Extra Residues Research Articles

Characterization of a novel y-type HMW-GS with eight cysteine residues from Triticum monococcum ssp. monococcum

The composition and number of high-molecular-weight glutenin subunits (HMW-GSs) play important roles in determining the grain-processing quality of common wheat. The Glu-1Ay allele is silent in common wheat. In this study, an active y-type HMW-GS allele termed 1Ay8.2 (GenBank No. KP137569) was identified from Triticum monococcum L. ssp. monococcum (AmAm, 2n=2x=14), a species with a genome related to the A-genome of common wheat. Compared with previously reported active 1Ay subunits, this novel subunit contained an extra cysteine residue at position 103 of the amino acid sequence in the N-terminal region, in addition to the six cysteines in the N- and C-terminal regions found in most active 1Ay subunits and the one in the repetitive region that appears in only a few 1Ay alleles. This subunit was expressed in an amphiploid (AAAmAmBB, 2n=6x=42) between Triticum turgidum L. ssp. dicoccon and T. monococcum ssp. monococcum. This amphiploid could be used as a bridge to transfer 1Ay8.2 into common wheat cultivars. Replacing the silenced 1Ay in common wheat with the active 1Ay8.2 allele harboring an extra cysteine residue is expected to improve the quality by increasing the number of HMW-GSs and promoting the formation of covalent interactions through disulfide bonds with the extra cysteine residue.

Distortion of the Major Histocompatibility Complex Class I Binding Groove to Accommodate an Insulin-derived 10-Mer Peptide

The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell-mediated destruction of pancreatic β-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8(+) T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to "open the back door" to accommodate extra C-terminal peptide residues.

VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

Methionine Misacylation of tRNA on CaMKII in Response to ER Stress

Methionine mistranslation is a recently discovered phenomenon that cells deliberately incorporate methionine residues at selected positions in proteins. Met-mistranslation is regulated by reactive oxygen species (ROS) and has been proposed to serve as an adaptive mechanism for oxidative stress response. CaMKII is a multifunctional protein that is necessary for Ca2+ homeostasis in cells. Recent evidence showed that CaMKII could be alternatively activated by ROS through methionine oxidation. Here we show that CaMKII activity could be regulated through methionine mistranslation triggered by Ca2+ stress in mammalian cells. For the first time we found that Ca2+ overload induced Met-mistranslation on CaMKII indicated by mass spectrometry. Mutant CaMKII proteins with extra Met residues have different kinase activity and autophosphorylation level from wild type CaMKII. In addition, mutant CaMKII (V208M) translocated into endoplasmic reticulum (ER) indicated by immunofluorescent staining. Furthermore, mammalian cells with mutant CaMKII has high level of cell apoptosis measured by TUNEL assay. In conclusion, our study provided additional evidence that Met-mistranslation of CaMKII protein could be used as a novel adaptive mechanism in response to ER stress upon elevated intracellular Ca2+ level in diverse disease processes.

Factor (G‐CSFa) Enhances Peripheral Platelet Recovery in C57 Radiotherapy Mice and No Immunogenicity in Rats

Using site-direct mutagenesis and recombinant DNA technology, we had previously obtained a structurally modified derivative of human G-CSF termed G-CSFa, which has three extra amino acid residues added at the amino-terminus. The novel rhG-CSFa displayed a lower plasma clearance rate, suggesting a much extended plasma functional half-life. Previous studies showed that G-CSFa is more potent than the wild-type counterpart in stimulating proliferation and differentiation of myeloid cells of the granulocytic lineage, both in vitro and in vivo. Here, we show that G-CSFa can significantly accelerate peripheral platelet recovery in C57BL/6 mice exposed to irradiation. In the G-CSFa treatment group, the peripheral platelet count has fully recovered on day 19( list the specific number),whereas in vehicle control group, it was on a dramatically low level.(list number) We further demonstrate that G-CSFa is not immunogenic in rats, by confirming the absence of any binding antibodies, or neutralizing antibodies, to G-CSFa in the sera of Sprague-Dawley rats following repeated G-CSFa administration. Taken together, these findings further support the benefits of G-CSFa for clinical therapy.

