Expression and secretion of human bone morphogenetic protein-7 in pichia pastoris

The synonymous codons are used in a highly nonrandom manner in hosts of widely divergent species, which is termed ‘codon usage bias’. Several reports suggest that codon usage bias sometimes obstructs attempts to express high levels of exogenous genes. In this study, an attempt was made to express mature peptide of human bone morphogenetic protein-7 with optimized codons in P. pastoris expression system. Three low-usage ARG codons (CGG or CGA) in hBmp7 mature domain have been successfully transformed into P. pastoris-preferred ARG codons (AGA) with overlap extension PCR-based multiple-site-directed mutagenesis for a high level expression of hBMP7 mature peptide. The results of this study showed that the production level (25.45 mg/L) of a codon-optimized strain increased 4.6-fold in comparison with that (5.5 mg/L) of noncodon-optimized strain. A strain harboring multicopy of codon-optimized hbmp7 expression cassette showed an even higher expression level, which was about 2-fold compared with that of the single-copy one. These recombinant hBMP7 mature peptides were produced as 18-kD monomer proteins and were easily purified from culture supernatants using ion-exchange chromatography. Functional assay demonstrated that rhBMP7 could induce ectopic cartilage formation, although its inductive ability was much less active than that of CHO cell-derived hBMP7.     

Synonymous codon usage bias and overexpression of a synthetic gene encoding Interferon α2b in yeast

To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut⁺ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×10⁵ IU/mL. Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)     

Impact of mate availability, population size, and spatial aggregation of morphs on sexual reproduction in a distylous, aquatic plant

In distylous, self-incompatible plants, clonal propagation, unbalanced floral morph frequencies, and reduced population size can interfere with the functioning of distyly by compromising legitimate intermorph pollinations, resulting in reduced reproductive output. Here, we examined the mating system and the impact of mate availability, population size, and spatial aggregation of morphs on reproductive output in the distylous, clonal, aquatic plant Hottonia palustris. Controlled pollinations under greenhouse conditions detected no spontaneous selfing without the action of a pollen vector (autonomous autogamy) and demonstrated very low fruit and seed development after self-pollination. Intermorph (legitimate) crossings resulted in high reproductive output in both floral morphs (long- and short-styled individuals), whereas intramorph (illegitimate) crossings decreased fruit and seed development by more than 50%, indicating that the species has partial intramorph-incompatibility. In natural populations, small population size and increasing deviation of floral morph frequencies negatively affected reproductive outcome. Individuals of the majority morph type developed significantly fewer fruit and seeds than individuals of the minority morph type. This rapid decline in fecundity was symmetrical, indicating that regardless of which morph was in the majority, the same patterns of negative frequency-dependent mating occurred. Increasing spatial isolation between compatible morphs significantly reduced fruit and seed set in both morphs similarly. This study provides clear indications of frequency- and context-dependent mating in natural populations of a distylous plant species.     

植物标本标签的计算机印制数据系统

本文讨论了主要用以印制标本采集记录标签的数据系统及其数据库建库方案与编程原理。该系统可在ＩＢＭ及其兼容系列个人计算机上使用,适合用于个人或科研与教学机构中小型标本采集信息管理;可用以数据的检索、标本标签的印制和植物名录的打印;也可用以地区性的植物区系与生态学研究。     

Under‐representation of avian studies in landscape genetics

Landscape genetics is a rapidly growing discipline that examines how heterogeneous landscapes and other environmental factors influence population genetic variation. We conducted a systematic review of the landscape genetic literature which demonstrates that birds are severely under‐represented relative to their species diversity and general publication prevalence. Most avian studies were on species that have relatively low dispersal ability, and we suggest that this reflects an assumed high vagility of birds that precludes spatial genetic variation relatable to landscape heterogeneity. However, spatial genetic variation exists in several bird species with very high dispersal ability, but this has not been considered in the context of landscape features. Genetic patterns may also relate to landscape due to breeding habitat selection and territorial behaviour, despite the fact that species may be able to move throughout different landscape elements with minimal movement costs. Habitat loss and fragmentation are continuing globally and are strongly related to declines in bird populations. Landscape genetic studies provide a means to understand, predict and mitigate the effects of anthropogenic landscape change on birds. This review promotes the need for landscape genetic studies of birds, such that a greater understanding of the drivers of their genetic structuring can be developed and generalizations can be made from landscape genetic studies that apply more broadly across taxa.     

Simplified method to obtain enhanced expression of tau protein from E. coli and one-step purification by direct boiling

Tau is an intrinsically disordered protein responsible for maintaining the structure and stability of axonal microtubules. However, in certain disease conditions including Alzheimer’s disease, tau protein may undergo biochemical and structural changes to form intracellular aggregates. Since tau is a proline- and arginine-rich eukaryotic protein, heterologous expression in Escherichia coli often results in poor yield and has been a major technical challenge. In the current work, we have improved the expressed yield of tau by overcoming codon bias problem and established a simplified protocol for efficient extraction. The reported method has two distinct features: (i) enhanced tau expression (upto eightfold) by supplementing deficient tRNAs that aid in rapid translation and (ii) direct boiling of expressed E. coli cells to extract tau with no separate cell lysis step. We further demonstrate that tau extracted by the direct boiling method is similar to tau purified by size-exclusion chromatography exhibiting similar structural and biophysical characteristics including aggregation propensity. Since morphologies and in vitro toxicity of fibrillar tau aggregates were also similar, tau extracted by the one-step direct boiling method can be used for tau aggregation assays without any additional purification.     

The use of three-dimensional model construction of virtual technology in orthopedic treatment

ObjectiveThe objective of this study is to explore the construction of a digital three-dimensional model of virtual technology that plays an auxiliary role in orthopedic treatment.MethodsThree fracture patients were selected, with no abnormality was observed in bone examination, no musculoskeletal disease in the past; and spiral CT scan of the spine and pelvis, upper limbs, and lower limbs was performed. The virtual technology was used to build a digital 3D model, mainly using the editing software Mimics10.0 software. In addition, the virtual three-dimensional model was verified by virtual surgery, data storage security, work efficiency of the model, model validity, three-dimensional characteristics of the model, the interaction mode of the model, and the data accuracy of the model were studied.ResultsThe digital 3D model was successfully established by Mimics10.0 software. The data fitting efficiency was very high. The data storage security of the 3D model was greatly improved compared with the 2D model, and the work efficiency was improved by at least 50%. There was also a significant change in the accuracy and interaction of data acquisition. Therefore, the detection of digital 3D model work through virtual surgery simulation fully demonstrated the positive auxiliary role of 3D model in orthopedic treatment.ConclusionThe digital 3D model based on Mimics10.0 software is efficient and accurate in obtaining data. It is very effective for subsequent adjuvant therapy in the field of orthopedics, reducing the probability of misdiagnosis by doctors, saving time and improving efficiency, reducing patient's physical pain and unnecessary economic expenses.     

基于结构信息的蛋白质相互作用规律的研究

蛋白质-蛋白质相互作用是蛋白质发挥其功能的重要途径之一。作者基于相互作用蛋白质数据库、基因本体数据库和蛋白质结构分类数据库（structural classification of proteins, SCOP）,结合SWISS-PROT等相关数据库中蛋白质功能注释信息,定义描述蛋白质相互作用倾向性的参数,对酿酒酵母定位于不同细胞器蛋白、部分膜蛋白,以及酿酒酵母、线虫和大肠杆菌按SCOP分类的不同结构类蛋白质之间相互作用规律进行研究。结果表明各种细胞器、膜和结构类蛋白质之间相互作用确实存在明显的偏向性。