Gordon Caroline L, Khalaj Vahid, Ram Arthur F J, Archer David B, Brookman Jayne L, Trinci Anthony P J, Jeenes David J, Doonan John H, Wells Brian, Punt Peter J, van den Hondel Cees A M J J, Robson Geoffrey D
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
Centre for Phytotechnology, Institute for Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 Al Leiden, The Netherlands2.
Microbiology (Reading). 2000 Feb;146 ( Pt 2):415-426. doi: 10.1099/00221287-146-2-415.
A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.
:绿色荧光蛋白融合体（GLA::sGFP），它能使绿色荧光蛋白用作黑曲霉中蛋白质分泌的体内报告分子。设计了两种分泌融合体用于GLA::sGFP的分泌，它们采用了长度略有不同的葡糖淀粉酶蛋白（GLA499和GLA514）。GLA::sGFP的表达显示荧光定位于菌丝细胞壁和隔膜，并且在菌丝顶端荧光最强。仅在以豆浆培养基培养的年轻培养物的上清液中，通过蛋白质印迹法可检测到细胞外的GLA::sGFP。在较老的培养物中，培养基酸化和蛋白酶的诱导可能是细胞外和细胞壁荧光丧失以及无法通过蛋白质印迹分析检测到GLA::sGFP的原因。对含有GLA::sGFP构建体的菌株进行紫外线诱变，并筛选一般分泌途径中的突变体幸存者。分离出三个在淀粉或明胶培养基上均无法形成晕圈的突变体。与亲本菌株相比，所有三个突变体的生长都很差。荧光显微镜检查显示，对于其中两个突变体，GLA::sGFP在细胞内积累，没有细胞壁荧光的迹象，而对于第三个突变体，观察到细胞壁荧光，没有细胞内积累的迹象。这些结果表明，GLA::sGFP融合构建体可作为方便的荧光标记物用于研究体内蛋白质分泌的动态过程，并作为分离一般分泌途径中突变体的工具。
