krithika karunakaran & yamoah agyei
TRANSCRIPT
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Krithika Karunakaran & Yamoah Agyei
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Background & Introduction
• Integral Membrane Proteins (IMPs) are proteins permanently attached to the cell membrane of biological organisms.
• IMPs have many vital biological functions • constitute approximately 1/3 of all proteins in humans as well as being the
targets of nearly 60% of all FDA-approved drugs • Serve as receptors, enzymes, channels, transporters etc.
• Despite their importance, structural and functional information is limited
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Introduction
• As little as 4% of the protein database corresponding to these IMPs. With a small portion of these being of eukaryotic origin.
• GPCRs are an example of IMPs and important drug targets • make up 5% of the human protein coding genome and have no prokaryotic homologs
• Most IMPs have extremely low natural abundance. • Hence the use of heterologous hosts like E. coli, Lactobacillus sp. and S. cerivisae have
been employed, which allow for its overexpression. • Some researchers have used mammalian and insect cell cultures among others but
these systems have produced varied successes
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Introduction
• IMPs are very unstable when solubilized in detergents • Different approaches were employed to give better expressing and stable
variants of IMPs. • Different approaches in yeast and E.coli had been developed in-house not
mentioned in the paper
• In this study, researchers used E.coli because its cheap, fast growing and produces isotope-labelled proteins for NMR investigation
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Introduction
• Eukaryotic hosts tolerate the expression of IMPs better than bacterial cells because it ends up being toxic to the prokaryotes.
• This toxicity is largely due to cellular stress from mistargeting and misfolding of the growing polypeptide of other proteins in the prokaryote and not necessarily the IMP only.
• Sec translocon, difference in membrane bilayer properties are factors that could cause the cell stress.
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Aim
• The AIM of the study was to improve understanding of heterologous expression of IMPs in E. coli to increase expression and functionality of the products.
• To identify E. coli genes with an influence on GPCR expression. Using the Keio collection
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Summary of Methodology
Western blot
Bacteria culture of transformed Keio Cells
fluorescence-activated cell sorting FACS
Whole Genome sequencing (Illumina MiSeq) to detect
mutationRNA sequencing
Bioinformatics
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Results
• E. coli strains with wild type expression of neurotensin-1 receptor (GPCR) successfully transformed with all Keio collections (Keio-NTR1).
• Some variations in the results of some strains in FACS were detected in 5 clones. ➢These clones had become enriched (deletion of certain genes) ➢There was no obvious direct relationship between the deletions and IMP
biosynthesis ➢Growth and cell density after 20hrs was similar at 37ºC but after 20hrs at 20ºC
incubation, cell density of 4 of the 5 Keio originals was markedly increased as compared to the positive control (BW25113).
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Results
• The four Keio collections (ΔybaA, ΔqseB, ΔhybD and ΔuxuB) were transformed with pRG-NTR.
