunosorbent assay. RESULTS: About 4.5 mg of purified recombinant oANG was obtained from 0.5 L of E. coli culture. The Michaelis constant and turnover number of human renin with recombinant oANG were 0.16 µM and 0.51 s-1, respectively. A linear relationship was obtained when renin activity was plotted as a function of renin concentration using recombinant oANG as the renin substrate. Picomolar amounts of renin can be measured from known renin activity using this method. CONCLUSION: This study established a novel assay system for measuring renin at picomolar level using cost effective recombinant oANG.

Escherichia coli-based production of recombinant ovine angiotensinogen and its characterization as a renin substrate.

BACKGROUND: Angiotensinogen (ANG) is a macromolecular precursor of angiotensin, which regulates blood pressure and electrolyte balance. ANG is specifically cleaved by renin, an aspartic protease, to initiate the angiotensin-processing cascade. Ovine ANG (oANG) from sheep plasma has been shown to be a better substrate for human renin, and it has been used in clinical renin assays. To expand the availability of oANG, we aimed to produce milligram levels of recombinant oANG using an Escherichia coli expression system. RESULTS: When recombinant oANG was expressed from a T7 promoter in various E. coli strains at 37 °C, it accumulated in the insoluble fraction. However, by expressing oANG at 37 °C from a tac promoter, which has weaker transcriptional activity than a T7 promoter, we significantly elevated the ratio of soluble to insoluble recombinant oANG. Using a novel culturing system and auto-induction culture medium, we purified tac-expressed recombinant oANG to homogeneity, with a yield of 4.0 mg per liter of culture. Based on size-exclusion gel filtration analysis and dynamic light scattering analysis, the resulting purified oANG is a monomer in solution. The circular dichroism spectrum of E. coli-expressed recombinant oANG was similar to that of oANG expressed in CHO cells. Differential scanning fluorimetry showed that both preparations undergo a two-state transition during thermal denaturation, and the melting temperatures of recombinant oANG expressed in E. coli and CHO cells were 49.4 ± 0.16 °C and 51.6 ± 0.19 °C, respectively. The K(m) values of both oANG preparations were similar; the k(cat) value of E. coli-expressed recombinant oANG was slightly higher than that of CHO-expressed oANG. CONCLUSIONS: Recombinant oANG expressed in E. coli functions as a human renin substrate. This study presents an E. coli-based system for the rapid production of milligram quantities of a human renin substrate, which will be useful for both fundamental and clinical studies on renin and hypertension.

Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum, a thermo-acidophilic archaeon.

Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G(+) by archaeosine synthase. However, tRNA(Leu) from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G(+)13 and G(+)15 modifications. In this study, we focused on the biosynthesis mechanism of G(+)13 and G(+)15 modifications in this tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.

Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase.

In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ0). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ0. We prepared a series of hypomodified tRNAs(Ser)(GGA) from Escherichia coli strains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAs(Ser)(GGA) as substrates showed that the Km decreased due to the lack of modified nucleosides. The tRNAs(Ser)(GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNA(Ser)(GGA) enhanced tRNA stability. Furthermore, the ArcTGT K(m) strongly correlated with the melting temperature (T(m)), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.

Synthesis of small interfering RNAs containing acetal-type nucleoside analogs at their 3'-ends and analysis of their silencing activity and their ability to bind to the Argonaute2 PAZ domain.

In this study, we aimed to create small interfering RNAs (siRNAs) with increased silencing activities and nuclease resistance properties. Therefore, we designed and synthesized five types of siRNA containing acetal-type nucleoside analogs at their 3'-dangling ends. We found that the siRNA containing 1-O-(2,2,2-trifluoroethyl)-ß-D-ribofuranose at the 3'-dangling end was the most potent among the synthesized siRNAs and showed more resistance to nucleolytic degradation by a 3' exonuclease than a natural RNA did. Thus, modification of siRNAs by addition of 1-O-(2,2,2-trifluoroethyl)-ß-D-ribofuranose may hold promise as a means of improving the silencing activity and nuclease resistance of siRNAs.

Improved solid-phase DNA probe method for tRNA purification: large-scale preparation and alteration of DNA fixation.

The solid-phase DNA probe method, in which a target transfer RNA (tRNA) is hybridized with a complementary DNA oligomer, is generally used for tRNA purification. However, purification of tRNAs from thermophiles by this method is not easy because of their high melting temperatures. To overcome this problem, the use of tetraalkylammonium salts was previously reported [Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., and Nishikawa, K. (2010) Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res. 38, e89]. In this study, we initially devised a large-scale purification system using tetraalkylammonium salts. The yield of tRNA was increased more than 10-fold and the manual steps were decreased as compared with the previous procedure. However, deterioration of column was very rapid owing to shedding of the biotinylated DNA probe. We therefore devised a method of covalent DNA fixation, in which a 5'-aminohexyl (dT)8 oligomer was fixed onto the N-hydroxysuccinimide-activated agarose, and then a DNA oligomer containing the tRNA and repeated A8 sequences was annealed. The probe sequence for tRNA purification was synthesized in column with Klenow enzyme. This DNA fixation method enabled us to use the column repeatedly and to wash the column with warmed buffers. Thus, this DNA fixation method is economical as compared with the previous method using the biotinylated DNA probe.

