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Testing the Specificity of the Tat Pathway Proofreading Mechanism in E.coli by interfering with the Protein Folding

Table of Contents

Abstract………………………………………………………………………………………………………..1

1.  Introduction…………………………………………………………………………..2
2.  Materials and Methods………………………………………………………………9
   1.         Materials………………………………………………………………………9
   2.         Bacterial Strains and Plasmids…………………………………………….9
   3.         Growth Conditions………………………………………………………….11
   4.         SDS-PAGE…………………………………………………………………13
   5.         Transfer and Immunoblotting……………………………………………..14
   6.         Imaging………………………………………………..……………………14
3.  Results………………………………………………………………………………15
   1.         Efficient export of hGH by Tat pathway in the absence of prior disulfide bond formation……………………………………………………………..15
   2.         Export of ScFv exclusively by Tat pathway in CyDisCo cells with prior disulfide bond formation and in wild-type strain with no prior disulfide bond formation……………………………………………………………..18
   3.         Export assays of ScFv mutants with substitution of a disulfide bond residue in the VL domain. ………………………………………………..20
   4.         Export assays of ScFv mutants with substitution of a disulfide bond residue in the VH domain. ………………………………………………..22
   5.         Export assays of ScFv mutants with substitution of a disulfide bond residue in the VH, VL, and both domains. ………………………………23
   6.         Tat-exported ScFv acquire disulfide bonds in the cytoplasm…………24
   7.         essays4">Export assays of BT6 protein maquettes expressed in BL21 (DE3) strain at 37°C and 30°C. ………………………………………………….26
   8.         essays5">Export assays of BT6 protein maquettes expressed in W3110 strain at 37°C and 30°C. ……………………………………………………………28
4. Discussion……………………………………………………………………………30
5. Conclusion…………………………………………………………………………..35
6. References…………………………………………………………………………..37

Figures:

Figure 1: Structure of the ScFv molecule…………………………………………….6

Figure 2: Structure of BT6 protein maquettes……………………………………….7

Figure 3: Export of hGH by Tat pathway in MC4100 strain……….……………….16

Figure 4: Export of hGH by Tat pathway in BL21 strain………….…..……………17

Figure 5: Export of the ScFvM construct by the Tat Pathway in cells with/without CyDisCo components……………..………..…………………………………………19

Figure 6: Export of the ScFvM construct by the Tat Pathway in cells       with/without CyDisCo…………………………………………………………………………………………………19

Figure 7: Export efficiency of ScFv mutants with substitutions in the VL domain…………………………………………………………………………………..20

Figure 8: Export efficiency of ScFv C162S mutant with substitutions in the VL domain expressed in the presence/absence of CyDisCo components…….……21

Figure 9: Export efficiency of ScFv C97S mutant with substitutions in the VL domain expressed in the presence/absence of CyDisCo components………….22

Figure 10: Export efficiency of ScFv mutants with substitutions in the VH, VL, and               both domains……….………..…………………………………………………………23

Figure 11: Export efficiency of ScFv with both substitutions in the VH and VL domains…………………………………………………………………………………24

Figure 12: Cytoplasmic ScFv acquiring its disulfide bonds prior to Tat-dependent export in cells with CyDisCo components…………………………………………..25

Figure 13: ScFv acquires its disulfide bonds in the periplasm after Tat-dependent   export in cells without CyDisCo components………………………………………..26

Figure 14: Export of BT6 protein maquettes by Tat pathway in BL21 (DE3) strain at 37°C…………………………………………………………………………………..27

Figure 15: Export of BT6 protein maquettes by Tat pathway in BL21 (DE3) strain at 30°C….………………………………………………………………………………28

Figure 16: Export of BT6 protein maquettes by Tat pathway in W3110 strain at 37°C………….…………………………………………………………………………29

Figure 17: Export of BT6 protein maquettes by Tat pathway in W3110 strain at 30°C……………………………………………………………………………………29

Tables:

Table 1: The bacterial strains used in this study………………………………………………9

Table 2: The plasmids used in this study……………………………………………………….10

Table 3: The composition of the buffers used in the fractionation procedure……………………………………………………………………………………….12

Table 4: Composition of culture media and agar used in this study……………………..12

 

 Abstract

The production of therapeutic products at a lower cost with a high yield remains one of the most challenging aspects in the industrial and pharmaceutical fields. E.coli is an extensively used host organism for the production of therapeutic proteins. Protein gets produced in the cytoplasm and gets exported in an unfolded state by the Sec pathway to the periplasm where it folds. The Tat pathway has been recently discovered to export proteins in a folded state thus providing more advantage over the Sec pathway for exporting complex proteins and proteins with quick folding capability. This pathway exports folded proteins upon linking them with a Tat specific TorA signal peptide. The Tat pathway has a proofreading ability. It is not fully understood how this proofreading mechanism works and this is the subject of this study. This pathway seems to export some proteins (hGH) from the cytoplasm with the absence of prior disulfide bond formation. However, the scFv used in this study is more readily exported in the oxidized state – shown by expressing in the presence and absence of CyDisCo components and mutational studies on the bridge-forming Cysteine residues. CyDisCo components enable prior disulfide bond formation in the cytoplasm. We thus show that the Tat system preferentially exports substrates that should be less dynamic, giving us insight and better understanding of the Tat proofreading ability.

