Matrajt Mariana, Nishi Manami, Fraunholz Martin J, Peter Oliver, Roos David S
Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA.
Mol Biochem Parasitol. 2002 Apr 9;120(2):285-9. doi: 10.1016/s0166-6851(02)00014-2.
Comparing the steady-state expression levels of recombinant proteins in Toxoplasma gondii parasites indicates considerable variability, and this has sometimes caused difficulties in the engineering of transgenic parasites. Anecdotal observations suggested that alteration of the N-terminus, e.g. by engineering as a fusion protein, permits stable expression of various transgenes that were previously difficult to express in their native form. We have exploited the sensitivity and quantitative nature of fire-fly luciferase (LUC) to examine expression levels in further detail. Fusing the 26 N-terminal residues derived from chloramphenicol acetyl transferase (DeltaCAT) to LUC permits efficient transient or stable luciferase expression in transgenic parasite tachyzoites, providing a useful reporter for studies in T. gondii. Site-directed mutagenesis was used to alter the second codon of DeltaCAT-LUC to encode all 20 possible amino acids, and these constructs showed that changes in the second amino acid can have dramatic effects on luciferase activity, with Ala, Glu, and Asp codons yielding the highest expression levels. Similar results were observed for the expression of both GFP and the T. gondii HXGPRT gene, demonstrating the generality of this effect.
比较重组蛋白在刚地弓形虫寄生虫中的稳态表达水平显示出相当大的变异性，这有时给转基因寄生虫的工程改造带来困难。轶事观察表明，改变N端，例如通过构建融合蛋白，可使各种以前难以以天然形式表达的转基因稳定表达。我们利用萤火虫荧光素酶（LUC）的敏感性和定量特性进一步详细研究表达水平。将源自氯霉素乙酰转移酶（DeltaCAT）的26个N端残基与LUC融合，可使转基因寄生虫速殖子高效瞬时或稳定表达荧光素酶，为刚地弓形虫研究提供了一个有用的报告基因。利用定点诱变改变DeltaCAT-LUC的第二个密码子以编码所有20种可能的氨基酸，这些构建体表明第二个氨基酸的变化可对荧光素酶活性产生显著影响，丙氨酸、谷氨酸和天冬氨酸密码子产生的表达水平最高。对于绿色荧光蛋白（GFP）和刚地弓形虫HXGPRT基因的表达也观察到类似结果，证明了这种效应的普遍性。