Protein fishing using magnetic nanobeads containing calmodulin site-specifically immobilized via an azido group.

We developed a novel method for capturing proteins that interact with a target protein. This method utilizes a protein containing a site-specifically incorporated 3-azidotyrosine (N3-Y) and FG beads for immobilization of the protein via an azido group. Using calmodulin (CaM) as the target protein, we introduced N3-Y at position 72 and conjugated it to FG beads by copper-free click chemistry. From the Ca(2+)/CaM-binding proteins captured from mouse brain cell lysate and analysis by mass spectrometry, we identified six proteins: alpha-enolase (ENOA), glucose-6-phosphate isomerase (GPI), annexin A5 (ANXA5), malate dehydrogenase 1 (MDH1), glyceraldehyde-3-phosphate dehydrogenase and the well-known CaM-binding protein phosphoglycerate kinase 1 (PGK1). The presence of photo-crosslinking products via N3-Y for all the captured proteins except GPI indicated that they bound directly to CaM. In this study, ENOA, ANXA5 and MDH1 were identified as novel CaM-binding proteins, and PGK1 was bound to Ca(2+)/CaM and also Ca(2+)-free CaM. This method should prove useful for capturing novel interacting proteins and serve as a useful tool for proteomic analyses.

A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli.

We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(()(CUA)), and made an orthogonal tRNA((CUA)) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA(()(CUA)) gene. Although the yields of full-length CaM increased ~3-fold, the ratio of N(3)-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N(3)-Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.

Involvement of plasmin-mediated extracellular activation of progalanin in angiogenesis.

Progalanin is released from the small cell lung carcinoma line SBC-3A and converted to its active form by plasmin. To elucidate the role of progalanin activation in the extracellular compartment, matrix metalloproteinase (MMP) activity was studied in SBC-3A cells treated with progalanin siRNA, and angiogenesis was measured in tumor tissue originating from SBC-3A cell transplantation into mice. Progalanin siRNA caused downregulation of progalanin expression for approximately 8 days. MMP activity and angiogenesis were reduced in tumors induced by transplantation of progalanin siRNA-treated SBC-3A cells. In contrast, MMP-9 and MMP-2 activity and angiogenesis increased in tumors originating from progalanin siRNA-treated SBC-3A cells in the presence of galanin and progalanin. Furthermore, injection of tranexamic acid, a plasmin inhibitor, more markedly reduced MMP-9 and MMP-2 activity and angiogenesis in tumors originating from progalanin siRNA-treated SBC-3A cells and in tumor tissue originating from progalanin siRNA-treated SBC-3A cells in the presence of progalanin. The reduction of MMP-9 and MMP-2 activity with tranexamic acid was restored by galanin, but not by progalanin. Moreover, tranexamic acid reduced angiogenesis in control siRNA-treated SBC-3A cells. These results suggest that the activation of progalanin by plasmin in the extracellular compartment was involved in MMP-9 and MMP-2 activation and in angiogenesis in tumor tissue.

Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.

Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.

Site-specific attachment of a protein to a carbon nanotube end without loss of protein function.

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.

Activation of large form galanin-LI by extracellular processing in small cell lung carcinoma tissue.

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. Some small cell lung carcinoma (SCLC) cell lines such as SBC-3A release only the high-molecular-mass form, with lower molecular mass forms being undetectable. To investigate the mechanism of processing of progalanin to active peptide, we studied galanin-LI in both the culture media of SBC-3A cells and in extracts from in vivo mouse SBC-3A tumors. SBC-3A cells were found to release high molecular mass galanin, but did not release active peptides. In contrast, tumor extract contained both high-molecular-mass galanin, and a cleaved lower-molecular-mass form of the peptide (8, 5 and 2 kDa). The lower-molecular-mass peptide was identified as galanin(1-20) by MALDI-TOF mass spectrometry. We then looked at MMP-2 and MMP-9 release from SBC-3A cells and tumor tissue treated with galanin and progalanin, as revealed by gelatin zymography. Galanin elicited pro-MMP-2 and pro-MMP-9 release from SBC-3A cells and tumor tissue; however, recombinant progalanin induced pro-MMP-2 and pro-MMP-9 release from tumor tissue only. This study has shown that the galanin-LI released from SCLC SBC-3A cells consisted of the high-molecular-mass peptide form, and was processed extracellularly to galanin(1-20). Furthermore, galanin was seen to induce pro-MMP-2 and pro-MMP-9 release from SBC-3A cells.

Incorporation of 3-azidotyrosine into proteins through engineering yeast tyrosyl-tRNA synthetase and its application to site-selective protein modification.

An efficient method for site-selective introduction of 3-azidotyrosine into proteins has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutant tyrosyl-tRNA synthetase (Y43G) pair as the carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system. Using rat calmodulin (CaM) as a model protein, we prepared an unnatural CaM molecule carrying a 3-azidotyrosine residue at the predetermined position 80. The synthesized CaM containing 3-azidotyrosine was site-specifically modified via azido group with a fluorescent alkyne derivative by click chemistry. This method will be useful to prepare not only a cross-linkable protein containing 3-azidotyrosine but also a fluorescent protein with a single fluorophore to facilitate the elucidation of molecular mechanisms of protein functions and protein-to-protein networks.