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Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark

ABSTRACT: Identification of gel bands from purified fractions from expression of RseP in E.coli and Lactiplantibacillus plantarum.

INSTRUMENT(S): Orbitrap QExactive

ORGANISM(S): Lactiplantibacillus Plantarum Escherichia Coli (ncbitaxon:562)

SUBMITTER: Marie Vestergaard Lukassen

PROVIDER: MSV000092087 | MassIVE | Fri Jun 02 07:39:00 BST 2023

SECONDARY ACCESSION(S): PXD042670

REPOSITORIES: MassIVE

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Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark.

Kristensen Sofie S SS Lukassen Marie V MV Siebenhaar Suzana S Diep Dzung B DB Morth J Preben JP Mathiesen Geir G

Scientific reports 20230901 1

The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were opti ...[more]

PMID: 37658186

Similar Datasets

Lactiplantibacillus plantarum as a novel platform for production and purification of integral membrane proteins using RseP as the benchmark.

Project description:The present study describes a detailed procedure for expressing and purifying the integral membrane protein RseP using the pSIP system and Lactiplantibacillus plantarum as an expression host. RseP is a membrane-bound site-2-protease and a known antibacterial target in multiple human pathogens. In the present study, we screened five RseP orthologs from Gram-positive bacteria and found RseP from Enterococcus faecium (EfmRseP) to yield the highest protein levels. The production conditions were optimized and EfmRseP was purified by immobilized metal ion affinity chromatography followed by size-exclusion chromatography. The purification resulted in an overall yield of approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. The structural integrity of the purified protein was confirmed using circular dichroism. We further assessed the expression and purification of RseP from E. faecium in the Gram-negative Escherichia coli. Detection of soluble protein failed in two of the three E. coli strains tested. Purification of EfmRseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to EfmRseP expressed in L. plantarum. To our knowledge, this is the first time L. plantarum and the pSIP expression system have been applied for the production of membrane proteins.

| S-EPMC10474122 | biostudies-literature

Identification and Characterization of HDM D.p. immunogenic RNA

Project description:We examined the sequence information of the gel-extracted and purified RNA bands #1,#2 and #3 derived from House Dust Mite (HDM) D.p. immunogenic RNA. The individula RNA band was utilized for RNA-seq analysis.

2019-08-01 | GSE102210 | GEO

BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures

Project description:We ran native 5% polyacrylamide-TBE gel for electro mobility shift assay (EMSA) for DNA/RNA G4 structures with or without recombinant human core-BLM (cBLM) protein, which was extracted and purified from E.coli in order to show binding of the RNA/DNA by this protein. To further evidence, we cut the equivalent gel area where the shifted band is located for all the wells (DNA only, DNA+cBLM, RNA, RNA+cBLM and cBLM only) and anlayzed by LC-MS/MS to identify human BLM and to see if there are some E.coli contaminations in those specific bands. Moreover, we analyzed the stock solution of the recombinant cBLM as general control.

2023-10-24 | PXD042983 | Pride

Purified FLAG-Hrp1 GeLC-MS/MS - CHD1 remodelers regulate nucleosome spacing in vitro and align nucleosomal arrays over gene coding regions in S

Project description:Proteomics on affinity purified and mock purified FLAG-Hrp1. Reduction with DTT, alkylation by iodoacetamide. 1D gel, tryptic in gel digestion of cut-out bands, GeLC-MS/MS.

2012-11-01 | PXD000031 | Pride

Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

Project description:The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

| S-EPMC5589448 | biostudies-literature

S.wolfei membrane proteins - Membrane Complexes of Syntrophomonas wolfei Involved in Syntrophic Butyrate Degradation and Hydrogen Formation

Project description:Analyze S. wolfei membrane proteins separated on Blue Native gel bands

2017-07-28 | PXD003633 | Pride

GNPS - MLF2-MBP purification (Table S4, Prophet et al.)

Project description:MLF2 was purified from mammalian Expi293 cells as a fusion with MBP. As MLF2 associates tightly with HSP70/HSPA1A, this resulted in a stoichiometric co-purification with HSP70 members. To identify which ones, we subjected the whole purification or a gel-extracted band around 70 kDa to mass spectrometry.

2022-08-23 | MSV000090188 | GNPS

Identification of the CmSp1-target candidate proteins

Project description:To identify the CmSp1 targets, total protein of S. sclerotiorum sclerotia was incubated with CmSp1 and separated by gel electrophoresis. The corresponding protein bands in the control treatment were purified from the gel and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis for protein identification.

2023-09-27 | PXD045751 |

MNase-Seq of isolated mono-nucleosomes from strains W303 (wild-type) and htz1∆.

Project description:Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mono-nucleosomes harbouring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes, and – as proof of principle - the nucleosome preference of the chromatin remodeling complex, RSC. In order to generate a library of native yeast nucleosomes, we developed a three-step purification protocol: first, purified yeast nuclei were incubated with micrococcal nuclease (MNase), which preferentially digests naked DNA to generate short chromatin fragments. The resulting fragments were extracted from the nuclei, then bound to and eluted from DEAE sepharose. This was followed by ultracentrifugation through a sucrose gradient to separate the fragments by length to further remove contaminating proteins and free DNA. We chose a simple disassembly assay, which involves incubating the nucleosome library with ATP and the histone chaperone Nap1, with or without RSC. In this assay, RSC binds to nucleosomes and transfers the histones to Nap1, thereby releasing ‘naked’ DNA. Under certain conditions, reaction intermediates can be observed (tetramers or hexasomes), but for simplicity we chose to compare the input nucleosomes with the final naked DNA product. To separate the RSC-dependent released DNA from the non-remodeled nucleosomes, the reactions were subjected to native agarose gel electrophoresis, and DNA of the four bands isolated by gel-extraction. The upper bands, harboring nucleosomes, were named NUC (no RSC) and NUCR (with RSC), whereas the lower, ‘naked’ DNA bands were named DNA (no RSC) and DNAR (with RSC).

2019-05-08 | E-MTAB-7926 | biostudies-arrayexpress

A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform.

Project description:BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.

| S-EPMC3168869 | biostudies-literature

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