The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway.

Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a further snapshot in the yet-to-be-achieved complete description of the highly dynamic cadherin dimerization pathway. Moreover, this is the first multidomain cadherin fragment to be crystallized and structurally characterized in its closed conformation that does not carry any extra N-terminal residues before the naturally occurring aspartic acid at position 1. Finally, two clear alternate conformations are observed for the critical Trp2 residue, suggestive of a transient, metastable state. The P-cadherin structure and packing arrangement shown here provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

Antimicrobial Peptide Piscidin Permeabilizes Bacterial Membranes and Binds Intracellular Targets at Sub-Lethal Concentrations

Piscidins 1 (P1) and 3 (P3) are cationic α-helical membrane active antimicrobial peptides originally isolated from the hybrid striped seabass. The peptides, which possess broad-spectrum activity against bacteria, fungi, and viruses, bind to anionic membranes and lyse them once a sufficient concentration has been reached. In this study, we used confocal microscopy and FITC-labeled piscidins to show that at sub-inhibitory concentrations (0.75 μM), P1 and P3 are able to translocate across the membrane of Gram-positive and -negative bacteria. The FITC signal of the peptides was co-localized with the DAPI signal of the nucleoid region of the bacteria. CD using a 15 base pair piece of DNA and a gel retardation assay using a 1782 base pair piece of DNA confirmed that P1 and P3 could bind to DNA in vitro. It appears that P3 binding was more disruptive to the structure of DNA, and this may be due to the extra arginine residue in P3's sequence. However, these results did not indicate whether the peptides translocated through the membrane or entered the cells via pores formed in the membrane. To probe this question, we used the bacterial strain E. coli ML35 in a permeabilization assay to determine at which peptide concentration the membrane became permeabilized. Our data indicate that at the lowest concentration tested (0.1 μM) the membrane of ML35 cells became permeabilized by P1 and P3. Our results agree with prior findings that permeabilization of bacterial cell membranes can occur prior to lysis and cell death. The implications of piscidin's ability to translocate into bacterial cells and bind to intracellular targets at sub-lethal concentrations will be discussed in terms of its mechanism of action, which was previously believed to be based on its membrane activity.

Crystal Structures of Streptococcus Pyogenes and Xanthomonas Oryzae Cas5d Proteins

Clustered regularly interspaced short palindromic repeats (CRISPRs) are repetitive genetic elements found in archaeal and bacterial genomes that are involved in RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids. CRISPR-associated (cas) genes are also found adjacent to CRISPR loci and encode Cas proteins with a variety of nucleic acid-related functions. Cas5d proteins are subtype I-C specific Cas5 proteins that constitute one of the most plevalent Cas protein families in CRISPR-Cas systems, and have been predicted to have RNA-recognition motif (RRM) domains. Here, we report crystal structures of Cas5d proteins from Streptococcus pyogenes and Xanthomonas oryzae, which represent two Cas5d subgroups. S. pyogenes Cas5d contains extra C-terminal residues while X.oryzae Cas5d has an extended helical region protruding from the N-terminal RRM domain, which has not been observed in other Cas5d structures previously. Despite structural differences, the two Cas5d proteins share common functional properties such as specific endoribonuclease activity for pre-crRNA and non-specific double-stranded DNA binding. These findings suggest that Cas5d may have multiple roles in CRISPR-mediated immunity system and provide an explanation for the conserved mode of the pre-CRISPR RNA processing.

