Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin

The defining property of core streptavidin (cSA) is not only its high binding affinity for biotin but also its pronounced thermal and chemical stability. Although potential applications of these properties including therapeutic methods have prompted much biological research, the high immunogenicity of this bacterial protein is a key obstacle to its clinical use. To this end, we have successfully constructed hypoimmunogenic cSA muteins in a previous report. However, the effects of these mutations on the physicochemical properties of muteins were still unclear. These mutations retained the similar electrostatic charges to those of wild‐type (WT) cSA, and functional moieties with similar hydrogen bond pattern. Herein, we performed isothermal titration calorimetry, differential scanning calorimetry, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis to gain insight into the physicochemical properties and functions of these modified versions of cSA. The results indicated that the hypoimmunogenic muteins retained the biotin‐binding function and the tetramer structure of WT cSA. In addition, we discuss the potential mechanisms underlying the success of these mutations in achieving both immune evasion and retention of function; these mechanisms might be incorporated into a new strategy for constructing hypoimmunogenic proteins.     

Alternative downstream processes for production of antibodies and antibody fragments

Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Ⅲ型效应子GALA对青枯菌在两种寄主植物上致病性的影响

[目的]研究Ⅲ型效应子GALAs对青枯菌OE1-1在不同寄主植物致病性上的影响。[方法]构建青枯菌OE1-1的多种GALA缺失突变体,通过根切和叶片注射等方法研究GALAs对青枯菌OE1-1致病力和细胞内增殖能力的影响。[结果]GALA多基因缺失突变体对寄主烟草的致病力减弱,在烟草体内细菌繁殖能力较野生型明显降低,但在寄主番茄上不影响其致病性。[结论]GALA效应子对青枯菌OE1-1在烟草植株致病性上展现协同作用。     

Complementary double-stranded helical oligomers bearing achiral bifunctional groups that catalyze asymmetric aldol reaction

Two novel chiral dimer and trimer strands composed of m-terphenyl groups linked through p-diethynylbenzene units with the chiral amidine group and achiral piperazine group introduced at the terminus or center of the strands, respectively, and its complementary achiral carboxylic acid dimer and trimer were synthesized. The complementary chiral/achiral strands form an excess-handed double-helical structure as supported by intense split-type Cotton effects in the absorption regions of the conjugated backbones biased by the chiral amidinium–carboxylate salt bridges. The double-helical trimer was found to catalyze the direct aldol reaction of cyclohexanone with 4-nitrobenzaldehyde and produce the products with a moderate enantioselectivity despite the fact that the catalytically active bifunctional piperazine/carboxylic acid pair introduced in the middle is achiral, indicating the key role of the one-handed double-helical framework for supramolecular bifunctional organocatalysis.     

Expression of Ralstonia solanacearum type III secretion system is dependent on a novel type 4 pili (T4P) assembly protein (TapV) but is T4P independent

Type IV pili (T4P) are virulence factors in various pathogenic bacteria of animals and plants that play important roles in twitching motility, swimming motility, biofilm formation, and adhesion to host cells. Here, we genetically characterized functional roles of a putative T4P assembly protein TapV (Rsc1986 in reference strain GMI1000) and its homologue Rsp0189, which shares 58% amino acid identity with TapV, in Ralstonia solanacearum. Deletion of tapV, but not rsp0189, resulted in significantly impaired twitching motility, swimming motility, and adhesion to tomato roots, which are consistent as phenotypes of the pilA mutant (a known R. solanacearum T4P-deficient mutant). However, unlike the pilA mutant, the tapV mutant produced more biofilm than the wild-type strain. Our gene expression studies revealed that TapV, but not Rsp0189, is important for expression of a type III secretion system (T3SS, a pathogenicity determinant of R. solanacearum) both in vitro and in planta, but it is T4P independent. We further revealed that TapV affected the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway, consistent with previous reports that PhcA positively regulates expression of pilA and prhG. Moreover, deletion of tapV, but not rsp0189, significantly impaired the ability to migrate into and colonize xylem vessels of host plants, but there was no alteration in intercellular proliferation of R. solanacearum in tobacco leaves, which is similar to the pilA mutant. The tapV mutant showed significantly impaired virulence in host plants. This is the first report on the impact of T4P components on the T3SS, providing novel insights into our understanding of various biological functions of T4P and the complex regulatory pathway of T3SS in R. solanacearum.     

Involvement of a PadR regulator PrhP on virulence of Ralstonia solanacearum by controlling detoxification of phenolic acids and type III secretion system

Ralstonia solanacearum can metabolize ferulic acid (FA) and salicylic acid (SA), two representative phenolic acids, to protect it from toxicity of phenolic acids. Here, we genetically demonstrated a novel phenolic acid decarboxylase regulator (PadR)-like regulator PrhP as a positive regulator on detoxification of SA and FA in R. solanacearum. Although the ability to degrade SA and FA enhances the infection process of R. solanacearum toward host plants, PrhP greatly contributes to the infection process besides degradation of SA and FA. Our results from the growth assay, promoter activity assay, RNA-seq and qRT-PCR revealed that PrhP plays multiple roles in the virulence of R. solanacearum: (1) positively regulates expression of genes for degradation of SA and FA; (2) positively regulates expression of genes encoding type III secretion system (T3SS) and type III effectors both in vitro and in planta; (3) positively regulates expression of many virulence-related genes, such as the flagella, type IV pili and cell wall degradation enzymes; and (4) is important for the extensive proliferation in planta. The T3SS is one of the essential pathogenicity determinants in many pathogenic bacteria, and PrhP positively regulates its expression mediated with the key regulator HrpB but through some novel pathway to HrpB in R. solanacearum. This is the first report on PadR regulators to regulate the T3SS and it could improve our understanding of the various biological functions of PadR regulators and the complex regulatory pathway on T3SS in R. solanacearum.