In bacteria, proteins localise at specific sites to form an elaborate subcellular architecture (Rudner and Losick, 2010). Gram negative bacteria such as E. coli have four protein locations: the outer membrane (OM), the periplasm, the inner membrane (IM) or the cytoplasm (Figure 1-1) (Costerton et al., 1974). Fractionation is an experimental process used to isolate proteins from their respective sub-cellular compartments. Moreover, the activity of the protein of interest can also be tested in the different isolated fractions. The risk with this technique is to have cross-fraction contamination which would result in artefactual data. Another method is to fuse GFP to the protein of interest, allowing direct observation by microscopy (Arigoni et al., 1995). This approach is limited because fusions may influence the localisation of the protein of interest through misfolding, aggregation or the GFP could be proteolytically clipped and therefore can no longer identify the localisation of the protein of interest. Moreover, the activity of the latter cannot be evaluated in its respective fractions using this approach. Hence, the fractionation method was considered for this project with a primary focus on fraction purity.
Although published fractionation protocols are not wholly different from each other, there appears to be no standardized method to extract all compartments with due-diligence against cross- contamination and inconsistency (Balasundaram et al., 2009). Sub-cellular fractionation begins with extracting the periplasm through the outer membrane. Then the resulting cells are lysed to release the cytoplasm which is separated from the insoluble fraction which includes membranes and potentially aggregated proteins by centrifugation. Further preparation to isolate the inner membrane from the outer membrane can also be performed (Thein et al., 2010) but this is out of the scope of this work. Periplasmic extraction is the most critical step of the process since disruption of the inner membrane can result in contamination from cytoplasmic proteins. Common methods to extract the periplasm use either an EDTA-lysozyme strategy (Thein et al., 2010, Pierce et al., 1997) or cold osmotic shock (Kang et al., 2012, Manoil and Beckwith, 1986), or a combination of both
Chapter 4 - Fractionation
60 (Alanen et al., 2015) (Figure 4-1). In the former method, EDTA is used to destabilise the outer membrane (Clifton et al., 2015), allowing the lysozyme to enter the periplasm and hydrolyse the peptidoglycan cell wall. This process subsequently releases the periplasm and leaves the cells as spheroplasts (Pierce et al., 1997). The second method generates an osmotic shock induced by successively adding a hypertonic solution to weaken the outer membrane, followed by a hypotonic solution to partially disrupt the outer membrane without compromising the integrity of the inner membrane (Kang et al., 2012).
Figure 4-1: Diagram of the action mechanisms of the main periplasmic extraction procedures
On the left, the EDTA-lysozyme strategy aims to destabilise the outer membrane (OM) by chelating the bivalent cations such as Mg2+ and Ca2+. The latter are necessary for the stability of the outer membrane and act as
cofactors for enzymes involved in cell wall building. Once the outer membrane is fragilized, lysozyme can penetrate inside the periplasm and degrade the peptidoglycan. The periplasm is hence released while the inner membrane (IM) remains intact. On the right, the cold osmotic shock partially disrupts the outer membrane by the successive cell resuspension in hypertonic and hypotonic solutions.
The second and third steps in the fractionation process result in the recovery of the cytoplasmic and insoluble fractions simultaneously. This primarily involves cell lysis to disrupt the inner membrane and subsequent centrifugation to separate the cytoplasmic and insoluble fractions. The risks are poor
Chapter 4 - Fractionation
61 soluble protein recovery due to incomplete lysis or the use of harsh conditions resulting in loss of soluble protein through aggregation which in turn contaminates the insoluble fraction. The most common cell-lysis techniques used in small scale are freeze/thaw cycling and ultrasonication (Balasundaram et al., 2009, Thein et al., 2010). Freeze/thaw cycling causes the formation of ice crystals ultimately leading to cell disruption. However, loss of enzymatic activity have been reported (Lambert et al., 1983) as well as low yields (Stanbury and Whitaker, 1984). Ultrasonication uses the cavitation principle to disrupt the membranes by ultrasonic vibration creating local disruption of pressure in the form of cavitation bubbles which by collapsing release mechanical energy disintegrating the membranes (Harrison, 1991). The risks from this technique are the heat produced by the absorption of the acoustic energy which might denature proteins and the ionisation which was reported to inactivate enzymes (Lilly and Dunnill, 1969). Moreover, freeze/thaw cycling offers the advantage of treating several samples simultaneously whereas ultrasonication can treat only one sample at a time. Following cell lysis, the samples are centrifuged to isolate the insoluble debris. The pellet contains the membrane fragments and, when present in the cell, protein inclusion bodies. To the best of our knowledge, no single study clearly describes in detail, a robust method to obtain uncontaminated soluble bacterial fractions. For instance, (Tao, 2008) and Thein et al. (2010) compared periplasmic fractions obtained by EDTA/lysozyme and cold osmotic shock and demonstrated cross-contamination in each case. Many publications show no due-diligence to fraction purity (Pechsrichuang et al., 2016, Alanen et al., 2015, Pierce et al., 1997, Kang et al., 2012, Matos et al., 2014). Some do utilize controls in the form of sub-compartment specific host proteins as purity markers, but these are incomplete. For instance, Zhang et al. (2017) merely controlled the fraction purity in only one of their experiments by Western-blot using an anti-β-lactamase antibody. β-lactamase is an E. coli natural periplasmic protein which, in this case, was found contaminating the cytoplasmic and outer membrane fractions. In another example, Fisher et al. (2008) used an anti- GroEL antibody for the same purpose. GroEL being a well-known cytoplasmic protein, was used to assess the purity of the periplasmic fractions by the lack of detectable GroEL proteins but in only one of their experiments.
Chapter 4 - Fractionation
62 Figure 4-2: Diagram of the action mechanisms of the main cell lysis procedures
On the left, the freeze/thaw cycling strategy leads to creation of ice crystals which disrupt the cells. On the right, the sonication technique applies ultrasonication vibration to the cells creating cavitation bubbles. Their collapse releases mechanical energy disintegrating the membranes. OM: outer membrane, IM: inner membrane.