A Structured Loop Modulates Coupling between the Substrate-binding and Dimerization Domains in the Multidrug Resistance Transporter EmrE

Secondary active transporters undergo large conformational changes to facilitate the efflux of substrates across the lipid bilayer. Among the smallest known transport proteins are members of the small multidrug resistance (SMR) family that are composed of four transmembrane (TM) domains and assemble into dimers. An unanswered question in the SMR field is how the dimerization domain (TM4) is coupled with the substrate-binding chamber (TM1-3). To provide insight for this essential aspect of ion-coupled transport, we carried out a structure-function study on the SMR protein EmrE using solid-state NMR spectroscopy in lipid bilayers and resistance assays in Escherichia coli. The chemical shifts for EmrE were consistent with β-strand secondary structure for the loop connecting TM3 and TM4. Based on these structural results, EmrE mutants were created to ascertain whether a specific loop length and composition were necessary for function. A linker encompassing six extra Gly residues relative to wild-type EmrE failed to give resistance; however, the number of residues in the loop was not the only criterion for a functional efflux pump. Replacement of the central hydrophobic residue with Gly (L83G) also conferred no ethidium resistance phenotype, which supported the conclusion that the structure and length of the loop were both essential for ion-coupled transport. Taken together with a bioinformatics analysis, a structured linker is likely conserved across the SMR family to play an active role in mediating the conformational switch between inward-open and outward-open states necessary for drug efflux. These findings underscore the important role loops can play in mediating efflux.

Pre-Clinical Studies of a Novel Recombinant Human Granulocyte Colony-Stimulating Factor

The human granulocyte colony-stimulating factor (G-CSF), a glycoprotein consisting of 174 amino acids, enhances the survival, proliferation, and differentiation of neutrophil precursors. G-CSF also induces the mobilization of hematopoietic stem cells and progenitors, which further stimulates recovery from neutropenia. Further studies indicates that Glu19 in the A helix of G-CSF molecule electrostatically inter-reacts with Arg288 of G-CSF-R. A recent study on the crystal structure of G-CSF, complexed with the cytokine homologous region of G-CSF-R, reveals that residues in the amino-terminus of G-CSF may act as additional contact sites with G-CSF-R.In this investigation, we designed and purified a novel recombinant human granulocyte colony-stimulating factor analog (rhG-CSFa), which has three extra amino acid residues (arginine, glycine, and serine) added at the amino-terminus. The structural modification resulted in more positive charge in the amino-terminus of G-CSFa, and was expected to further enhance the binding between the cytokine and its receptor. We optimized the purification procedure, achieving further improvements in refolding rate and overall yield. In preclinical studies, we evaluated the pharmacodynamics, pharmacokinetics, acute, subacute, and chronic toxicity of rhG-CSFa. Pharmacodynamics study in non-human primates demonstrated that intravenous rhG-CSFa induces typical peripheral neutrophil responses (with two peaks from treatment day 8 to day 20) and dramatic increases in the recovery rate in the animal model. More importantly, rhG-CSFa induced a higher peak of neutrophil recovery on day 7 than wild G-CSF did in monkeys, a result indicating that rhG-CSFa would provide greater benefit for patients receiving myeloid-suppressive therapies. In addition, rhG-CSFa maintained higher neutrophil counts than wild-type G-CSF did after cessation of G-CSF administration in monkeys; its effects persisted over 20 days post-treatment, which should be more helpful to patients for combating deleterious infections and preparing for the next round of treatment in clinic. In pharmacokinetics studies, the novel rhG-CSFa displayed a lower plasma clearance rate, 0.28 ml/min/kg (vs 0.5-0.7 ml/min/kg for wild-type G-CSF). This result suggests that rhG-CSFa may dissociate from its receptor at a slower rate than does wild-type G-CSF, which is possibly due to a tighter binding to its cognate receptor, and would result in a much extended plasma functional half-life. In further preclinical safety evaluation, no obvious toxicity or immunogenicity was observed, neither was any adverse drug reaction. In conclusion, we have generated a novel rhG-CSFa protein, with much higher yield, enhanced circulation half-life, improved thermal stability and greater bioactivity (without changing the overall conformation), compared with wild type G-CSF. Our preclinical studies strongly suggest that rhG-CSFa can be further explored for clinical trials for eventual applications in the clinic. DisclosuresRen:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Shi:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Apoptosis imaging studies in various animal models using radio-iodinated peptide.

Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40% decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200% increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.

The pCri System: a vector collection for recombinant protein expression and purification.

A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5′end and multiple cloning sites at the 3′end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.

Characterization of pre-molten globule state of yeast iso-1-cytochrome c and its deletants at pH 6.0 and 25 °C

To understand the role of five extra N-terminal residues, we prepared wild type (WT) yeast iso-1-cytochrome c (y-cyt-c) and its deletants by subsequently deleting these residues. Denaturation of all these proteins induced by LiCl was followed by observing changes in molar absorption coefficient at 405nm (Δɛ405), the mean residue ellipticity at 222nm ([θ]222), and the difference mean residue ellipticity at 409nm (Δ[θ]409) near physiological pH and temperature (pH 6.0 and 25°C). It was observed that in each case LiCl induces biphasic transition, N (native) state↔X (intermediate) state↔D (denatured) state. The intermediate (X) was characterized by the far-UV, near-UV and Soret circular dichroism, ANS (8-anilino-1-naphthalenesulfonic acid) binding and dynamic light scattering measurements. These measurements led us to conclude that X state of each protein has structural characteristics of PMG (pre-molten globule) state. Thermodynamic stability of all proteins was also determined. It was observed that the N-terminal extension stabilizes the native WT protein but it has no effect on the stability of PMG state. Another state was observed for each protein, in the presence of 0.33M Na2SO4 at pH 2.1, which when characterized showed all structural characteristics of MG (molten globule) state.

Chemical synthesis of a synthetic analogue of the sialic acid-binding lectin siglec-7.

As a basis for the development of an artificial carbohydrate-binding lectin, we chemically synthesized a domain of siglec-7, a well-characterized sialic-acid-binding lectin. The full polypeptide (127 amino acids) was constructed by sequential native chemical ligation (NCL) of five peptide segments. Because of poor cysteine availability for NCL, cysteine residues were introduced at suitable ligation sites; these cysteine residues were alkylated in order to mimic native glutamine or asparagine residues, or converted to an alanine residue by desulfurization after NCL. After folding the full-length polypeptide, the sialic-acid-binding activity of the synthetic siglec-7 was clearly demonstrated by STD NMR and ELISA experiments. We succeeded in the synthesis of siglec-7 by installing three extra cysteine residues with side-chain modifications and found that these modifications did not affect the binding activity.

In vitro and in silico studies of urea-induced denaturation of yeast iso-1-cytochrome c and its deletants at pH 6.0 and 25 °C

Yeast iso-1-cytochrome c (y-cyt-c) has five extra residues at N-terminus in comparison to the horse cytochrome c. These residues are numbered as –5 to –1. Here, these extra residues are sequentially removed from y-cyt-c to establish their role in folding and stability of the protein. We performed urea-induced denaturation of wild-type (WT) y-cyt-c and its deletants. Denaturation was followed by observing change in Δε405 (probe for measuring change in the heme environment within the protein), [θ]405 (probe for measuring the change in Phe82 and Met80 axial bonding), [θ]222 (probe for measuring change in secondary structure) and [θ]416 (probe for measuring change in the heme-methionine environment). The urea-induced reversible denaturation curves were used to estimate Δ, the value of Gibbs free energy change (ΔGD) in the absence of urea; Cm, the midpoint of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Our in vitro results clearly show that except Δ(–5/–4) all deletants are less stable than WT protein. Coincidence of normalized transition curves of all physical properties suggests that unfolding/refolding of WT protein and its deletants is a two-state process. To confirm our in vitro observations, we performed 40 ns MD simulation of both WT y-cyt-c and its deletants. MD simulation results clearly show that extra N-terminal residues play a role in stability but not in folding of the protein.

Datura stramonium agglutinin: Cloning, molecular characterization and recombinant production in Arabidopsis thaliana

Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.

