Generation of Recombinant E.coli Protein containing Functional wzb Phosphatase and Cyan Fluorescent Protein

Expression of a functional recombinant fusion protein via the directional sub-cloning of an E.coli derived tyrosine phosphatase gene (wzb) into a pT5(6H)CFP mutant expression vector.

Abstract: Application of fluorescent fusion proteins to the field of expression and interaction proteomics as a means of dynamic imaging proteins in vivo has allowed for rapid advancements in biotechnology research. Production of such proteins first involves the insertion of a given protein-coding gene transcriptionally in-frame with a fluorophore sequence under the control of a single promoter and terminator. With reference to this experiment, successful BamHI and PstI digestion of both pHSG-WZB donor (coding for wzb tyrosine phosphatase) and pT5(6H)CFP recipient vectors allowed for ligation formation of pT5WZB(6H)CFP (coding for desired fluorescent fusion protein). Transformation into competent E.coli and plating against TE buffer and pHSG-WZB controls experimentally disputed theoretical expectations as ampicillin resistance (50725 cfu/mL) and fluorescence (93%) along with kanamycin resistance (66800 cfu/mL) and non-fluorescence were displayed, suggesting ineffective fragment ligation. Using effectively ligated transformants, screening for positive clones via plasmid extraction and PstI and XhoI digestion displayed two expected fragments; pT5 (experimental: 2339 bp) and cfp-wzb (experimental: 1198 bp). SDS-PAGE analysis of auto-induced positive clone cell lysate revealed the expression of the cfp-wzb fusion protein (experimental: 44.3 kDa). Using a hexahistidine tag, successful fusion protein purification was achieved via nickel affinity chromatography and confirmed by SDS-PAGE. Finally, para-nitrophenyl phosphate assay of tyrosine phosphatase activity allowed for determination of wzb kinetic properties such as Km (6.34 mM) and Vmax (0.0644 µmol/min/mg). Gaining an experimental competency in formation, detection and use of specific fusion proteins will ultimately allow for the appreciation of their importance in research involving protein localisation and/or trafficking.

Introduction: Generation of fluorescent recombinant fusion proteins involves the upstream or downstream insertion of a protein-coding gene of interest that is transcriptionally in-frame with a fluorophore-coding sequence not endogenously present within the culturing organism. Investigation into the functionality surrounding fluorophore-tagged fusion proteins has been a focal point of concerted research in recent decades due to its extensive experimental potential in the visualisation of gene and protein expression in vivo. Given the variable nature of this process, its applications are widespread and may involve visualising; spindle assembly and cell division in real-time mitosis, protein-protein interactions, intracellular transport of proteins within the endomembrane system and cellular compartmentalisation in plants (Ehrenberg 2008; Hanson & Köhler 2001). With reference to this particular experiment, cyan mutant of green fluorescent protein coding sequence was transcribed in-frame with wzb tyrosine phosphatase gene of interest along with a hexahistidine purification tag to ultimately generate a fluorescent and enzymatically functional recombinant.

Originally derived from Aequorea victoria, green fluorescent protein has been utilised extensively in molecular biology as a means of dynamic imaging of proteins in vivo. The induction of mutation at the 65th and/or 66th codon position have been shown to shift the excitation and emission peaks of the associated polypeptide chromophore to give rise to phenotypic variants e.g. Y66W mutation produces cyan fluroescent protein (Tsein 1998). Fluorescence of these proteins is the result of energy transfer from aequorin Ca2+-activated photoprotein following ultraviolet excitation (Barnum 2005). When mRNA translation of the fusion protein coding sequence occurs, the autocatalytic (host and exogenous substrate independent) nature of cfp folding in chromophore formation allows for tracing of the vicinity and/or transport of the resultant protein without substrate accessibility restrictions unlike bacterial and firefly luciferase (Chalfie et. al. 1994). Given its distinct and uncommon presence in natural populations, a variety of host cells whether mammalian or bacterial can be utilised in the expression and visualisation (by means of fluorescence microscopy) of cfp-tagged fusion proteins with a very high signal-to-background ratio (Spector & Goldman 2006; Hicks 2002). The level of fusion protein expression within a given organism can then be determined through the construction of fluorescence versus purified protein concentration standard curve (Ratledge & Kristiansen 2006). Although not explored, the presence of phenotypically variant fluorescent fusion proteins e.g. cyan, blue and green within a single organism allow for the simultaneous imaging of multiple protein-related intracellular events as seen in the Brainbow process (Livet et. al. 2007).

