Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq.

KEY MESSAGE: The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.

Flavokawain B induced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB231 and inhibited the metastatic potential of MDA-MB231 via the regulation of several tyrosine kinases In vitro.

BACKGROUND: The kava-kava plant (Piper methysticum) is traditionally consumed by the pacific islanders and has been linked to be involved in several biological activities. Flavokawain B is a unique chalcone, which can be found in the roots of the kava-kava plant. In this study, the operational mechanism of the anti-cancer activity of a synthetic Flavokawain B (FKB) on two breast cancer cell lines, MCF-7 and MDA-MB231 was investigated. METHOD: Several in vitro assays were attempted such as MTT, flow cytometry of cell cycle analysis, annexin V analysis, and JC-1 analysis to detect apoptosis. Moreover, in vitro metastasis assays were also performed such as transwell migration assay, invasion assay, rat aorta ring and HUVEC tube formation. Molecular analysis of related genes and proteins were conducted using real-time PCR and proteome profiler analysis. RESULTS: Based on our results, apoptosis was induced when both MCF-7 and MDA-MB231 were treated with FKB. A significant G2/M arrest was seen in MDA-MB231 cells. Additionally, FKB also inhibited the in vitro migration and invasion in MDA-MB231 cells in a dose dependent manner. Moreover, FKB can be a potential inhibitor in angiogenesis as it suppressed the formation of vessels in HUVEC cells as well as in the ex-vivo rat aortic ring assay. CONCLUSION: Our findings suggested that FKB also regulated several receptor tyrosine kinases. Overall, FKB is not only a potential candidate to be an anti-cancer agent, but as an anti-metastatic agent as well.

Comparison of free amino acids, antioxidants, soluble phenolic acids, cytotoxicity and immunomodulation of fermented mung bean and soybean.

BACKGROUND: Mung bean and soybean have been individually reported previously to have antioxidant, cytotoxic and immunomodulatory effects, while fermentation is a well-known process to enhance the bioactive compounds that contribute to higher antioxidant, cytotoxic and immunomodulation effects. In this study, the free amino acids profile, soluble phenolic acids content, antioxidants, cytotoxic and immunomodulatory effects of fermented and non-fermented mung bean and soybean were compared. RESULTS: Fermented mung bean was recorded to have the highest level of free amino acids, soluble phenolic acids (especially protocatechuic acid) and antioxidant activities among all the tested products. Both fermented mung bean and soybean possessed cytotoxicity activities against breast cancer MCF-7 cells by arresting the G0/G1 phase followed by apoptosis. Moreover, fermented mung bean and soybean also induced splenocyte proliferation and enhanced the levels of serum interleukin-2 and interferon-γ. CONCLUSION: Augmented amounts of free amino acids and phenolic acids content after fermentation enhanced the antioxidants, cytotoxicity and immunomodulation effects of mung bean and soybean. More specifically, fermented mung bean showed the best effects among all the tested products. This study revealed the potential of fermented mung bean and soybean as functional foods for maintenance of good health.

Molecular characterisation of new organisation of plnEF and plw loci of bacteriocin genes harbour concomitantly in Lactobacillus plantarum I-UL4.

BACKGROUND: Bacteriocin-producing Lactic acid bacteria (LAB) have vast applications in human and animal health, as well as in food industry. The structural, immunity, regulatory, export and modification genes are required for effective bacteriocin biosynthesis. Variations in gene sequence, composition and organisation will affect the antimicrobial spectrum of bacteriocin greatly. Lactobacillus plantarum I-UL4 is a novel multiple bacteriocin producer that harbours both plw and plnEF structural genes simultaneous which has not been reported elsewhere. Therefore, molecular characterisation of bacteriocin genes that harboured in L. plantarum I-UL4 was conducted in this study. RESULTS AND DISCUSSION: Under optimised conditions, 8 genes (brnQ1, napA1, plnL, plnD, plnEF, plnI, plnG and plnH) of plnEF locus and 2 genes (plw and plwG) of plw locus were amplified successfully from genomic DNA extracted from L. plantarum I-UL4 using specific primers designed from 24 pln genes selected randomly from reported plw, plS, pln423 and plnEF loci. DNA sequence analysis of the flanking region of the amplified genes revealed the presence of two pln loci, UL4-plw and UL4-plnEF loci, which were chromosomally encoded as shown by Southern hybridisation. UL4-plw locus that contained three ORFs were arranged in one operon and possessed remarkable amino acid sequence of LMG2379-plw locus, suggesting it was highly conserved. Interestingly, the UL4-plnEF locus appeared to be a composite pln locus of JDM1-plnEF and J51-plnEF locus in terms of genetic composition and organisation, whereby twenty complete and one partial open reading frames (ORFs) were aligned and organised successfully into five operons. Furthermore, a mutation was detected in plnF structural gene which has contributed to a longer bacteriocin peptide. CONCLUSIONS: Plantaricin EF and plantaricin W encoded by plnEF and plnW loci are classified as class I bacteriocin and class II bacteriocin molecules respectively. The concurrent presence of two pln loci encoding bacteriocins from two different classes has contributed greatly to the broad inhibitory spectrum of L. plantarum I-UL4. The new genetic composition and organisation of plnEF locus and concurrent presence of plnEF and plnW loci indicated that L. plantarum I-UL4 is a novel multiple bacteriocin producer that possesses vast potentials in various industries.