Efficient production of native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein system in Escherichia coli.

To develop an efficient and cost-effective approach for the production of small preventive peptide lunasin with correct natural N terminus, a synthetic gene was designed by OPTIMIZER & Gene Designer and cloned into pTWIN1 vector at SapI and PstI sites. Thus, lunasin was N-terminally fused to the pH-induced self-cleavable Ssp DnaB mini-intein linked to a chitin binding domain (CBD) with no extra residues. The resultant fusion protein was highly expressed by lactose induction in Escherichia coli BL21 (DE3) in a 7-l bioreactor and bound to a chitin affinity column. After washing the impurities, the Ssp DnaB intein mediated on-column self-cleavage was easily triggered by shifting pH and temperature to allow the native lunasin released. The final purified lunasin yielded up to 75 mg/l medium. Tricine/SDS-PAGE and matrix-assisted laser desorption time-of-flight (MALDI-TOF)/mass spectrometry (MS) verified the structural authenticity of the product, implying the correct cleavage at the junction between Ssp DnaB intein and lunasin. MTT assay confirmed its potent proliferation inhibitory activity to human cancer cells HCT-116 and MDA-MB-231; however, no cytotoxicity to normal human lens epithelial cell SRA01/04 and hepatoma HepG2. Taken together, we provide a novel strategy to produce recombinant native lunasin with correct N-terminal processing by using the pH-induced self-cleavable Ssp DnaB mini-intein.

Characterization of high molecular weight glutenin subunit genes from the Ns genome of Psathyrostachys juncea.

The Ns genome of the genus Psathyrostachys possesses superior traits useful for wheat improvement. However, very little is known about the high molecular weight (HMW) subunits of glutenin encoded by the Ns genome. In this paper, we report the isolation of four alleles of HMW glutenin subunit gene from Psathyrostachys juncea. Sequence alignment data shows the four alleles have similar primary structure with those in wheat and other wheat-related grasses, with some unique modifications. All four sequences more closely resemble y-type, rather than x-type, glutenins. However, our results show three of the subunits (1Ns2-4) contain an extra glutamine residue in the N-terminal region not found on typical y-type subunits, as well as the x-type subunit specific sequence LAAQLPAMCRL. These three subunits likely represent an intermediate state in the divergence between x- and y-type subunits. Results also indicate that the Ns genome is more closely related to the St genome of Pseudoroegneria than any other Triticeae genomes.

Convergent evolution of IL-6 in two leporids (Oryctolagus and Pentalagus) originated an extended protein.

Interleukin 6 (IL-6) is a class-I helical cytokine with a broad spectrum of biological activities and a gene structure conserved throughout vertebrates, with five coding exons. IL-6 from European rabbits belonging to the subspecies Oryctolagus cuniculus cuniculus was previously shown to differ from other mammals by extending an additional 27 amino acids. However, in other leporids (Sylvilagus spp and Lepus spp) that diverged from the European rabbit ~12 million years ago this mutation was not present. In this study, we extended the study of IL-6 for the Oryctolagus cuniculus algirus subspecies and five additional lagomorphs' genera (Brachylagus, Bunolagus, Pentalagus, Romerolagus, and Ochotona). We confirmed the presence of the mutated stop codon in both O. c. cuniculus and O. c. algirus. We found that the typical stop codon is present in Sylvilagus bachmani and Lepus europaeus, in agreement with previous reports, but also in Bunolagus, Brachylagus, and Ochotona. Remarkably, in Pentalagus we detected a deletion of the stop codon causing an extension of IL-6 for 17 extra residues. Our results indicate that the IL-6 extension in those species occurred by two independent events: one occurred between 2 and 8 million years ago in the ancestral of the Oryctolagus subspecies, and the other occurred in a Pentalagus ancestral at a maximum of 9 million years ago. The absence of this IL-6 extension in Bunolagus, sister genus of Oryctolagus, shows that this evolutionary event happened by convergence suggesting some functional relevance.