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A MALDI-TOF MS based method for the fast screening of recombinant protein high-yield strains

ABSTRACT: 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 target 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.

ORGANISM(S): Escherichia Coli

SUBMITTER: Mingliang Ye

PROVIDER: PXD045691 | iProX | Tue Sep 26 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:Basal-like breast tumors are aggressive cancers associated with high proliferation and metastasis. Currently basal-like breast cancer patients can only be treated with chemotherapy options. However, resistance to chemotherapy often occurs, resulting in recurrence and patient death. Some extremely aggressive basal-like breast cancers are also associated with hypoxia, inflammation and high leukocyte infiltration. Herein we discovered that the neural-specific transcription factor Engrailed 1 (EN1) is exclusively overexpressed in basal-like breast cancers. ShRNA-mediated knockdown of EN1 in basal-like breast cancer cells triggered potent and selective cell death. In contrast, ectopic overexpression of EN1 in normal cells activated survival pathways and conferred resistance to chemotherapeutic agents. Exogenous expression of EN1 cDNA reprogrammed the breast epithelial cells towards a long-lived, neural-like phenotype displaying dopaminergic markers. Gene expression microarrays demonstrated that the EN1 cDNA altered transcription of a high number of inflammatory molecules, notably chemokines and chemokine receptors, which could mediate pro-survival pathways. To block EN1 function, we engineered synthetic interference peptides (iPeps) comprising the EN1-specific sequences mediating essential protein-protein interactions necessary for EN1 function and an N-terminal cell-penetrating peptide/nuclear localization sequence. These EN1-iPeps rapidly mediated a strong apoptotic response in tumor cells overexpressing EN1, with no toxicity to normal or non EN1-expressing cells. Delivery of EN1-iPeps into basal-like cancer cells significantly decreased the IC50 of chemotherapeutic drugs routinely used to treat breast cancer. Lastly, MALDI-TOF mass spectrometry and immunoprecipitation assays demonstrated that EN1-iPeps captured targets involved in transcriptional and post-transcriptional regulation. Importantly, the EN1-iPeps bound the Glutamyl-prolyl tRNA synthetase (EPRS) target, which has been associated with the transcript-specific translational control of inflammatory proteins and activation of amino acid stress pathways. These studies show that EN1 activates intrinsic inflammatory pathways associated with pro-survival in basal-like breast cancer and that peptides targeting EN1 function could potentially be used to combat these lethal forms of breast cancer. reference x sample

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A MALDI-TOF MS based method for the fast screening of recombinant protein high-yield strains

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