1. Introduction

The biotechnology industry started in the 1980s where it gave rise to monoclonal antibodies and recombinant insulin production[1]. The main focus of the rapidly growing industry is to bring new products to the marketplace to produce huge profits[1]. However, upon the expansion of this industry, the cost of resources and production started having a major influence and the main issue shifted to decreasing the cost of production while maintaining a high income. This was done through cost reductions of “operational efficiencies” and “process improvements” [1]. Examples of these include reduced downstream processing and achieving a higher yield with low contamination levels [2].

Recombinant proteins can be expressed in a variety of host systems including insect and mammalian cell cultures, yeast, and bacteria [3]. The bacterium Escherichia coli is used extensively for the production of therapeutic proteins on the industrial and commercial levels [4]. Mainly, because E. coli has the ability of rapid growth on cheap substrates at a high density, and it has an enormous amount of cloning vectors [5]. The simplicity of cultivation and low production cost make it a desired host organism for protein expression [6]. This host organism contributes to the production of almost 30% of the certified therapeutic proteins present in the market [7].

Most recombinant proteins are expressed in the cytoplasm of E.coli [8]. Despite countless effort of optimization, misfolding of the targeted protein still occurs, resulting in the generation of inclusion bodies [8]. An alternative approach is exporting the targeted protein to the periplasm of E.coli to obtain properly folded recombinant proteins [8]. This method has proven successful through the export of human growth hormone in a fully functional and active state [9]. Moreover, exporting proteins to the periplasm holds more advantages over intracellular production in E.coli [8]. Advantages include: N-terminal recognition of the targeted protein, simpler downstream processing, higher solubility and stability of the protein, and elevated biological activity [10]. For instance, it is easier to purify and separate the target protein from other cellular components in the periplasmic space [11].

Two transport pathways exist for translocating proteins from the cytoplasm, across the cytoplasmic membrane, and to the periplasm of E.coli [12]. The majority of cytoplasmic proteins are exported through the general secretory (Sec) pathway to the periplasm [13]. A separate pathway that functions in parallel to Sec is known as the twin-arginine translocation (Tat) pathway as it contains an Arg-Arg motif in the N-terminal signal peptide of the proteins which are exported by this pathway [13]. The Sec pathway is the main platform through which proteins get exported to the periplasm [14]. The proteins exported by Sec pathway are synthesized as “pre-proteins” and translocated to the periplasm in their unfolded state whereby they fold to their native structure [15] [16].

However, the Tat pathway exports proteins in their native state since the proteins achieve a fully folded structure in the cytoplasm before getting translocated [16] [17]. The E. coli Tat pathway consists of three subunits which are membrane integrated: TatA TatB and TatC [16]. These components act as translocation machinery for the transportation of Tat substrates across the inner cell membrane and into the periplasm [16]. The physiological role of the Tat pathway is to transport a subset of proteins that must fold before translocation such as cofactor binding proteins and those having very quick folding or fold too tightly to be transported by the Sec pathway [18][19].

Specific signal peptide sequences present at the N-terminal of the protein direct it to the Tat translocon [20]. Tat signal peptides are on average longer and more hydrophobic than Sec signal peptides [21] . Tat signal peptides are prominent by their twin-arginine motif at the N-terminus [21]. However, they share the A-x-A cleavage motif at the C-terminus, a cleavage site for signal peptidase to remove the signal peptide, with the Sec signal peptides [21]. TorA is a Tat signal peptide most commonly used in E.coli to mediate recombinant protein export solely through the Tat system [22].

The signal peptide leads the recombinant protein to the periplasm where the signal peptide gets cleaved; this periplasmic compartment enables the folding of the protein and the formation of disulfide bonds due to its oxidative state [23]. This imposes a problem on the export of proteins with disulfide bonds since these do not attain their native conformation until they reach the oxidative environment therefore are recognized as misfolded and not exported by the Tat pathway to the periplasm [24].

CyDisCo (Cytoplasmic Disulfide bond formation in E.coli) components enable disulfide bond formation in E.coli wildtype strains while still having the reducing pathways intact [25]. These cells enable the efficient production of disulfide bonded proteins in the cytoplasm of E.coli by mimicking the natural processes through the introduction of a catalyst for disulfide bond formation (Erv1 p sulfhydryl oxidase) and a catalyst for disulfide isomerization (PDI human protein  disulfide isomerase) [26]. A previous study has proved that the combination of CyDisCo components with the Tat pathway successfully and efficiently resulted in the export of disulfide bonded proteins [24].

The Tat system has a high proofreading mechanism for the proteins passing through it since it only recognizes and exports fully folded proteins [27]. Previous studies have shown that the Tat system only exports proteins with proper folding and disulfide bonded proteins as well where the bonds are formed in the oxidative environment of the cytoplasm [27]. Other studies have shown that the misfolded protein precursors do reach the Tat translocon however they fail to be translocated [28]. New studies have shown that the Tat system in E.coli mediates the transport of around 30 eukaryotic proteins upon fusing them to a Tat signal peptide where smaller proteins showed higher efficiencies of export [29]. However, further studies are required to understand how the substrates are recognized and classified by the Tat system as folded or misfolded whereby they are either accepted or rejected for export.

Furthermore, the proofreading of the Tat system is meticulous and only accepts slight changes in the structure of some proteins [30]. For instance, some disulfide bonded proteins such as the human growth hormone (hGH) and a single-chain variable fragment (scFv) can be exported by the Tat pathway in wild type cells in their reduced state [31]. These proteins are assumed to take a near-native structure keeping the misfolding to a minimum in the absence of disulfide bonds [30].

