Assignment Task

Introduction

Heroin is an illicit opiate that sees recreational usage worldwide and causes many harmful effects. Deaths related to heroin usage have doubled since 2012, with heroin mostly sourced from outside the UK (Home Office, 2020). Upon ingestion, heroin is rapidly hydrolysed into 6- monoacetylmorphine (MAM) and morphine (Kamendulis et al., 1996). Heroin is primarily trafficked in particulate form (O’Hagen and Carvetta, 2015), whilst its major metabolites 6-acetylmorphine and morphine are readily detected in urine samples (Cone et al., 1991). It is therefore within the interest of law enforcement agencies, particularly border control, to have efficient and accurate heroin detection systems.

Mass spectrometry currently remains the gold-standard method to identify heroin at border control (Harper, Powell and Pijl, 2017), however this can require training and is somewhat time-consuming. An alternative detection system for recognition of heroin and its metabolites based on enzymelinked bioluminescence has previously been proposed (Rathbone et al., 1996). This detection system utilises recombinant heroin esterase (HerE) originally isolated from Rhodococcus sp. strain H1 (Cameron et al., 1994) and an NADP-dependent morphine dehydrogenase (MDH) isolated from Pseudomonas putida M10 (Bruce et al., 1990). The NADPH produced upon hydrolysis of heroin to morphine and subsequently morphine to morphinone is coupled to bacterial luciferase from Vibrio.

Harvey to produce light in the presence of heroin (Holt et al., 1996). This putative biosensor therefore allows rapid detection of heroin and its metabolites with a detection limit of a single particle of heroin (Rathbone et al., 1996). This recombinant HerE can be produced in E. coli via transformation with a plasmid containing the HerE gene that is induced to express HerE following addition of isopropyl β-D-1- thiogalactopyranoside (IPTG) in a pET-22(a+) system (Rathbone et al., 1996). In order to bring this biosensor into industrial-scale usage, the production of HerE, alongside MDH, must be optimised. A range of factors are important for optimisation of recombinant protein production (Rosano and Ceccarelli., 2014).

Upon considering protein synthesis rates, incubation temperature is a key variable that ultimately governs protein yield (Schein, 1989). Higher temperatures accelerate E. coli growth and protein synthesis, but can reduce yield of soluble protein through formation of inclusion bodies (IBs): insoluble aggregates of mis-folded protein (Kane and Hartley, 1988). On the other hand, lower temperatures avoid this issue at the cost of reduced protein synthesis rates.

This study assesses the effect of induction temperature on HerE production in E. coli with the expectation that higher temperatures will facilitate higher protein yield. The aim of this work is to use this information to contribute towards optimisation of recombinant HerE production for use in the described illicit drugs biosensor (Rathbone et al., 1996).

Methodology

Plasmids used A pre-prepared pET-28a vector containing the heroin esterase gene from Rhodococcus sp. H1 (Cameron et al., 1994; Rathbone et al., 1997) was used for recombinant HerE expression. The heroin esterase gene is located downstream of a T7 promoter and possesses a Cterminal His-Tag to facilitate purification via affinity chromatography. This plasmid additionally contains a kanamycin-resistance gene as a selectable marker.

Bacterial Transformation

Bacterial transformation was performed via heat shock. 50 µL of E. coli BL21 cells were pipetted into a transformation tube on ice, and 5 µL plasmid DNA was added. Cells were placed on ice for 30 mins, followed by heat shock at 42 °C for 10 seconds. The mixture was then placed on ice for a further 5 mins, followed by addition of 950 µL room temperature SOC. The mixture was warmed at 37 °C for 60 mins, shaking at 250 rpm. 100 µL of the plasmid DNA + cells + SOC mixture was added and the resulting mixture spread onto selection plates consisting of Lb + Agar + Kanamycin (50 µg/mL) followed by incubation at 37 ⁰C overnight.

Bacterial Growth

A single colony from these plates was used to inoculate 6 x 50 mL LB media supplemented with 50 µg/mL kanamycin. Cultures were incubated overnight at 37 ⁰C with shaking at 175 rpm. The next day, 100 µL of each overnight sample was diluted 1:10 with MQ water and its absorbance at OD600 was measured using MQ water as blank. Following this, the 6 x 500 mL LB medium cultures were inoculated with X mL of each overnight culture such that X mL = (0.05 / overnight OD600) * (new culture volume, mL). These cultures were then incubated for growth at 37 ⁰C, shaking at 150 rpm. Absorbance at OD600 of each culture was measured every hour until OD600 ~ 0.6 (3.5 hours later). Cultures were then incubated at 20⁰C with shaking at 150 rpm until OD600 ~ 1.0 (1 hour post incubation). 1 mL aliquots of each culture were taken and centrifuged at 15000 rpm for 1 min, and pellet was stored at -20 ⁰C to be used in downstream SDS-page analysis.

Protein Expression

Following growth at 20 °C, IPTG was added to each culture at a final concentration of 1 mM and the cultures were left to shake at 150 rpm overnight at 20, 29 and 37 °C. Simultaneous duplicate experiments were performed. The next day, culture samples were split into falcon tubes and centrifuged at 3900 rpm for 20 mins at room temperature. Cell pellets were then stored at -20 ⁰C to await lysis. Again, 1 mL aliquots of supernatant from each treatment were taken to be centrifuged, with pellet kept for downstream SDS analysis.

Detection and Quantification

Sodium Dodecyl Sulphate Polyacrylamide Electrophoresis (SDS PAGE) was used to detect presence of HerE following purification with preprepared 10 % mini PROTEAN TGX gels (Biorad). Running buffer used was at a 1X solution Tris – SDS diluted with distilled water. 10 µL of 2X lamelli dye was added to 10 µL of sample and loaded into wells, along with lanes containing 10 µL molecular weight marker (ThermoFisher Scientific, #26616). Following SDS gels were stained with SimplyBlue™ Safestain (ThermoFisher Scientific # LC6060) and imaged with UV light via a transilluminator.

Statistical Analysis

Due to the relatively small number of samples (N = 2 for each treatment), non-parametric Kruskall-Wallis tests were used to assess significance of data (at p ≤ 0.05). All statistical analysis was performed in R studio (R Studio Team, 2020).

Effect of Induction Temperature on Her Expression

The effect of induction temperature on HerE expression was investigated by transformation of E. coli with HerE expression plasmids followed by growth at 37 °C. Induction of HerE expression with IPTG was then performed at a range of temperatures (20, 29 and 37 °C). All cultures displayed similar population size following initial growth at 37 °C (Supplementary Figure 1) and this was further controlled as explained in methodology. Figure 2A shows transformation was successful as demonstrated via multiple PCR validation experiments using primers for HerE and the T7 promoter. SDS-PAGE analysis revealed HerE to be expressed by all samples at an estimated molecular weight of ~ 39 kDa (Figure 2B - 2D). Duplicate SDS pages are shown in Supplementary Figure 2. A decline in band intensity from lane six to lane nine, along with presence of HerE in these lanes, suggests some HerE was lost during chromatography washing stages. Additionally, some HerE seems to have been lost from both samples induced at 29 °C in post-induction media supernatant (lane two), and to a lesser extent from samples included at 37 °C (Figure 2C, 2D; Supplementary Figure 2C, 2D).

Discussion

This work shows there was no significant effect of temperature on E. coli growth. However, it is widely known that the optimum temperature for E. coli growth is 37 °C (Doyle and Schoeni, 1984), which seems to be the case in this study despite lack of significance (Figure 1). Furthermore, there was no significant effect of induction temperature on recombinant HerE expression levels in E. coli. The lack of significant results are surprising given the plethora of studies showing the effect of temperature on expression of recombinant proteins (Farewell and Neidhardt, 1998; Mühlmann et al., 2017; Sadeghian-Rizi et al., 2019). Given the small number of samples used in this study, it would be fair to assume the lack of significance is largely due to small sample size offering restricted statistical power. Doubling the data gathered in R to replicate a higher sample size did generate significant differences between temperatures for both E. coli growth rate (Figure 1) and HerE [removed]Figure 2E), with higher and lower temperatures allowing highest growth rate and highest HerE expression levels, respectively. Therefore, although no significant differences were obtained in this study, these results will still be considered with caution in context of other studies.

It is well known that the rate of protein synthesis in E. coli increases proportionally with temperature, with higher temperatures allowing increased protein [removed]Farewell and Neidhardt, 1998). Additionally, lower temperatures are shown to reduce translation initiation rates by decreasing the pool of actively translating ribosomes (Farewell and Neidhardt, 1998). With this information, one would assume it would be standard practice to induce protein expression at high temperatures such as 37 °C in order to produce highest protein levels. However, high recombinant protein expression levels in E. coli coupled with production in a foreign host often leads to aggregation of proteins as IBs (Williams et al., 1982; Palmer and Wingfield, 2014; Rosano and Ceccarelli., 2014). A common strategy to avoid this is lowering temperature during protein [removed]Schein and Noteborn, 1988). This leads to lower expression rates, allowing more time for protein folding and reducing the number of hydrophobic interactions that drive aggregation (Upadhyay et al., 2012). Interestingly, addition of his-tags to recombinant proteins to facilitate purification has also been shown to drive IB formation (Li et al., 2009; Zhu et al., 2013). Here, it is proposed that His-tags may interrupt protein folding, thereby driving aggregation. To avoid this issue, it would be imperative to ensure the His-tag is inserted in a position that does not interrupt protein folding, suggesting further work could scrutinise the location of this His-tag. Additionally, the His-tag could be cleaved using endoproteases in order to reduce its effect on protein aggregation post-purification.

This would have the additional benefit of maximising protein purification via washing with endoproteases as a final chromatography step to ensure as much protein as possible is released from the chromatography column (Costa et al., 2014). Despite the aforementioned lack of significance, trends in our data indicate higher HerE concentrations were obtained at lower temperatures (Figure 2E). Although this does correlate with other studies (Sadeghian-Rizi et al., 2019), it may not indicate that more protein was expressed, rather that there was simply more soluble protein within buffer solution upon measuring. Indeed, samples incubated at higher temperatures within this study did contain insoluble fibres (Supplementary Figure 4), which could potentially be IBs. In a similar vein, heroin esterase displays reduced thermal stability at temperatures above 30 °C (Holt et al., 1996) This could explain results that higher induction temperatures seemingly lead to lower quantities of expressed protein, as some of the protein produced may have aggregated out of solution and subsequently may not have been quantified accurately.

Furthermore, the quantification results in Figure 2 represent protein from the second chromatography elution fraction, and in hindsight the first elution fraction should have been retained instead. Overall, these quantification results highlight the fact that temperature.

Analysis of SDS-PAGE results (Figures 2B – 2D) shows HerE was successfully expressed and purified from E.coli across the range of temperatures. The purified protein was estimated to be around 39 kDa, which is in line with similar reports (Cameron et al., 1994; Rathbone et al., 1996). Presence of HerE in the chromatography flow-through samples suggests some protein was lost during chromatography, which may explain the relatively small concentrations within the final eluent. This suggests the his-tagged HerE protein may not have fully bound to the column. There is a possibility this could be due to protein aggregation preventing access of His-tags to the column, though this loss in protein occurred in the low-temperature samples where aggregation may be unlikely (Schein and Noteborn, 1988). Alternatively, the His-tag may have been lost in some proteins, which could be assessed by performing Western blotting using anti-His antibody. The concentration of imidazole in the washing and flow-through buffers could have also been too high, allowing imidazole to outcompete and replace HerE binding to the column, thereby displacing it. Across both samples induced at 29 °C there was evidence of HerE present in surrounding growth media following induction (Figure 2C; Supplementary Figure 2B) and subsequently a low amount of HerE was present in the final eluent. This should not have happened as recombinant HerE is not typically secreted by E. coli. Presence of HerE in the growth media suggests some cells may have lysed and released the protein, potentially due to cell death.

The purified HerE displayed enzymatic activity across all samples and successfully metabolised phenyl acetate as a proxy for heroin (Figure 3). This activity was approximately the same for HerE expressed at each temperature. The time period required to obtain substantial metabolism of phenyl acetate was rather large compared to other assays (Holt et al., 1996) at 5.5 hours, though this could be due to the relatively small protein yield obtained in this study. Although the absorbance reading of the control sample was subtracted from all values for standardisation purposes, the fact that the control reading increased suggests experimental error. The spectrophotometer used could have been faulty, and this assay could be repeated with better equipment to obtain more accurate results. Further work should also be performed to test the activity of the purified HerE against heroin itself, as this was not performed in this study due to lack of availability.

In conclusion, optimisation of induction temperature is an important step towards industrial scaleup of HerE production for applications towards the illicit heroin biosensor described in this study (Rathbone et al., 1996). This study failed to detect a significant effect of induction temperature on HerE yield and thus the next logical step would be to perform more repeats for more statistical power. Furthermore, purified HerE should be tested for any insoluble aggregates and these should be quantified in order to truly assess protein production at higher temperatures. Lastly, optimisation of further variables should be investigated, along with similar experiments for MDH, to bring this biosensor into production.