The presence of fusion protein enzymatic activity, whether attributed to the protein product from the gene of interest or from a reporter marker, allows for a highly specific detection of catalysis and hence, localisation of the recombinant (Tuan 1997). With reference to this particular experiment, tyrosine phosphatase-coding wzb acts as the gene of interest whose presence can be ultimately confirmed through assessment of enzymatic activity of the resultant fusion protein. Vincent et al. (1999) demonstrated that wzb was pivotal in tyrosine phosphatase activity toward autophosphorylated wzc in vivo. The autophosphorylation of wzc induces the down-regulation of the biosynthesis of colanic acid; a polyanionic heteropolysaccharide comprised of glucose, galactose, fucose and glucaronic acid that, when secreted, is responsible for the formation of a capsule surrounding the cell surface during exposure to stresses such as osmotic pressure and elevated temperature (Whitfield 2006; Hanna et. al. 2003). And so, the removal of the phosphate from the tyrosine group of wzc by wzb action will result in the standard production of colanic acid (Vincent et. al. 2000). As a result, detection of the fusion protein containing wzb can be achieved in vitro through the treatment of the active phosphatase with a chromogenic substrate known as para-nitrophenyl phosphate. The removal of the phosphate group from this compound by the phosphatase will eventually result in the production of para-nitrophenol; a yellow substrate whose presence can be measured and quantified using a spectrophotometer. Selection of wzb tyrosine phosphatase for in-frame transcription of the fusion protein can be attributed to; high signal-to-background ratio of the catalysed reaction using purified recombinant protein, stability of enzymatic activity, sensitivity of detection via colorimetric assay, low molecular weight resulting in minimal steric interference of translated protein and short amino acid sequence allowing for effective expression vector ligation (Balbas & Lorence 2004; Aquilina 2014, pp. 69). Finally, the steric effects of the cyan fluorophore on the enzymatic activity of wzb can also be determined through comparison to scientific literature of wzb enzymatic kinetics outlined by Vincent et al. (1999).

Incorporating ligand-binding tags allows for manipulation of the specific natural affinities between two molecules in the detection and/or purification of recombinant proteins. Ideally, the fusion protein should contain an affinity for a specific ligand that is not endogenously present in E.coli to any appreciable extent. With reference to purification of fusion protein protocol, the specific ligand can be immobilised onto a chromatography column resin and the recombinant protein isolated from a whole cell lysate on the basis of affinity chromatography. The presence of a hexahistidine amino acid sequence in the final expression vector will ultimately allow for the purification of the fluorescent and enzymatically active fusion protein due its natural affinity for nickel cations allowing for nickel-nitrilotriacetate column affinity chromatography (Hicks 2002).

In summation, this project aims to express an enzymatically functional fluorescent fusion protein via the directional sub-cloning of E.coli W3110 genome-derived wzb tyrosine phosphatase coding sequence into a pT5(6H)CFP mutant expression vector and subsequent transformation and auto-induction of E.coli hosts. Gaining competency in performing the protocols surrounding the generation of fusion proteins will ultimately allow for an appreciation of their importance in the broad field of proteomics research.