• Expressing NTR1 in the clones reached higher final cell density after 20 hours at 20 °C, in comparison to the wt E. coli strain BW25113
• To identify the genetic modification that causes the difference in the observed phenotype, the DNA sequence of the whole genome of Keio (ΔqseB) and the wild-type strain BW25113 were determined
• In the ΔqseB Keio clone, a non-silent single nucleotide substitution in the rpoD gene was identified and confirmed by Sanger sequencing ➢This results in a valine instead of glutamate in amino acid position 575 (domain 4.2) of the
sigma 70 transcription factor. The primary sigma factor during exponential growth
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Results
Figure 1. Expression of NTR1 receptor in the selected E. coli strains from the Keio collection a. Showing cell growth after 20hrs b. Showing NTR1 expression levels
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Figure 2. Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations
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Figure 2. Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations
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Genetic background of other Keio clones
The rpoD-E575V mutation was not found in the other enriched clones or the WT BT25113 • Mutations in other Keio clones
• ΔhybD: deletion of 16 amino acids from position 172-197 • ΔuxuB: mutation I141S • ΔybaA: mutation D213Y
Figure 2. Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations
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E. Coli with & without rpoD mutation
• New E. coli strain carrying only the mutation rpoD-E575V created to confirm correlation of mutation with observed phenotype
• Through site-directed genome mutagenesis based on recombineering, two strains generated:
BW25113 rpoD-E575V Δmug::Km (abbreviated rpoD*) BW25113 wt rpoD(E575) Δmug::Km (abbreviated rpoD wt)
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Figure 3. Expression of NTR1 in different strains
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Other GPCRs• To test whether the observed phenotypes are obtained with hard to express
GPCRs are expressed, the original Keio clones were transformed with pRG plasmids encoding • Alpha-1b adrenergic receptor (ADRA1b) • Tachykinin receptor (TACR) • µ-opioid receptor (MOR)
Figure 4. Cell growth after 20h of GCPRs expression in different Keio clones
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Gene Expression
• Hypothesis: it is possible that this single-point mutation modifies the mRNA levels in bacteria, in particular the mRNA of the GPCR itself being expressed from the plasmid, but also that of other proteins
• Universal RNA Spike II (TATAA Biocenter) • All samples from the E. coli wt and
rpoD* exhibited nearly identical Ct values for RNA Spike II
• Expression of NTR1 gene is down-regulated in rpoD*
• Expression of NTR1 is down-regulated in all Keio clones
• Hypothesis: the mutation in rpoD may have, in addition to its direct effect on the GPCR gene, a more general effect on the expression of many genes
• Whole transcriptome analysis (RNA-seq) of E. coli wt and rpoD* both without pRG-NTR plasmid
Mutation E575V may have a role in the specific interaction between RNA polymerase and promoters during transcriptional activation
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Table 1. Summary of gene enrichment analyses from RNA-seq data
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Time course of NTR1 accumulation
• Cell samples of the wt and rpoD* mutant strains were taken as a function of time after induction of protein expression to understand NTR1 expression kinetics and whether the rpoD* mutation may influence it • Estimate cell number, functional receptors/cell, NTR1 expression, and
total NTR1 protein amount by Western Blot
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Figure 5. Kinetics of NTR1 expression. E. coli wt and the rpoDE575V mutant strain were transformed with pRG-NTR. NTR1 expression at 20°C was performed for 20 hours. Samples were taken at different time points
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Figure 5. Kinetics of NTR1 expression. E. coli wt and the rpoDE575V mutant strain were transformed with pRG-NTR. NTR1 expression at 20°C was performed for 20 hours. Samples were taken at different time points
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Discussion
• Phenotypes of the enriched strains ! favorable for expressing wt GPCRs as these strains overcome stress imposed by GPCR expression • E. coli mutations are stress dependent – possible that stress during
construction of the Keio strains could result in genetic instability of E. coli wt parent strain ! allowing rpoD mutation to occur • Mutations in sigma 70 may not only affect transcription initiation
rates, but also shift the balance between other sigma factors
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Discussion
• Mutations in sigma 70 lower the affinity of the RNA polymerase to the promoter ! • transcriptional initiation frequency is lowered !
• slower, longer lasting mRNA and GPCR production ! • less toxicity for the cell, longer cell growth !
• higher GPCR expression levels
• 20°C vs 37°C ! rpoD mutations must have additional beneficial effects for the cell at 20°C by affecting other genes
• Transcriptome analysis shows genes related to cold shock response are down-regulated in the rpoD mutant strain compared to wt E. coli strain at 20°C
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Conclusion
• The mutated sigma 70 alters the relative expression level of many genes, and thus also has a general effect on alleviating stress from the overexpression of the GPCR at several levels, contributing to the desirable phenotype
• Future direction: • Findings can be used for future strain engineering • Transcriptional factor fine tuning can be a new and different way to adjust and coordinate the
biogenesis of membrane proteins
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Questions?