Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37 degrees C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34 degrees C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl beta-D-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.

The nucleus is bordered by a double bilayer nuclear envelope, communicates with the cytoplasm via embedded nuclear pore complexes, and is structurally supported by an underlying nucleoskeleton. The nucleoskeleton includes nuclear intermediate filaments formed by lamin proteins, which provide major structural and mechanical support to the nucleus. However other structural proteins also contribute to the function of nucleoskeleton and help connect it to the cytoskeleton. This chapter reviews nucleoskeletal components beyond lamins, and summarizes specific methods and strategies useful for analyzing nuclear structural proteins including actin, spectrin, titin, LINC complex proteins and nuclear spindle matrix proteins. These components can localize to highly specific functional subdomains at the nuclear envelope or nuclear interior, and can interact either stably or dynamically with a variety of partners. These components confer upon the nucleoskeleton a functional diversity and mechanical resilience that appears to rival the cytoskeleton. To facilitate the exploration of this understudied area of biology, we summarize methods useful for localizing, solubilizing and immunoprecipitating nuclear structural proteins, and a state-of-the-art method to measure a newlyrecognized mechanical property of nucleus.

In this work, taking human beta-defensin-2 (HBD2) as a demonstrative molecule, the strategies for high efficient production of functional human beta-defensins in E. coli were studied. Fusion mature HBD2 (TrxA-mHBD2) showed high solubility and productivity without the need for lowering the cultivation temperature. The solubility of target fusion protein could attain 81.3% even at 37 degrees C with a volumetric productivity as high as 235 mg/L in a rich medium MBL at the same temperature and reached 346 mg/L at 28 degrees C. The His-Tag in the fusion protein enabled the application of affinity chromatography separation to obtain high purity of the overexpressed recombinant fusion protein. After digestion by enterokinase, purification via cationic exchange chromatography, and desalting by ultrafiltration, mature HBD2 product was obtained with a purity of 95% in an overall recovery of 29.2%. The antimicrobial activity of the recombinant mature HBD2 and the influence factors were tested using E. coli K12D31 as a sensitive strain.

Correlations between altered nuclear shape and disease are empirically observed, but the causes of nuclear dysmorphisms are poorly understood. The nucleoskeleton, which provides the majority of the mechanical stability of the nucleus, is composed primarily of intermediate filaments of lamin proteins. The nucleoskeleton forms a mostly-planar network between the inner nuclear membrane and chromatin. It is unclear if blebs and larger scale changes in nuclear morphology are consequences of reorganization of the nucleoskeleton alone or of other cellular processes. To test this, we computationally recapitulate the lamina network using a mechanical network model created as a network of Hookean springs. A- and B-type lamin filaments were distributed over a spherical surface into distinct networks linked to one another by lamin-associated proteins. Iterative force-based adjustment of the network structure, together with a stochastically modified Bell model of bond breakage and formation, simulates nucleoskeleton reorganization with blebs. The rate of bleb retraction into the nucleus depends on both initial size of the bleb and number of networks being deformed. Our results show that induced blebs are more stable when only one filament component is deformed or when the networks have no interconnections. Also, the kinetics of retraction is influenced by the composition of the bleb. These results match with our experiments and others.

A codon optimized mature human beta-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l(-1), i.e. 0.21 g mature HBD3 l(-1). Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli.