微生物学杂志
微生物學雜誌
미생물학잡지
JOURNAL OF MICROBIOLOGY
2015年
2期
14-21
,共8页
铜绿假单胞菌%胞外多糖%转运蛋白PslD%去垢剂%亲和层析
銅綠假單胞菌%胞外多糖%轉運蛋白PslD%去垢劑%親和層析
동록가단포균%포외다당%전운단백PslD%거구제%친화층석
Pseudomonas aeruginosa%extracellular polysaccharide%transporter PslD%detergent%affinity chromatogra-phy
条件性致病菌铜绿假单胞菌是细菌生物被膜研究的模式菌，其分泌的胞外多糖 Psl 在生物被膜形成中起关键作用。PslD 为 Psl 多糖的转运蛋白，由256个氨基酸构成，生物信息学分析揭示其有 N 端信号肽且为一次跨膜蛋白。分离纯化完整跨膜蛋白需要去垢剂的作用，去垢剂种类繁多且性质不一，研究设计了一套筛选溶解 PslD 的去垢剂的方案。通过抗组氨酸标签的 Western blot 分析，n-Decyl-β-D-maltopyranoside（DM）， n-Decyl-N，N-dimethylamine-N-oxide（DDAO）及 n-Dodecyl-N，N-dimethylamine-N-oxide（LDAO）被认为溶解 PslD的效率较高。通过改变总蛋白与去垢剂比例，进一步优化了去垢剂的溶解条件，即8 mg/ mL 总蛋白：质量分数为1%的 LDAO。在此溶解条件下，仅通过第一步 Ni 柱亲和纯化目的蛋白纯度可到达80%以上，这为进一步结晶尝试及其结构生物学研究奠定基础。
條件性緻病菌銅綠假單胞菌是細菌生物被膜研究的模式菌，其分泌的胞外多糖 Psl 在生物被膜形成中起關鍵作用。PslD 為 Psl 多糖的轉運蛋白，由256箇氨基痠構成，生物信息學分析揭示其有 N 耑信號肽且為一次跨膜蛋白。分離純化完整跨膜蛋白需要去垢劑的作用，去垢劑種類繁多且性質不一，研究設計瞭一套篩選溶解 PslD 的去垢劑的方案。通過抗組氨痠標籤的 Western blot 分析，n-Decyl-β-D-maltopyranoside（DM）， n-Decyl-N，N-dimethylamine-N-oxide（DDAO）及 n-Dodecyl-N，N-dimethylamine-N-oxide（LDAO）被認為溶解 PslD的效率較高。通過改變總蛋白與去垢劑比例，進一步優化瞭去垢劑的溶解條件，即8 mg/ mL 總蛋白：質量分數為1%的 LDAO。在此溶解條件下，僅通過第一步 Ni 柱親和純化目的蛋白純度可到達80%以上，這為進一步結晶嘗試及其結構生物學研究奠定基礎。
조건성치병균동록가단포균시세균생물피막연구적모식균，기분비적포외다당 Psl 재생물피막형성중기관건작용。PslD 위 Psl 다당적전운단백，유256개안기산구성，생물신식학분석게시기유 N 단신호태차위일차과막단백。분리순화완정과막단백수요거구제적작용，거구제충류번다차성질불일，연구설계료일투사선용해 PslD 적거구제적방안。통과항조안산표첨적 Western blot 분석，n-Decyl-β-D-maltopyranoside（DM）， n-Decyl-N，N-dimethylamine-N-oxide（DDAO）급 n-Dodecyl-N，N-dimethylamine-N-oxide（LDAO）피인위용해 PslD적효솔교고。통과개변총단백여거구제비례，진일보우화료거구제적용해조건，즉8 mg/ mL 총단백：질량분수위1%적 LDAO。재차용해조건하，부통과제일보 Ni 주친화순화목적단백순도가도체80%이상，저위진일보결정상시급기결구생물학연구전정기출。
The opportunistic Pseudomonas aeruginosa is a model organism for biofilm research. Its excreted extracel-lular polysaccharide Psl is a key component of biofilm matrix in P. aeruginosa. The Psl transporter PslD is composed of 256 amino acids,bio-informatics analysis has revealed that it comprised of one transmembrane protein having N ter-minal signal peptide. Detergents are required to isolate and purify transmembrane protein. However,detergents are various in kinds with different properties,in this study a set of scenario was designed for screening detergent to dis-solve Ps1D. Through anti-histidine labeled western blot analysis n-Decyl-β-D-maltopyranoside(DM),n-Decyl-N, Ndimethylamine-N-oxide( DDAO)and n-Dodecyl-N,N-dimethylamine-N-oxide( LDAO)were considered to have high extracting efficiency. By altering the ratio of total protein to the detergent,further optimal solubilization condition was determined as 8 mg/ mL total protein:1%(w/ v)LDAO. Under this soluble condition,over 80% purity can be achieved only through the first step of Ni column affinity chromatography to purify the goal protein,this had laid a foundation for study on its further crystallization attempt and its structural biology.
