Escherichia coli‐based cell free production of flagellin and ordered flagellin display on virus‐like particles

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli‐based cell‐free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell‐free expression. We then found that the D0 domain at the C‐terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease‐deficient cell extracts or deletion of the flagellin D0 domain. A human Toll‐Like Receptor 5 (hTLR5)‐specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non‐natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus‐like particles (VLPs) using bioorthogonal azide‐alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10‐fold. Biotechnol. Bioeng. 2013; 110: 2073–2085. © 2013 Wiley Periodicals, Inc.     

Polyamines in halophilic archaebacteria

Abstract The presence of stable mRNA in domant spores of several Streptomyces species is reported. The proteins encoded by these mRNAs are remarkably similar in all the species studied. A high degradation rate during the first hour of germination is a common feature to these proteins.     

mRNA疫苗在疾病预防与治疗中的研究进展与展望

基于信使RNA(messenger RNA, mRNA)的核酸疫苗是近年来兴起的一种mRNA技术。mRNA疫苗比传统疫苗有许多优点,能够实现快速、经济、高效的生产。单个mRNA疫苗可以编码多种抗原,增强对特定病原体的免疫反应,提高疾病的治疗效率,以单一配方针对多种病原微生物或疾病。mRNA疫苗相关技术在新型冠状病毒肺炎疫情防控中被视作一种革命性的疫苗技术,以创纪录的速度完成研发并成功应用。由于mRNA自身稳定性差,新型递送系统的开发与应用至关重要。随着mRNA相关药理学的深入研究,mRNA疫苗的临床应用进入了一个崭新的阶段。近年来。mRNA疫苗在传染性疾病预防、肿瘤治疗等方面获得充分发展并取得了一定的研究成果,对其进行概述并进行一定程度的展望。     

The multiple flavors of GoU pairs in RNA

Wobble GU pairs (or GoU) occur frequently within double‐stranded RNA helices interspersed within the standard G═C and A─U Watson‐Crick pairs. However, other types of GoU pairs interacting on their Watson‐Crick edges have been observed. The structural and functional roles of such alternative GoU pairs are surprisingly diverse and reflect the various pairings G and U can form by exploiting all the subtleties of their electronic configurations. Here, the structural characteristics of the GoU pairs are updated following the recent crystallographic structures of functional ribosomal complexes and the development in our understanding of ribosomal translation.