24-appendix-d.Rmd 64.6 KB
 mk11g11 committed Feb 19, 2020 1 # Expression of the nicotinic acetylcholine receptor extracellular domain  mk11g11 committed Sep 29, 2019 2 3 4 5 6 7 8 9  \newpage ## Introduction nAChRs are the molecular targets of neonicotinoid insecticides. The adverse effects of neonicotinoids on non-target insects led to the restricted use and an eventual ban of these chemicals in the EU, which highlights the need for the synthesis of more selective compounds. The first step towards the generation of compounds effective on pest and not beneficial insects, is the understanding of the interactions between the neonicotinoid and their targets based on the knowledge of the agonist binding site structure. ### Structral basis of acetylcholine and agonist binding  mk11g11 committed Feb 19, 2020 10 Crystal structure of the mollusc homologue of nAChR extracellular domain (ECD) AChBP bound to imidacloprid [@ihara2008; @talley2008] and genetic analysis of the insect resistant strains [@liu2005] highlighted the importance of residues in both the principal and complementary site of the ligand binding site. The key determinants for acetylcholine are well known (Section \@ref(pharmacophore)). Neonicotinoids forms similar interactions with the conserved residues in the binding pocket (Section \@ref(pharmacophoreofneonics)). There are cation-pi interactions with the aromatic amino acids, hydrogen bonds via water molecule and Van der Waals interactions with loop E residues - these are common to interactions made with other agonists of nAChRs. There are also some proposed unique interactions between imidacloprid and AChBP, such as a hydrogen bond with Gln 55 of loop D. However, this Gln is not conserved and corresponds to basic residue in many insect nAChR subunits, thus the unique interaction may be present in some, and not all insect receptors. The importance of this residue was highlighted in the genetic analysis of naturally occuring resistant strains. Mutation from basic arginine to threonine at the corresponding position of $\beta1$ nAChR subunit of *Myzus Persicae* confers imidacloprid resistance [@bass2011].  mk11g11 committed Sep 29, 2019 11   mk11g11 committed Feb 19, 2020 12 Although AChBP is a useful model to study nAChR-drug interactions, as indicated above, there are differences in the amino acid sequence in the ligand binding pocket of AChBP and nAChR (Appendix A), including those that appear as key determinants of drug sensitivity. Therefore, a high resolution structure of nAChRs are needed to fully understand the underpinnings of selectivity of neonicotinoids and the chemical space in which improved selectivity might be achieved. Structures of $\alpha4\beta2$ [@morales-perez2016] and nAChR extracellular domain (ECD) of $\alpha2$ [@kouvatsos2016] and mouse $\alpha1$ [@dellisanti2007] human $\alpha9$ [@zouridakis2014] are known. Structures of nAChR extracellular domain bacterial homologes GLIC [@hilf2009] and ELIC [@hilf2008] as well as pentameric ion channels GABA [@miller2014] and glycine [@huang2015] have also been described. However, the crystal structure of insect receptors is not available. This may be caused by the difficulties in expression and purification of folded and soluble recombinant proteins [@rosano2014], which are essential and first steps towards the structural characterisation.  mk11g11 committed Sep 29, 2019 13 14  ## Biological systems for recombinant protein expression  mk11g11 committed Feb 19, 2020 15 The requirements for the structural analysis of any protein is the high level expression of stable and correctly folded protein [@rosano2014]. There is an array of biological systems used for the recombinant protein expression, however there is no single system that subserves as the single route to the best production of all proteins. This often means a trial and error approach, until the right system is identified. Some of the commonly used systems are: isolated cell lines of mammals or insects, and whole organisms such as yeast, fungi or algae. *E. coli* is one of the most commonly used host organisms for heterologous soluble protein expression [@berman2000] due to its simplicity, low cost, relatively good knowledge of transcription, translation and protein folding processes as well as ease of manipulation. According to Protein Data Bank, almost 75 % of all structures have been derived from proteins expressed in *E. coli* [@berman2000]. This includes the structure of GLIC [@hilf2009] and ELIC [@hilf2008]. In addition, an expression of functional AChBPs [@abraham2016] in *E. coli* cells and full length human $\alpha7$ [@tillman2016] have been reported.  mk11g11 committed Sep 29, 2019 16   mk11g11 committed Feb 19, 2020 17 (ref:e-coli-structure) **The *E. coli* cell.** *E. coli* is a Gram negative bacteria shielded by a capsule and encapsulated by 2 layers of the membrane separated by the periplasmic space. Main organelles in the cytosol are nucleoid containing the genetic material, inclusion bodies typically containing protein aggregates and ribosomes for protein translation.  mk11g11 committed Sep 29, 2019 18   mk11g11 committed Feb 19, 2020 19 {r e-coli-structure-label, fig.cap="(ref:e-coli-structure)", fig.scap='Schematic representation of the \\textit{E. coli} cell.', fig.align='center', echo=FALSE}  mk11g11 committed Sep 29, 2019 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 knitr::include_graphics("fig/results5/png/e_coli.png")   mk11g11 committed Feb 19, 2020 49 ## Strategies used to express pentameric ligand gated ion channels in *Escherichia coli (E. coli)*.  mk11g11 committed Sep 29, 2019 50 51 52  Despite the instrinsic difficulties of expressing multimeric and membrane proteins in *E. coli*, they have been used to express and purify high quantity of folded nAChRs and related proteins. This includes AChBP [@abraham2016], ELIC [@nys2016; @hilf2008] and full length human $\alpha7$ nAChRs [@tillman2016], and the ECD of the rat $\alpha7$ [@fischer2001]. Strategies employed in these studies highlight ways of overcoming major difficulties faced when trying to express recombiant proteins in *E. coli*.  mk11g11 committed Feb 19, 2020 53 Eukaryotic secretory and membrane proteins are targeted to ER and Golgi where they undergo a process of maturation, before being sent their correct localisations. As part of the maturation process, vast number of these proteins undergo a process of post-transcriptional modification [@khoury2011], such as addition of carbohydrates (glycosylation) or formation of disulphide bonds. These modifications can contribute to the stability of the protein, aiding their folding [@xu2015]. The *E. coli* secretory system is much simpler, therefore the nature, the frequency and the mechanism of post-translational modifications differ from those in eukaryotic cells [@dell2010]. There are a number of strategies employed to overcome this problem. For example, proteins requiring formation of disulphide bonds can be targeted to the periplasmic space of the *E. coli*, where this process can take place, due to the presence of active alkaline phosphatases [ref]. To enable formation of disulphide bonds of recombinant human nAChR, $\alpha7$ was targeted to the periplasmic space of *E. coli* with the pelB sequence [@tillman2016].  mk11g11 committed Sep 29, 2019 54   mk11g11 committed Feb 19, 2020 55 Targeting to the periplasmic space may also have other advantages. There periplasmic environment is less crowded due to reduced number of secreted proteins, in comparison to the cytoplasm, therefore the likelihood of proteolysis of the recombinant protein is reduced [@makrides1996]. This may lead to potential increased stability of proteins in the periplasm vs cytoplasm. Expression in the periplasm may be advantageous for some proteins, even those that do not require formation of disulphide bonds, such as ELIC [@hilf2008; @nys2016].  mk11g11 committed Sep 29, 2019 56   mk11g11 committed Feb 19, 2020 57 One of the major obstacles when producing a recombinant protein in *E. coli* is it that many proteins tend to form insoluble aggregates [@peternel2011]. This issue can be addressed by a number of approaches. First, recombinant protein can be tagged by another protein, native to the biological host. MBP is an *E. coli* periplasmic protein [@bedouelle1983]. N-terminal fusion of the protein of interest with MBP increases solubility and stability of proteins expressed in both cytoplasmic and periplasmic space [@raran-kurussi2015], including those rich in disulphide bonds [@planson2003]. MBP fusion strategy was employed by @fischer2001 to express $\alpha7$ ECD and @hilf2008 to express ELIC. Another way to increase solubility is to to produce a fragment and not a full length of the protein. This is a common approach employed when studying the soluble domains of transmembrane proteins, such as ECD containing the agonist-binding pocket of nAChRs. ECD is soluble, therefore it may be easier to successfully express and purify than the full-lent protein containing hydrophobic sections [ref]. Importantly, ECDs expressed heterogeneously can form pentameric assemblies and folded binding sites [@kouvatsos2016; @dellisanti2007]. ECD of $\alpha7$ was expressed in *E. coli* by @fischer2001. Another approach is to modify the genetic code, to introduce amino acid mutations, some of which may favour expression. For example, mutation of 2 amino acids to increase solubility can increase expression by 264-fold [@dale1994]. Mutation of a single amino acid in $\alpha7$ ECD sequence increases stability and solubility of expressed protein [@tsetlin2002]. There is also another version of the $\alpha7$ ECD containing greater alterations generated by @zouridakis2009 (Figure \@ref(fig:alpha7-seq-mutant-label)). These mutations increased protein solubility and production yield of the protein.  mk11g11 committed Sep 29, 2019 58   mk11g11 committed Feb 19, 2020 59 (ref:alpha7-seq-mutant) **Sequences of ECD nAChR variant with increased solubility.** Sequences of ECD used in this study: human $\alpha7$ (hu a7), honey bee $\alpha5$ (Ap a5) and *C. elegans* ACR-21 (Cel ACR-21) have been mutated (residues in red) based on the mutant version of $\alpha7$ (mut-10) (@zouridakis2009). In addition, Cys-loop of the honey bee and *C. elegans* subunits have been replaced for the more soluble Cys-loop sequence of *Aplysia* AChBP.  mk11g11 committed Sep 29, 2019 60 61 62 63 64 65 66  {r alpha7-seq-mutant-label, fig.cap="(ref:alpha7-seq-mutant)", fig.scap = "Sequences of ECD nAChR variant with increased solubility", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/intro_alignment.png")   mk11g11 committed Feb 19, 2020 67 Summarising, there is no universal protocol for the expression of folded and stable recombinant proteins particularly in the challenging subdomain of the membrane protein that derives from protein with complex quaternary (pentameric) structure. Therefore, it is a common practise to try several approaches, optimising the *E. coli* growth and expression conditions along the process. Years of research developed the use of solubility enhancers, targeting signalling sequences and other approaches to allow for the expression of complex molecules, such as nAChRs and bacterial structurally related proteins. For example, to express and purified ELIC from *E. coli*, @hilf2008 expressed ELIC tagged by maltose binding protein (MBP) and targeted to the periplasmic space by pelB. pelB is native to *Erwinia carotovora* [@lei1987]. It is a 22-amino acid sequence: *MKY*LLPTAAAGLLLLAAQ**PAMA**. As many other signal sequences, it contains 3 basic residues at the N-terminus (italic), a string of hydrophobic amino acids, and a cleavage site (bold) [@perlman1983]. The cleavage site is recognisable by the membrane bound signal peptidase [@pugsley1993], releasing the downstream peptide into the periplasmic space. Although not native to *E. coli*, pelB directs tagged proteins to the periplasm of this bacterium [@yoon2010], via the Sec translocation pathway. Despite all these advances, the successful expression of nAChRs in *E. coli* has been achieved only in a handful of cases. The inability to produce high quantity and quality of nAChRs, hinders their structural analysis and understanding of the molecular basis of selectivity of important agricultural compounds, such as neonicotinoids.  