Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.     

To the Final Goal: Can We Predict and Suggest Mutations for Protein to Develop Desired Phenotype?

Directed evolution of proteins is a good approach to develop desired phenotypes from existing proteins. Fully experimental protein evolution usually utilizes randomization of a given protein sequence by error-prone PCR or gene shuffling followed by high-throughput selection or timeconsuming screening method. However, these random methods create mutant library full of deleterious mutations. In addition, they need high-throughput screening or selection method to search for positive clones from an enormous size of mutant library. Construction of a mutant library while retaining the original function is important for efficient protein evolution because it greatly reduces time and effort for the identification of positive mutants. Therefore, researchers have tried to reduce the size of mutant library by minimizing the occurrence of deleterious mutants. Such efforts have led to the creation of a concept of ‘small but smart library’. For this goal, neutral drift theory has been applied. Although smart library greatly reduces the library size, it is still the beyond the capacity of low-throughput assay. In parallel, computational analysis of protein structure and efforts to discriminate mutatable residues from all residues of a given protein have been consistently pursued. Accumulated knowledge of protein evolution through random mutation and selection has improved our understanding of functions of amino acids in protein structure. Protein evolution by rational design is being developed based on such understanding. In this review, we describe how the use of semi-rationally designed library rather than completely random one has impacted the overall procedure of directed evolution. We also describe efforts made to evaluate the effect of single mutation. Such efforts will bring lazy boys to the final goal - computational mutation suggestion system.     

The effect of resistant starch (RS) on the bovine rumen microflora and isolation of RS-degrading bacteria

Applied Microbiology and Biotechnology - Resistant starch (RS) in the diet reaches the large intestine without degradation, where it is decomposed by the commensal microbiota. The fermentation of...     

The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.     

The spatiotemporal development of adipose tissue

Adipose tissue is a structure highly specialized in energy storage. The adipocyte is the parenchymal component of adipose tissue and is known to be mesoderm or neuroectoderm in origin; however, adipocyte development remains poorly understood. Here, we investigated the development of adipose tissue by analyzing postnatal epididymal adipose tissue (EAT) in mouse. EAT was found to be generated from non-adipose structure during the first 14 postnatal days. From postnatal day 1 (P1) to P4, EAT is composed of multipotent progenitor cells that lack adipogenic differentiation capacity in vitro, and can be regarded as being in the 'undetermined' state. However, the progenitor cells isolated from P4 EAT obtain their adipogenic differentiation capacity by physical interaction generated by cell-to-matrix and cell-to-cell contact both in vitro and in vivo. In addition, we show that impaired angiogenesis caused by either VEGFA blockade or macrophage depletion in postnatal mice interferes with adipose tissue development. We conclude that appropriate interaction between the cellular and matrix components along with proper angiogenesis are mandatory for the development of adipose tissue.     

Homeoprotein Hbx4 represses the expression of the adhesion molecule DdCAD-1 governing cytokinesis and development

We investigated the function of homeodomain-containing protein Hbx4 in Dictyostelium discoideum. Hbx4-overexpressing cells (Hbx4(OE)) displayed defects in growth rate and cytokinesis and showed differences in slug motility and cell-type proportioning from KAx3. Furthermore, the overexpression of Hbx4 inhibited the induction of cadA, which encoded the Ca(2+)-dependent cell adhesion molecule DdCAD-1, despite expression of csaA and gpaB. The electrophoretic mobility shift assay showed that the promoter of cadA contained the Hbx4-binding site. Moreover, constitutively expressed DdCAD-1 in Hbx4(OE) rescued the defects in cytokinesis and development. These results suggest that Hbx4 modulates DdCAD-1-mediated cytokinesis and cell-type proportioning.     

Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa

Increasingly, mutations in genes causing Mendelian disease will be supported by individual and small families only; however, exome sequencing studies have thus far focused on syndromic phenotypes characterized by low locus heterogeneity. In contrast, retinitis pigmentosa (RP) is caused by >50 known genes, which still explain only half of the clinical cases. In a single, one-generation, nonsyndromic RP family, we have identified a gene, dehydrodolichol diphosphate synthase (DHDDS), demonstrating the power of combining whole-exome sequencing with rapid in vivo studies. DHDDS is a highly conserved essential enzyme for dolichol synthesis, permitting global N-linked glycosylation. Zebrafish studies showed virtually identical photoreceptor defects as observed with N-linked glycosylation-interfering mutations in the light-sensing protein rhodopsin. The identified Lys42Glu variant likely arose from an ancestral founder, because eight of the nine identified alleles in 27,174 control chromosomes were of confirmed Ashkenazi Jewish ethnicity. These findings demonstrate the power of exome sequencing linked to functional studies when faced with challenging study designs and, importantly, link RP to the pathways of N-linked glycosylation, which promise new avenues for therapeutic interventions.