Methods: Aquilina (2014, pp. 49-70) details the specific protocol surrounding the procedure detailed below except for the following modifications; secondary centrifugation (5000 rpm for 10 minutes at 4oC) to recover an increased amount of cultured cells (and therefore recombinant protein) was not performed due to time constraints, dilution of purified protein for assessment of wzb phosphatase activity to 1 mg/mL was not achievable due to the dilute nature of samples and so, subsequent steps were performed using 0.287 mg/mL purified protein and an adjustment of volume of chromogenic substrate (100 mM pNPP) from 150 µL to 300 µL during the serial dilution of the wzb colorimetric activity assay was undertaken to elicit a greater absorbance response.

Prior to initiation of the experiment, PCR was utilised to specifically amplify E.coli W3110 genome-derived wzb coding sequence and sub-cloned into a pHSG298 recipient vector to form pHSG-WZB as per Appendix 1. BamHI and XhoI restriction digestion of the pHSG-WZB donor vector along with the mutant pT5(6H)CFP vector (Appendix 2) subsequently allowed for the ligation and formation pT5WZB(6H)CFP (Appendix 3). The success of desired fragmentation was verified by gel electrophoresis. This plasmid will ultimately be responsible for the expression of the desired recombinant protein containing a cfp-coding sequence downstream of the T5 promoter and in-frame with wzb tyrosine phosphatase. Transformation of the ligated plasmid into E.coli along with plating onto ampicillin and kanamycin-containing media allowed for selection of recombinants (to be subsequently expressed via auto-induction of E.coli and extracted) against a TE buffer and pHSG-WZB ampicillin negative controls and pHSG-WZB kanamycin positive control. Plasmid extraction of potentially positive recombinant clones and subsequent gel electrophoresis ensured that the desired ligated plasmid was present within cultured colonies (as sharing of the same phenotype and ampicillin antibiotic resistance between the desired plasmid and self-ligated original mutant recipient vector means that physical observation cannot be used as a means of differentiation). Cultures containing the desired recombinant plasmid were subsequently auto-induced to allow for copious expression of the pT5WZB(6H)CFP-translated fusion protein. Cultured cells were then disrupted (using lysis buffer containing 1 mg/mL lysozyme) to allow for recombinant protein extraction and SDS-PAGE analysis confirmed the presence of the expressed fusion protein on the basis of relative migration and resultant molecular weight determination. The hexahistidine tag present within the polypeptide structure of the fusion protein allowed for the subsequent purification and separation of the recombinant from other proteins present within the whole cell lysate using nickel-nitrilotriacetate affinity chromatography. A series of reagent treatments allowed for the removal of non-specific unbound and weakly bound proteins from the column before final elution buffer treatment allowed for isolation of proteins that are strongly bound i.e. recombinant fusion protein. This purified protein fraction was finally assayed using a chromogenic substrate known as para-nitrophenyl phosphate (pNPP) to assess the functionality of the recombinant protein’s wzb tyrosine phosphatase domain. From this, the effects of the cfp fluorescent domain upon the enzymatic capabilities of the wzb can be determined.

Results:

Confirmation of restriction digestion of pHSG-WZB donor and pT5(6H)CFP recipient:
In order to allow for the ligation of wzb (tyrosine-phosphatase coding) gene from pHSG-WZB into the pT5(6H)CFP recipient vector, successful BamHI and XhoI digestion must first be verified. With reference to Figure 1, gel electrophoresis was conducted and HyperLadder 1TM marker was used to generate a standard curve for determination of unknown protein MW based on relative migration (as per Appendix 4). Confirmation of the presence of digested pT5(6H)CFP (experimental: 3045 bp / theoretical: 3076 bp) along with the pHSG-WZB remnant (experimental: 2609 bp / theoretical: 2676 bp) and desired wzb (experimental: 442 bp / theoretical: 445 bp) fragments were achieved. Theoretical band lengths were derived using NEBcutter v2.0 (Vincze et.al. 2003).