mk11g11 committed Sep 29, 2019 68 69 70  \newpage  mk11g11 committed Feb 19, 2020 71 ## Aims  mk11g11 committed Sep 29, 2019 72 73 74 75 76  The aim of this chapter is to develop an *E. coli* based expression platform for insect nAChRs to enable characterisation of the ligand binding site and determination of structural features underpinning their interactions with neonicotinoids. As the first step, the expression and purification of human $\alpha7$ was initiated as a test bed. This receptor was chosen because it forms homopentameric receptors in which the recombinant expression of a single subunit can potentially drive functional expression of the ECD.  mk11g11 committed Feb 19, 2020 77 To enable expression of proteins in *E. coli* cells, necessary DNA elements were cloned into the expression vector (Figure \@ref(fig:construct-diagram-label)). These elements are the START codon, pelB, sequence encoding for a string of 10 histidines (DecaHIS), maltose binding protein (MBP) and clevage site 3C.  mk11g11 committed Sep 29, 2019 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95  (ref:construct-diagram) **Schematic diagram of the DNA construct used for the expression of $\alpha7$ ECD in *E. coli*.** This is more text that belongs here. {r construct-diagram-label, fig.cap="(ref:construct-diagram)", fig.scap = "Schematic diagram of the DNA construct used for the expression of $\\alpha7$ ECD in \\textit{E. coli}.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/Final_construct.png")   mk11g11 committed Feb 19, 2020 96 START codon initiates protein translation, pelB targets the protein to the periplasm, DecaHIS and/or MBP enable purification with a nickel or dextrin chromatography column, respectively. MBP also acts as a solubility enhancer, whereas 3C is cleavage site enabling removal of pelB-HIS-MBP-3C from downstream peptide upon treatment with an appropriate protease. Downstream of these elements sequence encoding for the $\alpha7$ extracellular domain was cloned. There are several reasons why expression the ligand binding domain without the transmembrane domain was carried out. First, this study is concerned with the structure of the ligand binding site, which is contained in the ECD domain of the receptor. ECD is potentially soluble, therefore easier to successfully express and purify than the full-length protein containing hydrophobic sections. Although ECDs can form pentameric channels, the sequences within the TM region of the receptor may be also important in the process of receptor assembly [@wang1996a]. To account for this, the ECD was flanked by sequence encoding for 2GSC - a single subunit of a pentameric protein.  mk11g11 committed Sep 29, 2019 97 98 99 100 101 102 103 104 105 106 107  (ref:2gsc) **Comparison of the pentameric soluble bacterial protein transmembrane domain of the nicotinic acetylcholine receptor.** 2GSC is a 4-helical protein, assembling into a pentameric bundle (a). The general architecture and dimensions closely reassemble those of the nAChR transmembrane domain (b). Images and dimensions were derived in UCSA Chimera (PDB codes:2GSC and 2BG9 for muscle nAChR). Distances were derived by calculating distances from the most distal atoms on the polar ends of the structures. {r 2gsc-label, fig.cap="(ref:2gsc)", fig.scap="Comparison of the pentameric soluble bacterial protein transmembrane domain of the nicotinic acetylcholine receptor", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/2GSC+TM_nachr.png")  2GSC is a cytosolic protein endogenous to Gram-negative bacteria *Xanthomonas campestris*. Its structure was derived by X-ray crystallography, following the expression in and purification from *E. coli* (Figure \@ref(fig:2gsc-label)) [@lin2006]. 2GSC is a four-helical protein assembling into pentameric bundles. The overall architecture is similar to that of the nAChR membrane spanning domain (Figure \@ref(fig:2gsc-label)), however, 2 GSC is soluble. It is therefore hypothesised that the oligomerisation of the 2GSC could aid assemble of pentameric ECD of nAChRs. It needs to be noticed that although this chapter describes the expression of human $\alpha7$ receptor, the expression of several other genes have been tested.  mk11g11 committed Feb 19, 2020 108 Two further ECDs, namely honey bee $\alpha5$ and *C. elegans* ACR-21 subunit were cloned. Their expression was driven from plasmids containing 2GSC, as well as two other proteins: 1VR4 and 2GUV. 1VR4 and 2GUV are bacterial proteins of unknown function, shown to form pentamers in *E. coli*. Out of 9 constructs tested, the results from the $\alpha7$ were the most promising.  mk11g11 committed Sep 29, 2019 109   mk11g11 committed Oct 06, 2019 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127   mk11g11 committed Sep 29, 2019 128   mk11g11 committed Oct 06, 2019 129   mk11g11 committed Sep 29, 2019 130 131 132 133 134 135 136  \newpage ## Results ### Generation of the vector for the expression of human $\alpha7$ nAChR in *E. coli* periplasm.  mk11g11 committed Feb 19, 2020 137 The expression vector was generated in a two-step reaction. First, *pelB-HIS-MBP-3C* was cloned, followed by the *$\alpha7$-2GSC*. For simplicity, *pelB-HIS-MBP-3C* sequence will be refereed to as MBP-3C.  mk11g11 committed Sep 29, 2019 138   mk11g11 committed Feb 19, 2020 139 MBP-3C was PCR amplified (Figure \@ref(fig:pet27-mbp-label), Table \@ref(tab:MBP-amplification)) with *SalI*, *NdeI* flanking primers (Table \@ref(tab:primer-seq1)) from the vector used for expression of ELIC [@hilf2008]. Purified PCR product and the destination *pET27* vectors were sequentially digested with *SalI* and *NdeI* restriction enzymes to enable ligation (Figure \@ref(fig:pet27-mbp-label)). Ligation and colony selections were performed to generate *pET27-pelB-HIS-3C* (*pET27-MBP-3C* for short), suitable for expression of proteins in the periplasm. The success of cloning was provisionally indicated by *NdeI* and *SalI* digestion to produce the backbone and insert DNA fragments of purified plasmid. The positive clones were amplified and sequenced using primers flanking the insert (Appendix \@ref(fig:pelb-3c-lbl)). A single nucleotide mutation (C substituted by A) occurred between the *pelB* and *DecaHIS*, but this conservative mutation (GCC to GCA codon) encode for alanine (Appendix E).  mk11g11 committed Sep 29, 2019 140   mk11g11 committed Feb 19, 2020 141 (ref:pet27-mbp) **Generation of the vector for the expression of proteins in the periplasm of *E. coli*.** (a) Cartoon representation of the process of amplification of the gene by PCR (a) indicating the restriction sites of enzymes used for DNA digestion. *pelB-HIS-MBP-3C* sequence was amplified from *pET26-GLIC* vector, gel excised and purified. Digested with *SalI* and *NdeI* PCR fragment was cloned into digested *pET27*. 9b) Agarose gel of digested PCR template (*pET26-GLIC*), PCR products (2), *pET27* vector backbone (3) and cloned expression *pET27-pelB-HIS-MBP-3C* vector (4 and 5) against DNA ladder (M). The sizes of generated DNA fragments in bp are given under the DNA bands. The localisation of restriction sites within the DNA fragments are indicated in a.  mk11g11 committed Sep 29, 2019 142 143 144 145 146  {r pet27-mbp-label, fig.cap="(ref:pet27-mbp)", fig.scap = "Generation of the vector for the expression of proteins in the periplasm of \\textit{E. coli}.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/cloning_pet27_mbp.png")   mk11g11 committed Feb 19, 2020 147 $\alpha7$-ECD-2GSC was cloned into the *pET27-MBP-3C* expression vector (Figure \@ref(fig:pet27-hu-ligation-label)). *pBMH* plasmid containing coding *$\alpha7$-ECD-2GSC* sequence was synthesised. The *Hu$\alpha7$-2GSC* gene was PCR amplified with primers containing non-complementary sequences containing *SalI* and *NheI* restriction sites (Table \@ref(tab:primer-seq1) and Table \@ref(tab:human-lgd-amplification)). PCR product and *pET27-MBP-3C* were sequentially digested with *SalI* and *NheI* restriction enzymes. Purified DNA fragments were ligated, colonies selected and DNA purified. DNA was then analytically digested with *SalI* and *NheI* as well as send for sequencing. Cloned DNA sequence was error free (Appendix F). The generated *pET27-MBP-3C-$\alpha7$-ECD-2GSC* vector was used for the expression of $\alpha7$-ECD.  mk11g11 committed Sep 29, 2019 148   mk11g11 committed Feb 19, 2020 149 (ref:pet27-hu-ligation) **Generation of the vector for the expression of ligand binding domain of human $\alpha7$ nAChR.** Schematic representation (a) and DNA agarose gel (b) of generation of expression vector. $\alpha7$ was PCR amplified using primers flanked with restriction enzyme recognition sites, digested and cloned into digested *pET27-pelB-3C* vector.  mk11g11 committed Sep 29, 2019 150 151 152 153 154  {r pet27-hu-ligation-label, fig.cap= "(ref:pet27-hu-ligation)", fig.scap= "Generation of the vector for the expression of ligand binding domain of human $\\alpha7$ nAChR.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/generation-of-pet27-hua7-1vr4.png")   mk11g11 committed Feb 19, 2020 155 ### Expression of $\alpha7$ chimera in *E. coli*  mk11g11 committed Sep 29, 2019 156   mk11g11 committed Feb 19, 2020 157 The *pET27-pelB-3C-$\alpha7$-ECD-2GSC* was used to express the chimera protein in *E. coli* cells. To enable protein expression, *E. coli* cells were transformed with the expression plasmid and grown in the presence of antibiotic kanamycin. Bacteria were grown in LB growth media, which contains nutrient to support bacterial growth. The conditions at which bacteria are grown can be modified to optimize protein expression (PhD thesis of Ben Yarnall, data not shown). The factors were investigated with resoect to the induced expression of $\alpha7$ ECD chimera. Transformed *E. coli* culture was grown at 37 $^\circ$C until OD600nm≈1.0 before adition of 0.5 mM IPTG for 6 hours. This method allowed for high levels of protein expression over a short period of time.  mk11g11 committed Sep 29, 2019 158   mk11g11 committed Feb 19, 2020 159 In parallel, culture was grown at 37 $^\circ$C until it reached the exponentially growing phase (OD~600nm~ = 0.6). At that point, the temperature was lowered to 18 $^\circ$C, and the growth allowed to proceeded until OD~600nm~ = 1.0. At this point, 0.2 mM IPTG was added and the cultures incubated overnight at 18 $^\circ$C.  mk11g11 committed Sep 29, 2019 160 161 162 163 164  Pre- and post-induction samples were collected for both growth and IPTG induction conditions (Section \@ref(samples) and mixed with denaturing sample buffer. Proteins present in the SDS-bacterial cell extracts before and after IPTG induction were resolved with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualised with the Coomassie staining (Figure \@ref(fig:hua7-WB-label)). Addition of IPTG should lead to the expression of the $\alpha7$ construct. Indeed, a distinct band of 84 kDa can be seen on the gel. The size of this band corresponds to the predicted size of expressed coding sequence from the $\alpha7$ construct.  