A new technology has now been employed where antibody genes are being cloned and expressed as fragments in E.coli and other hosts [32]. These fragments still have the antigen binding site intact however the size of the antibody unit is being reduced and this offers better advantages for clinical purposes than fully sized antibodies [33]. The scFv is an outcome of these genetically engineered antibodies. It is composed of two variable regions, the heavy chain (VH) and light chain (VL), joined together by a peptide linker [33]. The linker does not affect the folding ability of the domains neither the formation of a functional antigen-binding site [33]. The scFv fragment used in this study (scFvM) has two disulfide bonds: one linking cysteine-23 residue to cysteine-97 and the other linking cystein-162 to cysteine-232 (Figure 1) (Colin Robinson Lab, unpublished).

Another protein used to assess the proofreading capability of the Tat system in this study is a maquette known as BT6. This has a 4 α-helical structure which supports the ligation of bis-histidine to hemes [34]. The 4-helix structure accommodates two hemes which stand perpendicularly to each other in relation to the histidine ligands to which they bind (Figure 2). Each heme is supported by two histidine ligands [34] [35]. The ligation of the histidines to the hemes creates a fully folded protein which is recognized by the Tat system. Mutants of this BT6 maquette (BT6h2) which binds two hemes were created to alter the number of heme binding sites. The BT6h1 mutant can only bind one heme whereas the BT6h0 cannot bind any heme (Figure 2) (Colin Robinson Lab, unpublished).

Figure 2: Structure of BT6 protein maquettes [36]. A) The structure of the BT6h2 protein maquette which binds to two hemes. B) The structure of BT6h1 binding to one heme. C) The structure of BT6h0 bound to no heme.

In this study, we investigate the export of hGH by the Tat system by expressing it in pET23/ptac and pEXT22 which are high and low copy plasmids, in two different strains BL21 and MC4100. Here we try to find out which plasmid results in greater export of hGH to the periplasm.

The second aim of this study was to test the proofreading ability of the Tat system by modifying the scFvM, known to be exported through Tat, and expressing it in cells with CyDisCo components. This was done through mutational studies of the bridge-forming cysteine residues in each of the heavy and light chains of the scFv and observe which variants the Tat system will accept or reject for export. The Cysteine residues at each of the C97 or C162 domains were substituted. Then checking whether the mutated proteins could be exported to the periplasm.

The final aim of this study was to test the extent to which the Tat system is able to recognize a protein as folded based on conformational stability. The BT6 protein maquettes, able to bind differing numbers of hemes have different conformational stabilities, were tested for export through the Tat system to prove the extent of their recognition.

1. Materials and methods

1. Materials

All chemicals and reagents were acquired from Sigma Chemical Company Ltd. (Dorset, UK) unless otherwise designated.

2.2 Bacterial Strains and Plasmids

The competent cells listed in Table 1, obtained from Colin Robinson Lab, were prepared according to the following protocol [37].

The purification of plasmids was carried out using the QIAprep spin miniprep kit (Qiagen) according to the manufacturer’s instructions [38].

All the bacterial strains and plasmids used in this study are represented and detailed in Tables 1-2.

Table 1: The bacterial strains used in this study.

 

Strain	Description	Source
MC4100, AraR	AraR, FaraD139DlacU169 rpsL150 relA1  flB5301 deoC1 ptsF25 rbsR	[39]
Δtatabcde	Like MC4100 AraR; ΔtatABCDE	[39]
BL21	fhuA2 [lon] ompT gal [dcm] ΔhsdS	[40]
BL21(DE3)	F2 ompT hsdSB(rB2 mB2) gal dcm (DE3)	[24]
W3110	F- mcrA mcrB IN(rrnD-rrnE)1 lambda-	[31]

 

Table 2: The plasmids used in this study.

 

Plasmids	Description	Antibiotic	Expressed In	Source
Controls
pKRK3	pET23/ptac TorAsp+4aa-hGH-His6 KanR	Kanamycin	BL21  MC4100 Δtat	Colin Robinson Lab, unpublished
pKRK7	pEXT22 TorAsp+4aa-hGH-His6 KanR	Kanamycin	BL21  MC4100 Δtat	Colin Robinson Lab, unpublished
ScFv
pHAK13	pET23/ptac TorAsp+4aa-ScFvM-His6 + CyDisCo AmpR	Ampicillin	BL21(DE3)  Δtat	[30]
pAJ15	pET23/ptac TorAsp+4aa-ScFv(M)-His6 AmpR	Ampicillin	BL21(DE3)  Δtat	[30]
pAJ19	pET23/ptac TorA-ScFvM C162S + CyDisCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
pAJ58	pET23/ptac TorA-ScFvM C162A + CyDisCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
pAJ18	pET23/ptac TorA-ScFvM C97S + CyDisCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
pAJ34	pET23/ptac TorA-ScFvM C97S C162S “Dual” + CyDisCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
pAJ18	pET23/ptac TorA-ScFvM C97S -CyDiscCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
pAJ19	pET23/ptac TorA-ScFvM C162S -CyDiscCo AmpR	Ampicillin	BL21(DE3)	Colin Robinson Lab, unpublished
BT6
pAJ21	pEXT22 TorA-BT6 (2 heme)	Kanamycin	BL21(DE3)  W3110	Colin Robinson Lab, unpublished
pAJ25	pEXT22 TorA-BT6M (no heme)	Kanamycin	BL21(DE3)  W3110	Colin Robinson Lab, unpublished
Un-named	pEXT22 TorA-BT6 (1 heme)	Kanamycin	BL21(DE3)  W3110	Colin Robinson Lab, unpublished