Figure 1. Verification of the success of pHSG-WZB donor and pT5(6H)CFP recipient vector digestion via gel electrophoresis. Lane 1, HyperLadder 1TM standard MW markers used to generate a standard curve of log(MW) versus relative migration (Rf) with strong linearity (R2 = 0.9911) as per Appendix 4; Lane 2, BamHI and XhoI successful digestion of pHSG-WZB detailing remnant (experimental: 2609 bp / theoretical: 2676 bp) and desired wzb (experimental: 442 bp / theoretical: 445 bp) fragments; Lane 3, BamHI and XhoI successful digestion and linearisation of pT5(6H)CFP (experimental: 3045 bp / theoretical: 3076 bp).
Plating of resultant ligated pT5WZB(6H)CFP plasmid transformants against a series of controls to determine ligation and transformation success:
In order to verify the transformation of these ligated recombinants, the transformants were plated on a series of ampicillin and kanamycin agar media. TE buffer and pHSG-WZB Ap100µg/mL controls were plated to ensure that any growth observed was due to the antibiotic selective marker present in the ligated transformants. Both these samples resulted in observation of no viable colonies (0 cfu/mL; no fluorescent colonies). However, pHSG-WZB Km50µg/mL resulted in an immeasurable amount of colonies with no fluorescence exhibited. Observation of the ligation sample plated on Km50µg/mL displayed an unexpected result of 66800 cfu/mL non-fluorescent colonies. The varying plating volumes of ligation samples Ap100µg/mL resulted in the following; 50 µL (44880 cfu/mL; 80% fluorescent colonies), 100 µL (54480 cfu/mL; 98% fluorescent colonies) and 200 µL (52815 cfu/mL; 100% fluorescent colonies). The ligation sample average was 50725 cfu/mL.

Table 1. Summation of ligated sample transformant plating results against TE buffer and pHSG-WZB controls. All media contained 40 µM IPTG to promote induction of colony growth.
JM109 Transformation:
Volume Plated:
Media:
Colony Forming Units:
% Fluorescence
TE Buffer
100µL
Ap100µg/mL
0 cfu/mL
No colonies observed. pHSG-WZB 100µL
Ap100µg/mL
0 cfu/mL
No colonies observed. pHSG-WZB 100µL
Km50µg/mL
Too many to determine.
0%
Ligation sample
50µL
Ap100µg/mL
44880 cfu/mL
80%
Ligation sample
100µL
Ap100µg/mL
54480 cfu/mL
98%
Ligation sample
200µL
Ap100µg/mL
52815 cfu/mL
100%
Ligation sample
100µL
Km50µg/mL
66800 cfu/mL
0%

Verification of successful ligation to form pT5WZB(6H)CFP via screening for positive clones:
Prior to the expression of the fusion protein that is to be derived from pT5WZB(6H)CFP, verification of the plasmid’s presence must first be determined via gel electrophoresis. Bands were then analysed to identify either negative (pT5(6H)CFP) or positive (pT5WZB(6H)CFP) clones. With reference to Figure 2, restriction digestion of pT5WZB(6H)CFP with PstI and XhoI displays two major fragments; remnant pT5 fragment (experimental: 2339 bp / theoretical: 2331 bp) and the cfp-wzb fragment (experimental: 1198 bp / theoretical: 1190 bp). The pattern consistent with a PstI and XhoI digestion of a negative clone (self-ligation) also contains two major fragments; remnant pT5 fragment (experimental: 2339 bp / theoretical: 2331 bp) and the cfp fragment (experimental: 725 bp / theoretical: 755 bp). Non-digested forms of each plasmid displayed laddering and/or smearing and acted as qualitative controls. Unknown protein MW was derived using the marker-derived standard curve (Appendix 5).
Figure 2. Gel electrophoresis confirming the successful screening of positive pT5WZB(6H)CFP clones against unsuccessful examples resulting in negative pT5(6H)CFP clones. PstI and XhoI restriction digestion and analysis of resultant bands allowed for success to be determined. Lane 1, HyperLadder 1TM standard MW markers used to generate a standard curve of log(MW) versus relative migration (Rf) with strong linearity (R2 = 0.9911) as per Appendix 5; Lane 2, Uncut positive clone; Lane 3, Digested positive clone showing two major fragments; remnant pT5 fragment (experimental: 2339 bp / theoretical: 2331 bp) and the cfp-wzb fragment (experimental: 1198 bp / theoretical: 1190 bp); Lane 4, Uncut negative clone; Lane 5, Digested negative clone displaying the pT5 fragment along with a cfp fragment (experimental: 725 bp / theoretical: 755 bp).