Proteome analysis of Escherichia coli periplasmic proteins in response to over-expression of recombinant human interferon α2b.

The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins.

The flavokawains: uprising medicinal chalcones.

Plant-based compounds have been in the spotlight in search of new and promising drugs. Flavokawain A, B and C are naturally occurring chalcones that have been isolated from several medicinal plants; namely the piper methysticum or commercially known as the kava-kava. Multiple researches have been done to evaluate the bioactivities of these compounds. It has been shown that all three flavokawains may hold promising anti-cancer effects. It has also been revealed that both flavokawain A and B are involved in the induction of cell cycle arrest in several cancer cell lines. Nevertheless, flavokawain B was shown to be more effective in treating in vitro cancer cell lines as compared to flavokawain A and C. Flavokawain B also exerts antinociceptive effects as well as anti-inflammation properties. This mini-review attempts to discuss the biological properties of all the flavokawains that have been reported.

A streamlined strategy for self-production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains.

Positive selection vectors carry a lethal gene encoding a toxic product that is harmful to most laboratory E. coli strains. Previously, we reported a strategy for in-house production of a commercial positive selection vector, the pJET1.2/blunt cloning vector, using common laboratory E. coli strains. However, the strategy involves lengthy gel electrophoresis and extraction procedures to purify the linearized vector after digestion. Here, we streamlined the strategy to eliminate the gel-purification step. A uniquely designed short fragment called the Nawawi fragment was inserted into the coding sequence of the lethal gene of the pJET1.2 plasmid, resulting in the pJET1.2N plasmid that can be propagated in the E. coli strain DH5α. Digestion of the pJET1.2N plasmid with EcoRV released the Nawawi fragment, and the resulting blunt-ended pJET1.2/blunt cloning vector can be used directly for DNA cloning without prior purification. Cloning of a DNA fragment was not hindered by the Nawawi fragments carried over from the digestion step. After transformation, the pJET1.2N-derived pJET1.2/blunt cloning vector produced > 98% positive clones. The streamlined strategy accelerates the in-house production of the pJET1.2/blunt cloning vector and enables DNA cloning at a lower cost. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03647-3.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes.

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.

The PAL2 promoter activities in relation to structural development and adaptation in Arabidopsis thaliana.

Phenylalanine ammonia lyase (PAL) plays a major role in plant growth, development and adaptation. In Arabidopsis thaliana, the enzyme is encoded by four genes, namely PAL1, PAL2, PAL3, and PAL4 with PAL1 and PAL2 being closely related phylogenetically and functionally. PAL1 promoter activities are associated with plant development and are inducible by various stress agents. However, PAL2 promoter activities have not been functionally analysed. Here, we show that the PAL2 promoter activities are associated with the structural development of a plant and its organs. This function was inducible in an organ-specific manner by the avirulent strain of Pseudomonas syringae pv. tomato (JL1065). The PAL2 promoter was active throughout the course of the plant development particularly in the root, rosette leaf, and inflorescence stem that provide the plant with structural support. In aerial organs, the levels of PAL2 promoter activities were negatively correlated with relative positions of the organs to the rosette leaves. The promoter was inducible in the root following an inoculation by JL1065 in the leaf suggesting PAL2 to be part of an induced defence system. Our results demonstrate how the PAL2 promoter activities are being coordinated and synchronised for the structural development of the plant and its organs based on the developmental programme. Under certain stress conditions the activity may be induced in favour of certain organs.

Nain-e Havandi Andrographis paniculata present yesterday, absent today: a plenary review on underutilized herb of Iran's pharmaceutical plants.