 

 2.3 Growth conditions

 

The bacterial transformation was performed according to the following protocol [41]. Then, a single colony was inoculated in 5ml of LB media with appropriate antibiotics (100  μg/mL Ampicillin, 50 μg/mL Kanamycin) and grown at 37°C 220 rpm overnight. The OD₆₀₀ of each overnight culture was measured in the morning then the following formula was used: [A₆₀₀/0.05=x, 50/x= y ml].

From the overnight culture, 50 ml of LB media with 100μg/ml for Ampicillin (50μg/mL for Kanamycin) was inoculated with the relevant y ml volume in a 250ml Erlenmeyer flask to an OD₆₀₀ of 0.4-0.6 at 37°C 220 rpm. When the OD₆₀₀ gets to 0.4-0.6, cells are induced for 3 hours with 5μl of 1 mM IPTG at 30°C 220 rpm. After induction, the OD₆₀₀ was taken, where (10/A₆₀₀) ml volume was centrifuged at 4°C 3000 rpm for 10 minutes. The supernatant was discarded then the pellet was re-suspended in 500μl of buffer 1 (refer to Table 3). It was then topped up with 500μl dH₂O, 40μl of 2 mg/ml lysozyme and incubated on ice for 5 minutes after which 20μl of 1M MgSO₄ (Fisher Scientific) was added. The samples were then centrifuged at 4°C 14000 rpm for 2 minutes. 750μl of the supernatant which corresponds to the Periplasmic fraction (P) was transferred to an Eppendorf and frozen at -20°C. The pellet was re-suspended in 500μl of buffer 2. The samples were then centrifuged at 4°C 14000 rpm for 5 minutes after which the supernatant was discarded. The pellet was re-suspended in 1ml of buffer 3 and sonicated on ice at 8μm amplitude for 4-6 x 10 seconds, with 10 seconds between each sonication (Soniprep 150 plus, Sanyo Gallenkamp, UK). Samples are then transferred to centrifuge tubes and centrifuged at 4°C 70000 rpm for 30 minutes using the ultracentrifuge (TL-100 ultracentrifuge, Beckman). 750μl of the supernatant which corresponds to the Cytoplasmic fraction (C) was transferred to an Eppendorf and frozen at -20°C. The pellet, corresponding to the membrane and insoluble fraction, was re-suspended in 1 ml of buffer 3 and frozen at -20°C.

Table 3: The composition of the buffers used in the fractionation procedure.

Buffers	Composition
Buffer 1	100 mM Tris-Acetate (pH 8.2), 500 mM sucrose, and 5mM EDTA (pH 8.0)
Buffer 2	50 mM Tris-Acetate (pH 8.2), 250 mM sucrose, and 10mM MgSO4
Buffer 3	50 mM Tris-Acetate (pH 8.2) and 2.5mM EDTA (pH 8.0)

Table 4: Composition of culture media and agar used in this study

Media	Composition (g/L)
Lysogeny broth (LB)	NaCl                 10  Tryptone           10 Yeast Extract    5
LB Agar	NaCl                 10  Tryptone           10 Yeast Extract    5 Bacto Agar       10
1xPBS-T	99.3g of PBS (oxoid) dissolved in 1L of ddH2O  Then diluted with 8990ml of ddH2O and 10ml of Tween.

The media and their compositions are represented in Table 4. For preparing LBA plates with antibiotic, we dissolve 400ul of the appropriate antibiotic in 400ml of LBA (cooled to 50°C) and pour them into plates.

 

2.4 SDS-PAGE

 

15% SDS gels with 10 wells were used. The gels were placed in the XCELL Surelock gel box and filled with MOPS SDS running buffer. Beta-mercaptoethanol (5ul) was added to the 5x PGLB loading buffer (950ul). 20μl of the loading buffer was added to 80μl of each of the samples to a final volume of 100μl and placed in the heat block at 90°C for 10 minutes. For the immunoblots run on non-reducing gels, no beta-mercaptoethanol (reducing agent) was added to the loading buffer and the samples were not boiled for 10minutes. The samples were then loaded accordingly: Protein Marker(10μl), Cytoplasmic fraction(10μl), Membrane/Insoluble fraction(10μl), and Periplasmic fraction(20μl).The SDS-PAGE was run at a 200V constant voltage, 200mA current, for around 45 minutes until the dye line runs out of the gel. The gels are then placed, one in a staining box and the other in a separate box to be used for western blotting, and rinsed with distilled water for 30 seconds. The gel in the staining box was covered with Coomassie blue stain and placed on a gel rocker for 1 hour, and then de-stained twice for 1 hour.