Confirmation of fusion protein expression via SDS-PAGE analysis of whole cell lysate:
Before further investigation into the fusion protein can occur and to verify the success of the expression methodology, SDS-PAGE analysis of whole cell lysate was undertaken to allow for identification and semi-quantification of the recombinant protein. Protein concentration of the auto-induced whole cell lysate was found to be 0.468 mg/mL. With reference to Figure 3, Thermo-Fischer Scientific (TFS) PageRuler PlusTM MW pre-stained marker was used to construct a standard curve for determination of unknown protein molecular weight on the basis of relative migration (as per Appendix 6). Comparison between auto-induced and un-induced E.coli whole cell lysate allows for identification of the fusion protein (experimental: 44.3 kDa / theoretical: 44.6 kDa) and a semi-quantitative comparison of protein expression between samples. The relative amount of protein within the induced whole cell lysate was found to be much larger than the un-induced sample.

Figure 3. SDS-PAGE fractionation of whole cell lysates to confirm the presence of fusion protein. Lane 1, Thermo-Fischer Scientific (TFS) PageRuler PlusTM pre-stained marker used to construct a standard curve for determination of unknown protein MW as per Appendix 6; Lane 2, un-induced whole cell lysate contain prominent lysozyme band (experimental: 14.3 kDa / theoretical: 14.4 kDa); Lane 3, auto-induced whole cell lysate with prominent pT5WZB(6H)CFP fusion protein band (experimental: 44.3 kDa / theoretical: 44.6 kDa).

Evaluation of the effectiveness of fusion protein purification via Ni2+ affinity chromatography:
Following the confirmation of the cfp-wzb fusion protein within the whole cell lysate, purification must ensue prior to enzymatic assay (to ensure only wzb catalysis is responsible for chromogenic para-nitrophenyl phosphate changes). The presence of a hexahistidine peptide sequence within the final recombinant protein has a considerably high affinity for nickel cations and so, allows for affinity chromatography purification. With reference to the Figure 4, it details the fraction elution plot and a very high absorbance value was expected for the unbound protein fraction as this takes into account all non-specific proteins present within the whole cell lysate. Several binding buffer washes ensured further elution of undesired proteins from the nickel affinity column occurred until minimal absorbance at 280 nm was achieved. Subsequent washes with low imidazole concentrations induced a small initial change in absorbance which subsequently returned to baseline. Finally, addition of elution buffer resulted in a considerable change in absorbance consistent with the removal of specific proteins from the affinity chromatography column. In relation to Figure 5, SDS-PAGE analysis of nickel-nitrilotriacetate affinity chromatography fractions allowed for the determination of which fractions contain the purified desired fusion protein to be subsequently assayed. Flow through detailed the fractionation of non-specific unbound E.coli proteins whilst the wash lanes contained the non-specific yet weakly bound proteins. It was found that elution buffer fractions with high concentrations of imidazole contained the specific fusion protein (experimental: 44.3 kDa / theoretical: 44.6 kDa).

Figure 4. Absorbance readings of nickel-affinity chromatography flow through, wash and elution fractions during fusion protein purification. Spikes in absorbances are consistent with protein elution from column.

Figure 5. SDS-PAGE analysis of nickel-affinity chromatography flow through, wash and elution fractions during the purification of fusion protein. Lane 1, flow through of whole cell lysate; Lane 2, column wash three; Lane 3, column wash seven; Lane 4, elution one; Lane 5, elution two; Lane 6, elution three; Lane 7, TFS PageRuler PlusTM pre-stained marker used to construct a standard curve for determination of unknown protein MW as per Appendix 6. Wash fractions remove weakly bound non-specific proteins from the column. Elution fraction isolated bound fusion protein (experimental: 44.3 kDa / theoretical: 44.6 kDa).