mk11g11 committed Feb 19, 2020 165 To authenticate the band as the protein of interest Western blot of the samples run on the SDS-PAGE was performed (Section \@ref(western)). Western blot uses antibodies with high affinity for the N-terminal DecaHIS tag (Section \@ref(abs)). As seen in Figure \@ref(fig:hua7-WB-label), no protein was detected in the pre-induction sample. Whereas addition of IPTG inducer resulted in production of HIS-tagged protein. This protein is of expected size of ~ 84 kDa.  mk11g11 committed Sep 29, 2019 166   mk11g11 committed Feb 19, 2020 167 Western blot was examined closely to establish which condition resulted in the production the highest amount of the recombinant protein. Samples run on the SDS-PAGE gel were volume-normalised, thus the intensity of bands on Western blot can be compared. The expression of $\alpha7$ chimera at 37 $^\circ$C and induction with 0.5 mM IPTG was the highest after 2 hours and decreased over time. Interestingly, multiple bands were detected in sample collected after the overnight protein expression at lower temperature and lower IPTG concentration. This suggests multiple sized HIS-tagged proteins are present in the sample, including truncated or proteolysed ones. Based on band intensities, the highest level of expression was achieved after overnight expression with low IPTG concentration and at low temperature.  mk11g11 committed Sep 29, 2019 168   mk11g11 committed Feb 19, 2020 169 (ref:hua7-WB) **Expression of the $\alpha7$ ECD chimera protein in *E. coli*.** Coomassie stained SDS-PAGE gels (a) and corresponding Western blot (b) of proteins obtained from the lysed whole cell samples. Transformed with $\alpha7$ ECD containing plasmid *E . coli* were grown in 1 L of LB. Protein expression was induced with either 0.5 mM IPTG and proceeded to be grown at 37 $^\circ$C or with 0.2 mM IPTG and proceeded to be raised at 18 $^\circ$C. Samples of the cellular suspension prior to induction (pre-induction) and at 2, 4, 6 hours after the induction with high IPTG concentration were taken. Alongside, a sample after overnight (16-hour expression) driven by low concentrations of IPTG (0.2 mM) were taken. Samples were prepared as described in Section \@ref(samples).  mk11g11 committed Sep 29, 2019 170 171 172 173 174  {r hua7-WB-label, fig.cap="(ref:hua7-WB)", fig.scap= "Expression of the $\\alpha7$ ECD chimera protein in \\textit{E. coli}.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/induction-of-hua7-2gsc-WB-and-SDS.png")   mk11g11 committed Feb 19, 2020 175 Following overnight protein expression at 18 $^\circ$C, induction by 0.2 mM IPTG, and 6-hour expression at 37 $^\circ$C induction with 0.5 mM IPTG, the protein was purified. (Section \@ref(purification-general-methods)). At each stage of purification the samples were collected to run them on the SDS-PAGE gel. Briefly, purification was done is a three-step process. The cells were precipitated and broken down by sonication to release their content. Homogenised cells were then spun down (low speed spin) to remove the unbroken cells, nucleic acids, organelles and large insoluble cellular particles, such as inclusion bodies (precipitant was collected as a whole cell sample). The supernatant from this low speed spin was then spun at 100 000g to precipitate cellular organelles. $\alpha7$ ECD in the 100 000g soluble fraction was subsequently resolved using solid phase Ni^2+^-NTA IMAC purification (Section \@ref(his)). Briefly, the soluble fraction was incubated with Ni^2+^-NTA resin for 2 hours at 4 $^\circ$C to allow binding of the expressed HIS-tagged $\alpha7$ ECD chimera to beads. The mixture was decanted into the chromatography column. The unbound proteins were collected in the flow-through, before the the beads were washed three times. The wash fractions were pooled and run as “Wash” on the gel. The resin-bound protein was then eluted by washing the column with 5 mL of 0.2 mM imidazole, to displace the HIS-tagged protein from the imobilized Nickel by competition. The eluted proteins were collected in two eluate fractions (Eluate 1 and 2).  mk11g11 committed Sep 29, 2019 176   mk11g11 committed Feb 19, 2020 177 Representative samples from each of the fractions indicated above were resolved on the SDS-PAGE gel (Figure \@ref(fig:expression-conditions-test-label)) and the presence of recombinant protein was detected by a Western blot with anti-HIS-tag antibodies (Figure \@ref(fig:expression-conditions-test-label) b). Since most of the purification features are common following expression at 18 and 37 $^\circ$C, some general comments are be made first. Then the comparison between the total purified protein will be made between the two.  mk11g11 committed Sep 29, 2019 178   mk11g11 committed Feb 19, 2020 179 The whole cell sample is the precipitate collected after the low-speed spin of the sonicated cells. As expected, a large number of proteins of various sizes were present in this sample, as visible on the Coomassie stained SDS-PAGE. There is a high intensity band of 84 kDa (size corresponding to the expression product of $\alpha7$ ECD chimera) on both the Coomassie stained SDS-PAGE and the Western blot. This suggests that following harvest, the cells were either not broken up entirely and the recombinant protein retained intra-cellularly, or the protein was present in the inclusion bodies. To account for this, the sonication steps were extended from 6 to 8 minutes in the future experiments.  