2.5 Transfer and Immunoblotting

2 pieces of 10x10cm 3MM paper and one 10x10cm nitrocellulose membrane were soaked in transfer buffer for 5 minutes, then placed on the blotter (Pierce power blotter, Thermo Scientific) in the following order: 3MM paper, nitrocellulose membrane, gel, 3MM paper; where air bubbles were rolled out. The blotter was assembled and run for 15 minutes at 5V. After transfer, the nitrocellulose membrane was blocked overnight in a box with 5% semi-skimmed milk powder in 1xPBS-T. The membrane was washed in 1xPBS-T before incubation with the 1.09mg/ml Anti-His (C-terminal) primary antibody (invitrogen) for one hour. Washing was done based on the following protocol [42]. The membrane is washed again before incubation with the secondary antibody 1mg/ml Anti-Mouse IgG (Promega) for one hour.

2.6 Imaging

The membrane is washed again and stained with ECL substrates (enhanced chemiluminescence) (Clarity Western ECL Substrate, BioRad, UK) following the manufacturer’s instructions [43], in order to detect immuno-reactive bands. The images were taken using the ChemiDoc XRS+ (BioRad, UK) with Image Lab Software. The coomassie gels were imaged using the same machine with the use of a white filter.

 

3. Results

 

3.1 Efficient export of hGH by Tat pathway in the absence of prior disulfide bond formation

The hGH protein was used as a control, as it is known to be exported by the Tat system. The aim of this study was to show the difference between the export of hGH using high-copy and low-copy plasmids. The precursor protein was expressed in a high copy number expression vector (pET23) and a low copy number expression vector (pEXT22). The constructs used contain a C-terminal His tag to facilitate identifying the proteins, and a Tat-specific TorA signal peptide at the N-terminal to mediate export by Tat pathway.

Figure 3 illustrates the export assay of TorA-hGH in MC4100 strain whereas figure 4 illustrates the export in BL21 strain. In addition, the vectors were transformed into Δtat, used as a control, to show if export occurs solemnly through the Tat pathway. After 3 hour induction, cells were fractionated into cytoplasm, membrane, and periplasm fractions (C, M, and P) which were then immunoblotted with His-tag antibodies corresponding to the His-tag on the hGH construct. Figure 3 shows that most of the hGH in pET23 and pEXT22 was exported to the periplasm (P) and refined to its mature size (22.2kDa). Whereas, a good amount of the precursor protein is shown in the membrane fraction and a small amount in the cytoplasm which also contains a moderate amount of mature-sized hGH. However, in Δtat no protein was present neither in its mature form nor as a precursor in any of the fractions.  The pEXT22 low copy vector and the pET23 high copy vector, expressed in MC4100 strain, showed similar protein export to the periplasm. The coomassie gel of this immunoblot shows that the membrane fraction of the pET23 is very faint, indicating that some of the membrane fraction might have been lost. The sample with Δtat was not fractionated properly since the periplasmic fraction has a high protein molecular weight and the membrane has a very low protein molecular weight. The pEXT22 fractionation was right since the cytoplasm having a high protein molecular weight, the membrane having less, and the periplasm having the least.

 

Figure 3: Export of hGH by Tat pathway in MC4100 strain.

hGH was expressed in E.coli with a TorA signal peptide which is Tat specific. It was expressed in a high copy plasmid (pET23) and a low copy plasmid (pEXT22) transformed into MC4100 strain. It was also expressed in Δtat strain used as a control to show that export is solemnly through the Tat pathway. A) Following induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the hGH construct. B) Coomassie gels were also run and analyzed for the following fractions. The mature size hGH is located at 22.2 kDa whereas its precursor having the TorA intact is located at around 27 kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

Figure 4 shows good export of hGH to the periplasm in pET23 whereas pEXT22 shows less export. The pET23 contains a small amount of the mature protein in the membrane and a good amount of it in the cytoplasm. Whereas pEXT22 has both the precursor and mature sized protein in the membrane and cytoplasmic fractions with the membrane having a thick band of the precursor and a small amount of the mature sized protein. However, the cytoplasmic fraction showed high protein expression for both the precursor and the mature sized hGH. Protein expression in Δtat is exhibited by a tiny amount in the cytoplasm, none in the membrane, and no export at all to the periplasm. The coomassie gels show that the fractionation was successful for the following samples except for Δtat which showed an odd molecular weight (similar to Figure 3)., In BL21 strain, the pET23 plasmid shows better protein export of the hGH compared to pEXT22.

Comparison of protein expression between the two strains shows that hGH was highly expressed in the BL21 strain.

 

 

Figure 4: Export of hGH by Tat pathway in BL21 strain.

hGH was fused to a TorA signal peptide in E.coli which is Tat specific. It was expressed in a high copy plasmid (pET23) and a low copy plasmid (pEXT22) transformed into BL21 strain.  It was further expressed in Δtat strain to show that export is solemnly through the Tat pathway. A) Following 3 hour induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the hGH construct. B) Coomassie gels were also run and analyzed for the following fractions. The mature size hGH is located at 22.2kDa whereas its precursor having the TorA intact is located at around 27kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

 

 

 

3.2 Export of scFvM exclusively by Tat pathway in CyDisCo cells with prior disulfide bond formation and in wild-type strain with no prior disulfide bond formation

 

The aim of this study was to test whether scFvM is exported solely by the Tat pathway in the BL21 (DE3) strain and if the export of this protein requires prior disulfide bond formation. This wildtype scFv having a size of 28.1 kDa is recognized as fully folded by the Tat system and gets exported in the presence of CyDisCo components [31]. scFvM was expressed with CyDisCo (+) components which enable prior disulfide bond formation and in wild type cells lacking the CyDisCo components (-).

