Krithika Karunakaran & Yamoah Agyei

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

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

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

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.

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

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

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).

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

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

Figure 2. Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations

Figure 2. Structural interactions of sigma 70 factor in the RNA polymerase transcription initiation complex and location of selected mutations

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

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)

Figure 3. Expression of NTR1 in different strains

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

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

Table 1. Summary of gene enrichment analyses from RNA-seq data

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

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

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

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

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

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

Questions?