End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation

We report an enzymatic end-point modification and immobilization of recombinant human thrombomodulin (TM), a cofactor for activation of anticoagulant protein C pathway via thrombin. First, a truncated TM mutant consisting of epidermal growth factor-like domains 4-6 (TM(456)) with a conserved pentapeptide LPETG motif at its C-terminal was expressed and purified in E. coli. Next, the truncated TM(456) derivative was site-specifically modified with N-terminal diglycine containing molecules such as biotin and the fluorescent probe dansyl via sortase A (SrtA) mediated ligation (SML). The successful ligations were confirmed by SDS-PAGE and fluorescence imaging. Finally, the truncated TM(456) was immobilized onto an N-terminal diglycine-functionalized glass slide surface via SML directly. Alternatively, the truncated TM(456) was biotinylated via SML and then immobilized onto a streptavidin-functionalized glass slide surface indirectly. The successful immobilizations were confirmed by fluorescence imaging. The bioactivity of the immobilized truncated TM(456) was further confirmed by protein C activation assay, in which enhanced activation of protein C by immobilized recombinant TM was observed. The sortase A-catalyzed surface ligation took place under mild conditions and occurs rapidly in a single step without prior chemical modification of the target protein. This site-specific covalent modification leads to molecules being arranged in a definitively ordered fashion and facilitating the preservation of the protein's biological activity.     

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06–120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in the development of bioluminescent indicators of the covalent modification of proteins.     

Functional integration of a semi-synthetic azido-queuosine derivative into translation and a tRNA modification circuit

Substitution of the queuine nucleobase precursor preQ₁ by an azide-containing derivative (azido-propyl-preQ₁) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNA^Asp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.     

Expression and Biological Properties of a Novel Methionine Sulfoxide Reductase A in Tobacco (Nicotiana tabacum)

Methionine (Met) residues in proteins/peptides are extremely susceptible to oxidation mediated by reactive oxygen species, resulting in the formation of methionine sulfoxide, which could be inversely reduced back to Met by methionine sulfoxide reductase (MSR). In the present study, an A-type MSR gene, termed NtMSRA4, was isolated from tobacco (Nicotiana tabacum). Sequence analysis of NtMSRA4 amino acid sequence indicated that the gene, encoded a polypeptide with a molecular weight of 21 kDa, possessed the highly conserved motif, ‘GCFWG’ in the N-terminus and ‘KGCNDPIRCY’ motif in the C-terminus respectively. Substrate specific analysis revealed that recombinant NtMSRA4 protein could reduce specifically S-isomer of Dabsyl-MetSO to Dabsyl-Met in vitro using dithiothreitol as an electron donor. Enzymatic properties analysis showed that the temperature of 42 °C and pH 9.0 were optimum for NtMSRA4 activity. The K _m and K _cat values of NtMSRA4 were determined to be 40.04 μM and 0.048 S^?1 in the thioredoxin dependent reduction system. Overexpression of NtMSRA4 in E. coli cells enhanced resistance to H₂O₂ toxicity. Subcellular localization result showed that NtMSRA4 was located in the chloroplast. The expression level of NtMSRA4 was affected differently after exposure to various abiotic stresses.     

A rapid and selective methionine oxidative modification strategy

Considering the fact that site-selective late-stage diversification of peptides and proteins remains a challenge for biochemistry, strategies targeting low-abundance natural amino acids need to be further developed. As an extremely oxidation-sensitive and low-abundance amino acid, methionine emerges as a promising target for chemo- and site-selective modification. Herein we report an efficient and highly selective modification on methionine residues by one-pot O- and N-transfer reaction, generating sulfoximine-modified peptides with near-perfect conversion within 10 min. Moreover, the great tolerance to other natural amino acids has been demonstrated in reactions with various peptide substrates. To demonstrate the generality of this protocol, we have modified natural peptides and obtained sulfoximination products with high conversion rates. This methodology provides a novel strategy as the expansion of the methionine-based peptide functionalization toolbox.     

Preparation of nitrophorin 7(Δ1–3) from Rhodnius prolixus without start–methionine using recombinant expression in Escherichia coli

The heterologous recombinant expression of proteins in Escherichia coli without start–methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB–nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl₂ gradient.     

Recombinant granulocyte colony-stimulating factor (filgrastim): Optimization of conjugation conditions with polyethylene glycol

With the purpose of creating an active prolonged-release pharmaceutical substance, modification of the recombinant human granulocyte colony-stimulating factor G-CSF (filgrastim) with polyethylene glycol (PEG, molecular mass 21.5 kDa) has been performed. The method for the preparation of the filgrastim PEG derivative intended to develop and scale-up the technological manufacturing process is described. Protein modification with PEG was performed by selective covalent attachment of the ??-methyl-PEG-propionaldehyde molecule to the ??-amino group of the N-terminal of the methionine amino acid residue of the recombinant G-CSF. The selected reaction conditions provide no less than 85% product yield of the total protein, a high protein concentration in the reaction mixture (more than 9 mg/mL) and allow us to reduce PEG consumption on the protein terminal ??-amino group basis. RP HPLC and MALDI mass spectrometry data demonstrate that the preparation is modified by PEG at the N-terminal residue and contains no more than 10% of products with the higher degree of modification.     

Simplified,Enhanced Protein Purification Using an Inducible,Autoprocessing Enzyme Tag

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP₆), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His₆-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP₆ to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.     

Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39

To clarify the physiological function of pseudovitamin B₁₂ (or adeninylcobamide; AdeCba) in Spirulina platensis NIES-39, cobalamin-dependent methionine synthase (MS) was characterized. We cloned the full-length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin-, zinc-, S-adenosylmethionine-, and homocysteine-binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan as effective gene carriers: Effect of degree of substitution

The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan; TM-Bz-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells). The factors affecting the transfection efficiency e.g. degree of quaternization (DQ), the degree of dimethylaminobenzyl substitution (DS) and polymer/DNA weight ratio, have been evaluated. The results revealed that all TM-Bz-CS derivatives were able to condense with DNA. Illustrated by agarose gel electrophoresis, complete complexes of TM₅₇-Bz₄₂-CS/DNA were formed at weight ratio of above 0.5, whereas those of TM₄₇-Bz₄₂-CS/DNA and TM₅₇- Bz₁₇-CS/DNA were above 1. The rank of transfection efficiency of the chitosan derivatives were TM₅₇-Bz₄₂-CS > TM₄₇-Bz₄₂-CS > TM₅₇-Bz₁₈-CS. The pH of culture medium did not affect the transfection efficiency of TM₅₇-Bz₄₂-CS/DNA complex, whereas it affected the transfection efficiency of chitosan/DNA complex. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on chitosan which promoted the interaction and condensation with DNA as well as N-quaternization which increased chitosan water solubility and enhance gene expression. For cytotoxicity studies, TM-Bz-CS was safe at the concentration of the highest transfection. In conclusion, this novel chitosan derivative, TM₅₇-Bz₄₂-CS showed elevated potential as gene carrier by efficient DNA condensation and mediated highest level of gene transfection with negligible cytotoxicity in Huh7 cells.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

Surface-Modified Liposomal Formulation of Amphotericin B: <Emphasis Type="Italic">In vitro</Emphasis> Evaluation of Potential Against Visceral Leishmaniasis

Surface modification of liposomes with targeting ligands is known to improve the efficacy with reduced untoward effects in treating infective diseases like visceral leishmaniasis (VL). In the present study, modified ligand (ML), designed by modifying polysaccharide with a long chain lipid was incorporated in liposomes with the objective to target amphotericin B (Amp B) to reticuloendothelial system and macrophages. Conventional liposomes (CL) and surface modified liposomes (SML) were characterized for size, shape, and entrapment efficiency (E.E.). Amp B SML with 3% w/w of ML retained the vesicular nature with particle size of ～205 nm, E.E. of ～95% and good stability. SML showed increased cellular uptake in RAW 264.7 cells which could be attributed to receptor-mediated endocytosis. Compared to Amp B solution, Amp B liposomes exhibited tenfold increased safety in vitro in RAW 264.7 and J774A.1 cell lines. Pharmacokinetics and biodistribution studies revealed high t _1/2, area under the curve (AUC)_0–24, reduced clearance and prolonged retention in liver and spleen with Amp B SML compared to other formulations. In promastigote and amastigote models, Amp B SML showed enhanced performance with low 50% inhibitory concentration (IC₅₀) compared to Amp B solution and Amp B CL. Thus, due to the targeting ability of ML, SML has the potential to achieve enhanced efficacy in treating VL.     

Serial Expression and Activity Analysis of LNK-16: A Bovine Antimicrobial Peptide Analogue

Indolicidin is a broad-spectrum antimicrobial peptide (AMP) with great therapeutic potential; however, high manufacturing costs associated with industrial-scale chemical synthesis have limited its delivery. Therefore, the use of recombinant DNA technology to produce this peptide is urgently needed. In this study, a new methodology for the large-scale production of a novel bovine AMP was developed. LNK-16 is an analogue of indolicidin that contains a kallikrein protease site at its C-terminus. The amino acid sequence of LNK-16 was synthesized using Escherichia coli-preferred codons. Three copies of the target gene were assembled in series by overlapping PCR and cloned into pET-30a(+) for the expression of His-(LNK-16)₃ in E. coli BL21 (DE3) cells. The expressed fusion protein His-(LNK-16)₃ was purified by Ni²⁺-chelating chromatography and then cleaved by kallikrein to release LNK-16. The recombinant LNK-16 peptide showed antimicrobial activity similar to that of chemically synthesized LNK-16 and indolicidin. Together, these data indicate that the use of serial expression can improve the large-scale production of AMPs for clinical and research applications.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

TILLING to detect induced mutations in soybean

Background

Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.

Results

In higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of theArabidopsis CgS (CgS _Δ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein theCgS _Δ90was co-transformed with the methionine-rich15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgS_Δ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.

Conclusion

Ectopic expression of CgS_Δ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgS_Δ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgS_Δ90 and can not significantly improve the nutritional value of tubers. The level ofPAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgS_Δ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.     

The synthesis of 5-amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-Heptonic acid and its polycondensation to oligomers

5-Amino-2,6-anhydro-5-deoxy-D-glycero-D-gulo-heptonic acid has been synthesized by conventional introduction of an amino function via azide displacement, starting with a suitable derivative of 2,6-anhydro-D-glycero-L-manno-heptonic acid. The amino acid was converted into the methyl ester hydrochloride which, in methanolic sodium methoxide, gave oligomeric and polymeric amides, depending on the conditions applied. Four oligomeric esters, as well as the corresponding N-(2,4-dinitrophenyl) derivatives of the amino acids, could be separated by paper chromatography. The oligomers could be saponified under mild, basic conditions.