Figures 5 and 6 illustrate the export assay of the scFvM, which is the wild type, expressed in cells with (+)/without (-) CyDisCo components and in tat null (Δ tat) mutant strain lacking the tatABCDE genes.

Figure 5 and 6 show high export of the ScFvM to the periplasm of the E.coli strain having CyDisCo components. Meanwhile, the export of the ScFvM protein was moderately low in cells lacking CyDisCo components where disulfide bonds are unable to form in the cytoplasm prior to export. Only mature sized ScFv is present in the periplasm (28.1 kDa). Both membrane fractions of the ScFvM+ and ScFvM- have a slight amount of the ScFv precursor at around 33 kDa. The cytoplasmic fractions contain both the protein precursor and the mature sized protein in good amount. The ∆tat mutant strains transformed with ScFvM+ or ScFvM- show the precursor and mature protein in the cytoplasm but none in the membrane or periplasm indicating no export. A band appears in the membrane fraction of ScFvM+ and ScFvM+/Δtat at around 15kDa and a faint band appears in all of the corresponding membrane fractions at 10kDa.

 

 

Figure 5: Export of the ScFvM construct by the Tat Pathway in cells with/without CyDisCo components.

The ScFvM having a TorA signal peptide at the N-terminal was expressed in cells with (+) and without (-) CyDisCo components. The construct was also expressed in tat null mutant cells where the tatABCDE genes are absent, in CyDisCo (+). A) Following induction for 3hrs, the cells were fractionated and samples of the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted with His-tag antibodies corresponding to the C-terminal His-tag on the ScFvM. B) Coomassie gels were also run and analyzed for the following fractions. The precursor with the TorA signal peptide and mature size ScFvM are indicated on the right of the immunoblot. Whereas, the protein marker corresponding to the molecular mass (in kDa) is shown to the left.

 

Figure 6: Export of the ScFvM construct by the Tat Pathway in cells with/without CyDisCo.

The ScFvM having a TorA signal peptide at the N-terminal was expressed in cells with (+) and without (-) CyDisCo components. The construct was expressed in tat null mutant cells where the tatABCDE genes are absent, in CyDisCo (-). A) Following 3 hours of induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted with His-tag antibodies corresponding to the His-tag on the ScFvM. B) Coomassie gels were also run and analyzed for the following fractions. The precursor containing the TorA signal peptide and the mature size ScFvM are indicated on the right of the immunoblot. Whereas, the protein marker corresponding to the molecular mass (in kDa) is shown on the left.

3.3 Export assays of ScFv mutants with substitution of a disulfide bond residue in the VL domain.

ScFvM contains two disulfide bonds: one in the VH domain linking C23 to C97, and the other in the VL domain linking C162 to C232. The Cysteine residue at position 162 of the VL domain was substituted with Serine (S) or Alanine (A) and both expressed in cells with CyDisCo components (Figure 7). ScFvM+ shows export to the periplasm as seen in previous assays. C162S+ shows the precursor protein in the cytoplasm, a band at around 15 kDa in the membrane corresponding to degradation, and no export to the periplasm. Moreover, C162A+ shows both the precursor and the mature size protein in the cytoplasm, the precursor in the membrane, and efficient protein export to the periplasm represented by mature sized protein. Therefore, the Tat pathway identifies C162A+ as a fully folded protein and enables its export whereas C162S+ is not recognized by the Tat pathway due to being misfolded. In the Coomassie gels, the protein profiles of the C, M, and P indicate a correct fractionation of the adjacent samples.

 

Figure 7: Export efficiency of ScFv mutants with substitutions in the VL domain.

 ScFvM+ was expressed in the presence of CyDisCo components. Both C162S+ and C162A+ have the Cysteine in the C162 of the VL domain substituted by either Serine (S) or Alanine (A), and were expressed in cells with CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. Coomassie gels were also run and analyzed for the following fractions. The ScFv precursor containing the TorA signal peptide and the mature size ScFv are indicated on the right of the immunoblot. Whereas, the protein marker (in kDa) is shown on the left.

Then, the C162S was expressed in cells without CyDisCo to verify that the CyDisCo components were not causing the incorrect disulfide bond formation in the C162 residue of the VL domain (Figure 8). Both the C162S+ and C162S- show protein expression in the cytoplasm but no protein export to the periplasm. A faint band is present in the membrane fraction of the C162S-. Bands are shown in the membrane fractions of both ScFvM+ and C162S+ at 15 kDa, and at 10 kDa in all the membrane fractions.

Figure 8: Export efficiency of ScFv C162S mutant with substitutions in the VL domain expressed in the presence/absence of CyDisCo components.

C162S has the Cysteine in the C162 of the VL domain substituted by Serine (S) and was expressed in cells with (+) or without (-) CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. Coomassie gels were also run and analyzed for the following fractions. The precursor containing the TorA signal peptide and mature size ScFv are indicated on the right of the immunoblot. Whereas, the protein marker (in kDa) is shown on the left.

3.4 Export assays of ScFv mutants with substitution of a disulfide bond residue in the VH domain.

In figure 9, the Cysteine (C) in the C97 residue of the VH domain was substituted with a Serine (S) and expressed in cells with/without CyDisCo components. ScFvM+ shows export to the periplasm as seen in previous assays. Both C97S+ and C97S- show the precursor and mature sized protein in the cytoplasm and a faint band corresponding to the precursor in the membrane. C97S+ has a very faint band of mature sized protein in the periplasm, exhibiting slight protein export whereas C97S- has no export to the periplasm. Therefore, the Tat pathway identifies the C97S+ as a folded protein and enables its export to the periplasm when expressed with CyDisCo components whereas the C97S- is recognized as unfolded and no export is seen. Therefore, substitution in the C97 residue of the VH domain does not affect the recognition of the protein by the Tat system and its export when expressed in cells with CyDisCo. However, C97S when expressed in cells without CyDisCo components is not recognized by the Tat system for export.