Confirmation of wzb activity of fusion protein via para-nitrophenyl phosphate assay:
Using para-nitrophenyl phosphate chromogenic substrate, the enzymatic activity associated with the wzb phosphatase component of the fusion protein was determined via colorimetric absorbance assay. Subsequent comparison of derived Km and Vmax values to literature will allow for evaluation of the effects of cfp fusion on wzb enzymatic activity. Linear regression of the reciprocals of substrate concentration and reaction velocity was undertaken in construction of the Lineweaver-Burk plot. Statistical analysis was performed in the elimination of two outlier data points on the basis of the generalised statistical rule that any point further than two standard deviations (of the associated residuals) above or below the least squares line are considered to be outliers. This allowed for a line of best fit with a decent linearity, as measured by the squared correlation coefficient (R2 = 0.9735). Taking advantage of the fact that the reciprocal of Vmax is detailed by the y-intercept and that the gradient is equal to Km divided by Vmax, the following values were derived (to three significant figures); Km (6.34 mM) and Vmax (0.0644 µmol/min/mg). The associated Michaelis-Menten plot was also generated as per Appendix 7.

Figure 6. Lineweaver-Burk plot detailing enzymatic kinetics associated the wzb tyrosine phosphatase component of the final fusion protein. Para-nitrophenyl phosphate chromogenic substrate was used in the colorimetric absorbance assay to derive the plotted values. Results were as follows; Km (6.34 mM) and Vmax (0.0644 µmol/min/mg).