mk11g11 committed Sep 29, 2019 180   mk11g11 committed Feb 19, 2020 181 "Flow through" and "Wash" were samples collected during the first two steps of IMAC, and are expected to contain proteins with no- or weak - affinity to Ni^2+^-NTA resin. Indeed, a large number of proteins of various sizes can be seen on the Coommassie stained gel, particularly in the "Flow through". Additionally, there is also Immunoreactive proteins of the expected $\alpha7$ ECD chimera protein size present in both the Flow Through and Wash, suggesting it failed to bind to the Ni^2+^-NTA resin with high affinity. This could indicate that the insufficient amount of resin was present, therefore not all HIS-tagged protein managed to bind. Alternatively, the HIS-tag was buried in the tertiary and/or quaternary structure of the protein and was thus not accessible for interactions. Therefore, the amount of resin was increased for future experiments from 0.5 mL to 1 mL used for purification of the protein from 1 L of culture. Additionally the incubation time of the incubation of the soluble fraction with resin was increased from 2 hours to overnight.  mk11g11 committed Sep 29, 2019 182   mk11g11 committed Feb 19, 2020 183 Eluate samples are expected to contain proteins with high affinity to Ni^2+^-NTA resin. However, no immunoreactive protein was detected in the eluate following expression induced at 37 $^\circ$C and 0.5 mM IPTG. In contrast, there is a band on the Western blot in the eluate collected after expression at 18 $^\circ$C and 0.2 mM IPTG, corresponding to the $\alpha7$ ECD, based on its size of 84 kDa. Thus, more protein is being successfuly purified following extended expression at lower temperature. Therefore, based on the expression and purification, more protein is being expressied at and purified following expression at 18 $^\circ$C.  mk11g11 committed Sep 29, 2019 184   mk11g11 committed Feb 19, 2020 185 (ref:expression-conditions-test) **The effects of the temperature and inducer concentration on the expression of $\alpha7$ nAChR chimera in *E. coli*.** SDS-PAGE gel (a) and corresponding Western blot (b) of samples collected during the purification of $\alpha7$ nAChR chimera following the side by side expression of the protein at two different conditions. The expression was proceeded as explained in Figure \@ref(fig:hua7-WB-label). Following expression, cells were harvested and homogenised. Protein purification proceeded as described in methods (ref). The expected size of monomeric $\alpha7$ nAChR chimera is 84 kDa.  mk11g11 committed Sep 29, 2019 186 187 188 189 190  {r expression-conditions-test-label, fig.cap = "(ref:expression-conditions-test)", fig.scap = "The effects of the temperature and inducer concentration on the expression of $\\alpha7$ nAChR chimera in \\textit{E. coli}.", fig.align='center', fig.align='center', out.height = '60%', echo=FALSE} knitr::include_graphics("fig/results5/png/expression_condition_comparison_2.png")   mk11g11 committed Feb 19, 2020 191 ### Purification of the $\alpha7$ chimera protein  mk11g11 committed Sep 29, 2019 192 193 194  The expression and purification process was repeated with the modified conditions. That is, 1. lower IPTG concentration and low temperature during the expression, 2. extended sonication time, 3. overnight equilibration of the soluble fraction with resin and 4. increased amount of resin used were.  mk11g11 committed Feb 19, 2020 195 To determine whether modified conditions have an effect on the protein purification efficiency, samples were collected during the purification procedure. Following expression of the protein at 37 $^\circ$C induced with 0.2 mM IPTG, cells harvested from 1 L of culture were sonicated and centrifuged at 16000g. The centrifugation precipitate sample was run on the SDS-PAGE (Whole cells). The supernatant was spun down again at 100000g to collected a soluble fraction (Load) which was subsequently incubated with 1 mL of Ni^2+^-NTA resin (binding capacity of up to 40 mg) overnight. The mix was decanted onto the chromatography column. This was followed by 3 washes in 10 mL of buffer and 1 mL of 0.2 mM imidazole-containing buffer to generate 5 distinct eluate fractions obtained from the Ni2+-NTA IMAC samples. Collected samples were prepared and run on the SDS-PAGE gel (Figure \@ref(fig:hua7-expression-gel-label)).  mk11g11 committed Sep 29, 2019 196 197 198  This showed a band of the expected size of 84 kDa was present in eluate samples suggesting successful purification of $\alpha7$-ECD chimera together with few other contaminants, the 50 kDa one being the most prominent, as judged by the staining intensity.  mk11g11 committed Feb 19, 2020 199 Intensely stained band corresponding to the size of the $\alpha7$-ECD chimera is also seen in the whole cell and the load fractions, suggesting that the significant proportion of the induced protein was lost during centrifugation steps.  mk11g11 committed Sep 29, 2019 200 201 202 203 204 205 206 207 208  (ref:hua7-expression-gel) **Coomassie stained SDS-PAGE gel of samples collected during purification of $\alpha7$ ECD chimera protein.** *E. coli* cells were grown in 1L of TB. Protein expression was induced with 0.2 mM IPTG and proceeded overnight. {r hua7-expression-gel-label, fig.cap="(ref:hua7-expression-gel)", out.height = '30%', fig.scap = "Coomassie stained SDS-PAGE gel of samples collected during purification of $\\alpha7$ ECD chimera protein.", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/Hua7_expression.png")   mk11g11 committed Feb 19, 2020 209 During preparation of samples for the SDS-PAGE, samples are heated resulting in protein denaturation and disintegration of individual subunits in multimeric complexes. Therefore, based on obtained SDS-PAGE results, it is impossible to state whether the expressed $\alpha7$ ECD chimera is monomeric or pentameric. It is crucial for the recombinant protein to form multimeric complexes, because the nAChR ligand binding sites are on the interface of two neighbouring subunits. A blue native PAGE gel of non-denatured and non-reduced samples was run to allows for separation of proteins based on their mass and charge. The bands were visualised using a Coomassie stain.  