Figure 9: Export efficiency of ScFv C97S mutant with substitutions in the VL domain expressed in the presence/absence of CyDisCo components.

C97S has the Cysteine in the C97 of the VL domain substituted by Serine (S) and expressed in cells with/without CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. Coomassie gels were also run and analyzed for the following fractions. The mature size ScFv and the ScFv precursor containing the TorA signal peptide are indicated on the right of the immunoblot. Whereas, the protein marker (in kDa) is shown on the left.

 

3.5 Export assays of ScFv mutants with substitution of a disulfide bond residue in the VH, VL, and both domains.

Immunoblots were used to show protein export in three different mutants: substitution of the Cysteine with Serine in the C162 residue of the VL domain, same substitution to the C97 residue in the VH domain, and C97S C162S+ expressing both substitutions. Figure 10 shows that export to the periplasm is only seen in C97S+. Therefore indicating that substitution of cysteine with serine in the C162 residue of the VL domain tampers with the protein folding and makes it unrecognizable by the Tat pathway for export.

 

Figure 10: Export efficiency of ScFv mutants with substitutions in the VH, VL, and both domains.

C97S has a Cysteine to Serine substitution in the C97 of the VH domain, C162S has Cysteine to Serine substitution in the C162 of the VL domain, and C97S C162S contains both substitutions; all expressed in cells with CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. Coomassie gels were also run and analyzed for the following fractions. The mature size ScFv and the ScFv precursor with TorA signal peptide are indicated on the right of the immunoblot. Whereas, the protein marker (in kDa) is shown on the left.

To verify previous findings, the C97S C162S+ was immunoblotted against ScFvM+ and ScFvM- (Figure 11). This mutant showed no protein export to the periplasm. Therefore, verifying that this mutant is recognized as unfolded and not exported by the Tat pathway.

 

 

Figure 11: Export efficiency of ScFv with both substitutions in the VH and VL domains.

ScFvM is expressed is cells with/without CyDisCo components. C97S C162S+ contains both substitutions of Cysteine with Serine in the C97 and C162 domains, expressed in cells with (+) CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. Coomassie gels were also run and analyzed for the following fractions. The mature size ScFv and the ScFv precursor containing the TorA signal peptide are indicated on the right of the immunoblot. Whereas, the protein marker (in kDa) is shown on the left.

 

 

3.6 Tat-exported ScFv acquire disulfide bonds in the cytoplasm in the presence of CyDisCo components

The protein samples were analyzed on non-reducing SDS polyacrylamide gels. Proteins with disulfide bonds usually migrate in a different way under non-reducing conditions. On non-reducing gels, the unfolding of the disulfide bonded proteins is not hindered by SDS therefore these proteins migrate faster. This was done to verify if the protein samples had acquired their disulfide bonds in the cytoplasm and were in their oxidized form upon being available for export.

Figures 11 and 12 show that the cytoplasmic and periplasmic fractions of the ScFvM+ contain the protein in the oxidized form meaning that the protein has acquired its disulfide bonds in the cytoplasm.

C162S+ and C97S C162S+ cytoplasmic fractions only show the protein in its reduced state meaning that no disulfide bonds have been formed in the cytoplasm. The cytoplasmic fraction of the ScFvM- showed the protein in its reduced state meaning that the protein didn’t acquire the disulfide bonds in the cytoplasm; however, the periplasmic fraction showed the protein in its oxidized form implying that the protein acquired its disulfide bonds in the periplasm. For C97S+, the protein is in its reduced form in the cytoplasm, however it was not detectable in the periplasm although it was exported by the Tat system as implicated before.

 

 

Figure 12: Cytoplasmic ScFv acquiring its disulfide bonds prior to Tat-dependent export in cells with CyDisCo components.

ScFvM, C97S and C97S C162S were all expressed in cells with CyDisCo components. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. The samples were run on non-reducing gels where the disulfide bonded proteins migrate faster. The polyacrylamide gel was subjected to electrophoresis in the absence of reducing agent. The motilities of the reduced and oxidized ScFv are indicated on the right of the immunoblot. Coomassie gels were also run and analyzed for the following fractions. Whereas, the protein marker (in kDa) is shown on the left.

 

 

 

 

Figure 13: ScFv acquires its disulfide bonds in the periplasm after Tat-dependent export in cells without CyDisCo components.

ScFvM+, ScFvM-, and C97S C162S+ were expressed in cells with/without CyDisCo components as indicated. After 3 hours induction, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted to reveal the proteins. The samples were run on non-reducing gels where the disulfide bonded proteins migrate faster. The polyacrylamide gel was subjected to electrophoresis in the absence of reducing agent. The motilities of the reduced and oxidized ScFv are indicated on the right of the immunoblot. Coomassie gels were also run and analyzed for the following fractions. Whereas, the protein marker (in kDa) is shown on the left.