Nain-e Havandi (Andrographis paniculata Nees.) (AP) is an annual herbaceous plant belonging to the family Acanthacea. Only a few species of Andrographis genus out of 28 are medicinally concerned of which AP is the most important. Knowledge about the arrival of AP to Iran is extremely lacking but most probably it has been imported from India. However, evidence implies the familiarity of Iran's folkloric medicine with this plant, but it has been disappeared from contemporary medicine for unknown reasons. Presence of active ingredients from diterpenoids group such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide has given incredible unique medicinal properties to the plant. Traditionally, Nain-e Havandi has been used in the role of a non-farm plant as a remedy for skin problems, flu, respiratory disease, and snakebite in East and Southeast Asia for centuries. Recently, it has been utilized as a treatment for HIV, hepatitis, diabetes, cancer and kidney disorders. Intensive cultivation of the herb started only in the past decade in countries such as China, India, Thailand, Indonesia, West Indies, Mauritius and to some extent, in Malaysia. Availability of different ecological zones in Iran complies with reestablishment of AP in tropical and temperate regions of the country. This is killing two birds with one stone, supporting the conservational and economic aspects.

Functional expression of an orchid fragrance gene in Lactococcus lactis.

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of ß-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5'-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.

A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.

Key message: In-house production of a positive selection cloning vector could be simple, efficient and low cost. Abstract: DNA cloning technology requires a vector to harbour a gene of interest for multiplication of the gene in bacterial cells. Positive selection vector has become a popular type of cloning vector due to the simplicity and efficiency of the positive selection system. Due to the presence of a toxic gene, propagation of a commercial positive selection vector in common laboratory E. coli strains is infeasible. This study demonstrated a strategy for propagation and in-house production of a commercial positive selection vector, i.e., pJET1.2/blunt cloning vector, at low cost. This was done by insertion of a specially designed DNA fragment (MCS fragment), which can be easily removed later by EcoRV digestion, into the pJET1.2/blunt cloning vector to allow the propagation of the modified plasmid (termed pJET1.2M) in common E. coli strains. Removal of the MCS fragment from the pJET1.2M plasmid produces the pJET1.2/blunt cloning vector ready for gene cloning. The self-made pJET1.2/blunt cloning vector exhibited a cloning efficiency of 100%. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03289-x.

Genome-wide analysis of the CAD gene family reveals two bona fide CAD genes in oil palm.

Cinnamyl alcohol dehydrogenase (CAD) is the key enzyme for lignin biosynthesis in plants. In this study, genome-wide analysis was performed to identify CAD genes in oil palm (Elaeis guineensis). Phylogenetic analysis was then conducted to select the bona fide EgCADs. The bona fide EgCAD genes and their respective 5' flanking regions were cloned and analysed. Their expression profiles were evaluated in various organs using RT-PCR. Seven EgCAD genes (EgCAD1-7) were identified and divided into four phylogenetic groups. EgCAD1 and EgCAD2 display high sequence similarities with other bona fide CADs and possess all the signature motifs of the bona fide CAD. They also display similar 3D protein structures. Gene expression analysis showed that EgCAD1 was expressed most abundantly in the root tissues, while EgCAD2 was expressed constitutively in all the tissues studied. EgCAD1 possesses only one transcription start site, while EgCAD2 has five. Interestingly, a TC microsatellite was found in the 5' flanking region of EgCAD2. The 5' flanking regions of EgCAD1 and EgCAD2 contain lignin-associated regulatory elements i.e. AC-elements, and other defence-related motifs, including W-box, GT-1 motif and CGTCA-motif. Altogether, these results imply that EgCAD1 and EgCAD2 are bona fide CAD involved in lignin biosynthesis during the normal development of oil palm and in response to stresses. Our findings shed some light on the roles of the bona fide CAD genes in oil palm and pave the way for manipulating lignin content in oil palm through a genetic approach. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03208-0.

Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

Optimization of an induction strategy for improving interferon-alpha2b production in the periplasm of Escherichia coli using response surface methodology.

Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively.

Rapid and effective method of RNA isolation from green microalga Ankistrodesmus convolutus.

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.

Interactions of hepatitis B core antigen and peptide inhibitors.

The core protein (HBcAg) of hepatitis B virus (HBV) has been shown to interact with the large surface antigen during HBV morphogenesis, and these interactions can be blocked by small peptides selected from either linear or constrained phage display peptide libraries. The association of HBcAg with peptide inhibitors was quantitatively evaluated by isothermal titration calorimetry. The thermodynamic data show that the interaction between HBcAg and peptide MHRSLLGRMKGA is enthalpy-driven and occurs at a 3:1 stoichiometry and dissociation constant (Kd) value of 79.4 muM. However, peptide WSFFSNI displays a higher binding affinity for HBcAg with a Kd value of 18.5 muM when compared to peptide MHRSLLGRMKGA. A combinatorial approach using chemical cross-linking and surface-enhanced laser desorption/ionization-time-of-flight-mass spectrometry shows that the Lys of peptide MHRSLLGRMKGA interacted either with D64, E77, or D78 of HBcAg.