mk11g11 committed Sep 29, 2019 210   mk11g11 committed Feb 19, 2020 211 The collected eluate (Figure \@ref(fig:hua7-expression-gel-label)) was pooled. Two samples were prepared, one of which was boiled for ~ 5 minutes to denature any multimeric proteins into their constituent subunits. The boiled sample should contain proteins only in the monomeric form, whereas non-boiled sample should contain proteins in their native state. The boiled sample was cooled and together with the un-boiled one, run on the native gel with the aim to determine whether there are any high molecular weight bands selectively present in the un-boiled sample (Figure \@ref(fig:native-gel-label).  mk11g11 committed Sep 29, 2019 212 213 214 215 216 217 218 219 220 221  Boiled sample contains a single band of ~ 55kDa. There is also a corresponding band in the unboiled sample. A clear and strong staining present at the top of the gel produced from the un-boiled sample is also evident. This could represent a multimeric form of the $\alpha7$ ECD chimera. The size of a pentamer is 420 kDa, therefore it is possible that the electrophoresis was run not for long enough to allow the protein to enter the gel. Alternatively, the staining could represent protein aggregates.  mk11g11 committed Feb 19, 2020 222 (ref:native-gel) **Commasie stained Native Blue PAGE gel of $\alpha7$ ECD chimera eluates.** Boiled and unboiled eluate samples of eluted $\alpha7$ ECD chimera protein (Figure \@ref(fig:hua7-expression-gel-label)) were run on native non-denaturing gel alongside molecular weight markers (M) and un-boiled Bovine Serum Albumin (BSA) sample of 66.5 kDa.  mk11g11 committed Sep 29, 2019 223 224 225 226 227  {r native-gel-label, fig.cap="(ref:native-gel)", fig.scap='Commasie stained Native Blue PAGE of denatured and native elaute samples collected following the $\\alpha7$ ECD chimera purification', fig.align='center',out.width='30%',echo=FALSE} knitr::include_graphics("fig/results5/png/annotated_native_gel.png")   mk11g11 committed Feb 19, 2020 228 Size-exclusion chromatography, also known as gel filtration, is a complementary method for accessing the size of protein. The advantage of this method over PAGE is that the size estimation is much more accurate and the separation range is much greater (in this case, 10 - 600 kDa). This procedure uses a matrix filled column, containing pores of defined size. Loaded proteins can travel through the column at a defined speed, depending on their molecular weight. There is a reversal relationship between the molecular weight and the motility rate. That is, smaller molecules travel slower and are eluted later from the column, in comparison to the larger ones. Proteins are detected by spectroscopy because their amide bonds absorb at 280 nM. The result is a spectra of the absorbance against the eluted volume (Figure \@ref(fig:standard-curve-gel-filtration-label)).  mk11g11 committed Sep 29, 2019 229 230 231 232 233 234 235 236 237 238 239 240 241  To estimate the size of proteins present in a sample, the standard curve was generated using (Section \@ref(calibration)). The homogeneous solutions of proteins of known sizes were run to derive their spectra. The proteins used were: trypsin of 23.3 kDa, chicken serum albumin of 47.5 kDa, bovine serum albumin of 66.5 kDa and Dextrin which forms large aggregates. The peak position as a function of volume eluted was derived and normalised to the peak position of the void (aka protein which does not enter the column pores, but passes straight through). The the normalised peak positions for blue dextran, BSA, Chicken Serum Albumin and Trypsin were 0, 5.25, 6.90 and 7.75, respectively (Figure \@ref(fig:protein-standard-label)). These values were plotted on a logMw against normalised peak position graph and to produce an equation of a staright line of y = -0.17 x + 2.78, where y is the log molecular weight of the protein and x is the normalised peak position. This equation was then used to calculate the size of the proteins present in $\alpha7$ ECD chimera. (ref:standard-curve-gel-filtration) **Calibration curve for molecular weight determination by gel filtration.** 1 mL of standard proteins were applied to the column. Blue dextran was used to determine the void volume. {r standard-curve-gel-filtration-label, fig.cap="(ref:standard-curve-gel-filtration)", out.width= '80%', fig.scap= "Calibration curve for molecular weight determination by gel filtration." , fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/standard_curve.png")  To prepare samples, protein was expressed in 1 L of the growth medium and purified using optimized protocol. Eluate samples collected following Ni^2+^-NTA IMAC were pooled and concentrated to 500 $\mu$L. The final concentration of the sample was 3 mg / mL, as measured by spectoscropy. This sample was run on the SDS-PAGE gel (Figure \@ref(fig:gel-filtration-eluate-label) a).  mk11g11 committed Oct 06, 2019 242 (ref:filtration-gel) **Expression and purification of $\alpha7$ ECD chimera for size-exclusion chromatography**. SDS-PAGE gel of samples collected during protein expression and purification.  mk11g11 committed Feb 19, 2020 243 {r filtration-gel-label, fig.cap="(ref:filtration-gel)", fig.scap="Expression and purification of $\\alpha7$ ECD chimera for size-exclusion chromatography", fig.align='center', echo=FALSE}  mk11g11 committed Sep 29, 2019 244 245 246 247 248 249 knitr::include_graphics("fig/results5/png/160512_SDS_purification_ACR21-2GUV.png")  A protein of desired size of 84 kDa was present, as well 50 and 25 kDa bands. The sample was run through the filtration column and the peak spectra was derived (Figure \@ref(fig:gel-filtration-eluate-label) b). The highest peak was eluted at 16.20 mL. Normalised to void, this is 6.72 mL. This equates to 42.6 kDa. There are also small peaks: one eluted at 22 mL, and the other at 26.70 mL. These proteins are below 10 kDa. A small peak can be also seen at ~ 10 mL which overlaps with the void peak and may represent aggregated proteins.  