Jin Jing-Hao,Zhou Xue,Liu Wang,Zhang Zi-Hui,Huang Shen-Xin,Zhao Wen-Jing,Chen Xiao-Ren
Objective To examine the influence of widely used protein affinity tags and the tobacco PR1a signal peptide (SP) on detection, purification and bioactivity analyses of the small oomycete apoplastic effector SCR96 in planta . Results Through agroinfiltration, the phytotoxic effector SCR96 of Phytophthora cactorum was expressed in Nicotiana benthamiana leaf apoplast as a fusion protein carrying single affinity tag (His, HA or FLAG) at either C- or N-terminus. Leaf necrosis caused by different affinity-tagged SCR96 varied among tags and replicates. All of tagged proteins can be detected by antibodies against SCR96. All of SCR96 fusions except N-terminally fused 6His-tagged protein were detected using tag antibodies, indicating that 6His tag may be degraded when fused at N-terminus. Interestingly, C-terminal His- and FLAG-tagged SCR96 maintained the biological activity after purification. In the substitution assay of SCR96 SP, we observed that PR1a SP can lead chimeric SCR96 expression in N. benthamiana , but the replacement totally disrupted its bioactivity. Conclusion C-terminal His or FLAG tag, along with its original SP, is efficient enough to enable detection and purification of functional SCR96 from N. benthamiana leaf apoplast, which would facilitate plant–pathogen interaction studies.
Xiaoyan Liu,He Zhu,Yanan Li,Hongqiang Qin,Mingliang Ye
In this study, we presented a MALDI-TOF MS based approach for the screening of recombinant protein high-yield strains. It relies on the MALDI MS analysis of the affinity tag containing peptide that released from the targe t recombinant proteins expressed in host strains. This approach was illustrated by quantifying the His-tag containing target peptide which was removed from the recombinant protein of Src Homology 2 superbinder by tobacco etch virus protease . After the specific enrichment and detection of target peptide with the theoretical molecular weight of 1895.09 Da, we could achieve the identification of strains with different expression levels of Src Homology 2 superbinder. Furthermore, the screening of strains at the absolute quantitative level was realized by adding the synthesized heavy labeled peptide as internal standards, and the quantitative results have a good linear correlation. In general, based on the virtue of high speed, accuracy and sensitivity of MALDI-TOF MS, this approach has the potential to achieve high throughput detection, and then provide powerful technical support for the screening of recombinant protein high-yield strains.
危险品化学品经营许可证（带存储）