Discussion: Application of fluorophore-tagged fusion proteins to research settings has allowed for the development of understanding surrounding dynamic intracellular activities, especially those involved in proteostasis-related pathologies such as Huntington’s disease (Yang et. al., 2008). As a result, knowledge and experimental competency of the methodologies behind the production of specific fluroescent fusion proteins is of upmost importance in biotechnology research. Initially, the experimental procedure successfully digested pHSG-WZB donor and pT5(6H)CFP recipient expression vectors using type II (ATP-independent) BamHI and XhoI restriction endonucleases (Pingoud & Jeltsch 2001). The choice of pT5(6H)CFP as the wzb tyrosine phosphatase coding sequence recipient vector was due to; recognition of its promoter by JM109 E.coli RNA polymerase, presence of fluorescence reporter gene, hexahistidine tag for purification of translated product, high expression nature and the presence of a lactose operator for auto-induction of recombinant protein expression (Russell 2006). Directional sub-cloning of the wzb fragment into the digested pT5(6H)CFP to form pT5WZB(6H)CFP was subsequently achieved via T4 DNA ligase action. The success of this ligation process was determined via transformation of these recombinant plasmids into calcium chloride induced competent E.coli and plating on IPTG agar media (to promote colony growth and protein expression) against a TE buffer and pHSG-WZB ampicillin negative controls and a pHSG-WZB kanamycin positive control. Due to the sterile technique and lack of plasmid present in the TE negative control, E.coli plated onto agar containing ampicillin resulted in no observable colonies. The presence of a β-lactamase coding sequence allows for the induction of ampicillin resistance due its ability to catalyse the hydrolysis of ampicillin’s β-lactam ring (resulting in anti-biotic neutralisation). The lack of β-lactamase coding sequence within pHSG-WZB allows it to effectively act as a negative control. However, the ligated samples were found to be both ampicillin resistant and fluorescent meaning that transformants could contain either pT5WZB(6H)CFP or self-ligated pT5(6H)CFP. In order to establish the success of the ligation process to form the desired plasmid and hence differentiate between the two above plasmids, ligation samples were plated in the presence of kanamycin. Resistance is primarily conferred through action of neomycin phosphotransferase II (coded by neo gene) and involves the transference of a gamma-phosphate derived from ATP to the free hydroxyl group of the kanamycin aminoglycoside (Russell 2006; Misumi & Tanaka 1980). Although expected to a minor degree, the thriving of non-fluorescent transformed colonies is indicative of large-scale self-ligation of digested pHSG-WZB. Consequently, it can be safely concluded that the ligation process was extremely inefficient and prior to advancing through the experimental protocol, transformants containing the desired recombinant plasmid must be obtained. In future, dephosphorylation of the 5’end of digested DNA would perhaps assist in the ligation efficiency through prevention of self-ligation. To provide definitive evidence of ligated plasmid presence, extraction of mutant plasmids from the bacterial cultures ensued. Alkaline lysis was utilised for the differential extraction of plasmid DNA with minimal chromosomal contamination (Becker & Caldwell 1996). Reagents include; glucose (maintain osmolarity to prevent cell lysis), Tris-Cl (buffering agent to maintain re-suspension buffer at pH 8.0), EDTA (chelate divalent metals to destabilise bacterial cell membranes and inhibit DNase action), NaOH (creation of highly alkaline solution and denaturation of chromosomal DNA whilst leaving plasmid DNA intact), SDS (solubilise plasma membranes to assist in DNA release), potassium acetate and glacial acetic acid (re-establishment of pH resulting in precipitation of protein and genomic DNA). Subsequent treatment of extracted plasmids with XhoI and PstI restriction enzymes allowed for major fragments to be identified. As expected, the positive clones contained two distinct bands correlating with the pT5 remnant and cfp-wzb fragments as opposed to the negative clones containing pT5 remnant and cfp fragments. Once confirmed, the culture containing the desired recombinant plasmid underwent auto-induction as per the methods outlined by Studier (2005). The method of auto-induction is based upon the ability of certain media to induce protein expression following the attainment cell culture saturation. The ratio of glucose to lactose in the culturing E.coli has been identified as the crucial determinant in ensuring the occurrence of auto-induction. When compared to IPTG induction, auto-induction has various beneficial characteristics such as; elimination of the need to continuously monitor OD600 as an indicator of growth progression, requiring no monitoring and hence can be left overnight, running multiple culture inductions with minimal confusion and increased protein yields due to the associated increase in cellular density. Despite these range of benefits, issues associated with target protein degradation have been known to occur. Simply returning to the use of IPTG induction for that expressed protein will most likely rectify the complication (Sweeney 2014). Auto-induced cultures were incubated with ampicillin (to prevent contaminant bacterial growth) at thirty degrees overnight in order to facilitate the correct folding the cfp chromophore within the fusion protein. Following over-expression of the fusion protein by the auto-induced cultures, lysozyme cell lysis to release cellular proteins was performed. Lysozyme functions in the catalysis of 1,4-β-linkage hydrolysis between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycans and in doing so, cause the degradation of bacterial cell walls. The presence of imidazole within the lysis buffer will bind to untagged, histidine-rich contaminating proteins and so, any purification steps undertaken can be completed using fewer column washing steps. SDS-PAGE analysis of the auto-induced cell lysate allowed for the identification of the recombinant fusion protein via the measurement of relative migration to determine molecular weight using a standard curve. The lack of protein bands present in the un-induced sample could perhaps be explained by poor cell lysis and/or a very limited cellular density within the culture. Isolation and purification of the recombinant fusion protein from auto-induced samples can be achieved through taking advantage of the affinity between nickel cation binding sites and the charged histidine amino acid side chains. The affinity chromatography column is structured such that the tetradentate chelating ligand, nitrilotriacetic acid immobilises nickel cations on an agarose bead solid support whilst still allowing for histidine interactions. The initial flow through of the whole cell lysate through the nickel-nitriloacetate column will contain non-specific unbound proteins (due to no column interactions). The increase in imidazole concentration during the step elution washing procedure will allow for weakly bound non-specific proteins to be eluted. This can be attributed to the action of imidazole in competitively binding to the nickel cation binding sites and hence preventing proteins from interacting. In addition, high concentration of sodium chloride salt promotes the elution of non-specific weakly bound proteins. After several washes have been performed, a substantial increase in imidazole concentration allows for the elution of tightly bound and desired fusion proteins. High concentrations and/or poor preparation of imidazole in elution buffer has been shown to contribute to the absorbance readings of eluted fractions (Kelly, Jess & Price 2005). SDS-PAGE analysis of fractions allowed for identification of the fusion protein and the success of the affinity chromatography process. Following purification, dialysis of proteins to allow for buffer exchange was performed to ensure that the phosphatase activity assay was performed in optimal conditions. Finally, in order to assess the wzb phosphatase activity of the fusion protein, a pNPP substrate colorimetric assay was established. Phosphatase action in the cleavage of the phosphate group from the colourless pNPP to render para-nitrophenol will occur. In non-alkaline solutions, this by-product will also be colourless due to its exhibiting electronic excitation only at a wavelength of approximately 318 nm. However, the removal of a hydrogen ion to form the phenolate anion in alkaline solutions will result in a bathochromic shift in absorbance to 405 nm. And so, in the presence of an appropriate assay buffer, observation of this compound will now render a yellow by-product. If performed without experimental error, derivation of wzb kinetic values such as Km (6.34 mM) and Vmax (0.0644 µmol/min/mg) from the colorimetric assay will allow for confirmation as to whether the cfp polypeptide will negatively affect wzb activity through steric hindrance. When compared to theoretical kinetic values (Km: 1 mM and Vmax: 4.6 µmol/min/mg), it can be inferred that either the steric hindrance associated with the presence of cfp negatively impacts wzb to a major extent or the presence of systematic/modelling errors induced this result. One such inaccuracy can be attributed to the inefficacy associated with the dialysis process in exchanging buffers and creating the alkaline environment required for appropriate resolution of the chromogenic by-product. Another source of imprecision relates to the well-known degree of error associated with reciprocation of very small values when using linear regression analysis. To rectify this, kinetic values could be determined via the use of the non-linear progress-curve analysis detailed by Duggleby (1995). With relation to overall experimental procedure, an exponential increase in protein band accuracy of determination can be achieved through in-gel digestion (to liberate proteins) followed by mass spectrometric analysis (especially that of MALDI TOF/TOF allowing for derivation of peptide analysis). In summation, generation of fluorophore-tagged fusion proteins has allowed for the rapid expansion of the expression and interaction proteomics field by allowing for accurate and real-time imaging of proteins in vivo via fluorescence microscopy.
Appendices:

Appendix 1: Major restriction sites and open-reading frames of pHSG-WZB; starting plasmid with which original wzb tyrosine phosphatase coding sequence was derived (Aquilina 2014).

Appendix 2: Major restriction sites and open-reading frames of pT5(6H)GFP expression vector. Cyan mutant form was utilised in the experiment as a recipient plasmid for the insertion of wzb gene of interest (Aquilina 2014).

Appendix 3: Major restriction sites and open-reading frames of pT5WZB(6H)CFP. This recombinant plasmid is responsible for the translation of the desired fusion protein (Aquilina 2014).

Appendix 4: Standard curve for determination of the base pair length of unknown DNA fragments resulting from the BamHI and PstI digestion of both pHSG-WZB donor (coding for wzb tyrosine phosphatase) and pT5(6H)CFP recipient vectors.

Appendix 5: Standard curve for determination of the base pair length of unknown DNA fragments resulting from the screening of extracted plasmids for the presence of positive and negative clones.

Appendix 6: Standard curve for determination of kilodalton size of unknown protein bands resulting from the lysis and SDS-PAGE analysis of auto-induced E.coli cultures.

Appendix 7: Michaelis-Menten curve derived from the para-nitrophenyl phosphate assay of wzb activity of the expressed fusion protein. Plateau is consistent with a maximal reaction velocity.

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