mk11g11 committed Feb 19, 2020 250 (ref:gel-filtration-eluate) **Estimation of proteins sizes following $\alpha7$ chimera expression and purification.** SDS-PAGE gel (a) and gel filtration spectra of concentrated $\alpha7$ ECD chimera eluate.  mk11g11 committed Sep 29, 2019 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278  {r gel-filtration-eluate-label, fig.cap="(ref:gel-filtration-eluate)", fig.scap = "Estimation of size of proteins present following $\\alpha7$ ECD chimera expression and purification", fig.align='center', echo=FALSE} knitr::include_graphics("fig/results5/png/gel_filtration_eluate.png")  \clearpage ## Discussion  mk11g11 committed Feb 19, 2020 279 This chapter aims to determine whether *E. coli* BL21(DE3) cells are appropriate for the expression of ECD of nAChRs. This was done with a view to characterize candidate neonicotinoid binding sites. The difficulties in expression and purification of recombinant proteins, hinders their structural analysis and hence identification of molecular interactions between the target and the ligand.  mk11g11 committed Sep 29, 2019 280   mk11g11 committed Feb 19, 2020 281 There are several host systems available for the production of nAChRs and related proteins. For example, yeast cells have been successfully used to express assembled and folded mammalian $\alpha2$ [@kouvatsos2016], $\alpha1$ [@dellisanti2007] and $\alpha9$ [@zouridakis2014] LBD of nAChRs and nAChR LBD structural surrogate mollusc AChBP [@hilf2008; @hilf2009]. Functional mammalian $\alpha4\beta2$ receptors were successfully expressed and subsequently purified from both mammalian [@morales-perez2016] and insect cell lines [@kouvatsos2014]. *E. coli* is an attractive alternative due to the relative low cost of use and ease of manipulation. The successful expression of folded AChBPs [@abraham2016] and full length human $\alpha7$ [@tillman2016] was achieved in *E. coli* cells.  mk11g11 committed Sep 29, 2019 282 283 284  The suitability of *E. coli* as an expression system for nAChR ECD was tested by expressing and purifying human $\alpha7$ ECD - chimera protein.  mk11g11 committed Feb 19, 2020 285 ### Expression and purification of $\alpha7$ ECD chimera yields product of the correct size  mk11g11 committed Sep 29, 2019 286   mk11g11 committed Feb 19, 2020 287 288 The initial experiments were carried out to determine whether and under what conditions can the expression of $\alpha7$ ECD - chimera can be achieved. Two conditions were tested: rapid expression at 37 $^\circ$C and 0.5 mM IPTG and slower expression at 18 $^\circ$C and 0.2 mM IPTG. The cells expressing $\alpha7$ LBD - chimera were collected from both conditions. Pre- and post-induction samples were run on the SDS-PAGE gel and Coomassie stained to resolve and visualise proteins (Figure \@ref(fig:hua7-WB-label)). $\alpha7$ LBD - chimera was authenticated by Western blot using anti-HIS antibodies. Clear band of 84 kDa present in the induced samples, confirming successful expression. Greater intensity of the band from 18 $^\circ$C and 0.2 mM IPTG suggest this is a favourable condition for the expression of $\alpha7$ LBD - chimera. Lower temperature and IPTG concentrations were also beneficial for the expression of other $\alpha7$ LBD construct [@abraham2016]. In addition, to the band representing $\alpha7$ LBD - chimera there was also an induction of the 50 kDa protein, which is likely a proteolytic fragment with the HIS-tag, based on immunoreactivity.  mk11g11 committed Sep 29, 2019 289 290 291   mk11g11 committed Feb 19, 2020 292 Western blot with anti-HIS antibodies detected the presence of HIS-tagged proteins with immunoreactivity consistent with the expressed size of $\alpha7$ ECD - chimera. However, purification results showed that the proportion of induced protein availble for binding was disappointing. The expressed protein was lost during the purifcation procedure, some was precipitated following centrifugation of sonicated cells (Figure \@ref(fig:expression-conditions-test-label) Whole Cell sample), suggesting the formation of inclusion bodies. In addition, some expressed protein failed to bind to the nickel resin, potentially due to misfolding or aggregation. The expression of remaining contstruct was even more challenging, with a smaller proportion of the ECD - chimera purified.  mk11g11 committed Sep 29, 2019 293   mk11g11 committed Oct 06, 2019 294 ### Analysis of the quaternary structure.  mk11g11 committed Sep 29, 2019 295   mk11g11 committed Feb 19, 2020 296 To determine whether the protein was purified as a pentamer, a native-PAGE gel was run, which enables separation of folded and assembled proteins on the gel. Two samples were prepared: one containing denatured by boiling proteins and the other containing non-denatured, un-boiled proteins. A clear staining of high molecular weight proteins was observed in the un-boiled sample, but not in the boiled sample, suggesting purification of multimeric proteins. The presence of high molecular weight protein was not confirmed by gel filtration. Therefore further experiments are needed to investigate whether expressed $\alpha7$ ECD chimera forms pentameric structures. This could include binding of radio labelled ligands, such as $\alpha-bgtx$ [@barnard1971; @carbonetto1979; @clarke1985].  mk11g11 committed Sep 29, 2019 297 298 299   mk11g11 committed Feb 19, 2020 300   mk11g11 committed Sep 29, 2019 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366  In summary, this chapter validates the use of *E. coli* as a system for the expression of $\alpha7$ ECD and highlights the need for further optimisation to improve stability and purification efficiency of the recombinant protein. \newpage