 

 

3.7 Export assays of BT6 protein maquettes expressed in BL21 (DE3) strain at 37°C and 30°C.

We further went on to test the proofreading mechanism of the Tat system by hindering the heme binding sites in a completely synthetic protein maquette known as BT6. BT6h2 has two heme binding sites. The hemes bind to the histidines on the protein molecule and thus form a properly folded structure which is recognized by the Tat system for export. BT6h1 has one heme binding site which only enables partial folding of the protein and is recognized by the Tat system for export by not as efficiently (unpublished data). However, the BT6h0 has no heme binding sites which keeps the protein in an unfolded state and is not able to be recognized and exported by the Tat system.

In Figure 14, the BT6 protein maquettes were expressed in BL21 (DE3) strain at 37°C which is the temperature normally used for protein culture and incubation. The BT6h2 shows a good expression of the mature protein (17 kDa) in the cytoplasm and a higher expression in the periplasm which indicates that the protein was efficiently exported by the Tat system. The coomassie gels show that the fractionation was successful.

 

 

 

Figure 14: Export of BT6 protein maquettes by Tat pathway in BL21 (DE3) strain at 37°C.

BT6 protein maquettes were expressed with a TorA (Tat specific) signal peptide in E.coli. This protein has three forms: BT6h0 with no heme binding sites, BT6h1 with one heme binding site, and BT6h2 with two heme binding sites. A) Following inoculation and induction at 37°C, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the BT6. B) Coomassie gels were also run and analyzed for the following fractions. The mature size BT6 is located at 17 kDa; whereas its precursor linked to TorA is located at around 22 kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

 

Next, the BT6 protein maquettes were expressed at 30°C. Previous studies have shown that lowering the temperature improves recombinant protein yield [44]. However, in this study, no protein expression was detected in any of the fractions of the respective protein maquettes (Figure 15). Moreover, faint bands are seen in the membrane fractions at around 10 kDa.

 

 

Figure 15: Export of BT6 protein maquettes by Tat pathway in BL21 (DE3) strain at 30°C.

BT6 protein maquettes were expressed with a TorA (Tat specific) signal peptide in E.coli. This protein has three forms: BT6h0 with no heme binding sites, BT6h1 with one heme binding site, and BT6h2 with two heme binding sites. A) Following inoculation and induction at 30°C, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the BT6. B) Coomassie gels were also run and analyzed for the following fractions. The mature size BT6 is located at 17 kDa; whereas its precursor having the TorA intact is located at around 22 kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

 

3.8 Export assays of BT6 protein maquettes expressed in W3110 strain at 37°C and 30°C.

In Figure 16, the BT6 protein maquettes were expressed in W3110 strain at 37°C. The BT6h2 shows a high expression of the mature protein (17kDa) in the periplasm indicating efficient export by the Tat system. The coomassie gels show that the fractionation was successful. When expressed at 30°C, the mature sized protein is detected in the periplasm indicating efficient protein export (Figure 17).

Therefore, we can infer that the BT6 protein maquettes are better expressed in the W3110 strain as compared to the BL21 (DE3) strain.

 

Figure 16: Export of BT6 protein maquettes by Tat pathway in W3110 strain at 37°C.

BT6 protein maquettes were expressed with a TorA (Tat specific) signal peptide in E.coli. This protein has three forms: BT6h0 with no heme binding sites, BT6h1 with one heme binding site, and BT6h2 with two heme binding sites. A) Following inoculation and induction at 37°C, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the BT6. B) Coomassie gels were also run and analyzed for the following fractions. The mature size BT6 is located at 17 kDa whereas its precursor, linked to TorA, is located at around 22 kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

 

 

 

Figure 17: Export of BT6 protein maquettes by Tat pathway in W3110 strain at 30°C.

BT6 protein maquettes were expressed with a TorA (Tat specific) signal peptide in E.coli. This protein has three forms: BT6h0 with no heme binding sites, BT6h1 with one heme binding site, and BT6h2 with two heme binding sites. A) Following inoculation and induction at 30°C, the cells were fractionated and the cytoplasm, membrane, and periplasm fractions (C, M, and P) were immunoblotted His-tag antibodies corresponding to the His-tag on the BT6. B) Coomassie gels were also run and analyzed for the following fractions. The mature size BT6 is located at 17kDa whereas its precursor linked to TorA is located at around 22 kDa. The protein marker corresponding to the molecular mass (in kDa) is indicated on the left.

4. Discussion

 

The pET23/ptac has the T7 promoter replaced with a tac promoter. The tac promoter directs transcription at a higher efficiency than the parental promoters from which it is hybridized (trp and lac UV5 promoters) [45]. This promoter enables high expression of foreign genes in E.coli. The pEXT22 has a lac promoter which operates at high basal levels of expression[46]. Both of these plasmids are induced with isopropylthio-β-d-galactoside (IPTG) where the level of expression of the ptac promoter in pET23 is proportional to the concentration of IPTG upon induction [47]. Whereas the expression for pEXT22 is enhanced by two folds along induction with IPTG [46].

This study started by expressing the hGH, already proven to be exported by the Tat system with no prior disulfide bond formation, upon linking it with a TorA signal peptide [31].This protein was previously expressed in pET23/ptac vector and export was efficient[31]. In this study, the hGH was expressed in pET23/ptac high copy plasmid and in pEXT22 low copy plasmid. In the BL21 strain, expression of pET23/ptac seemed to show better export compared to the pEXT22 vector. We hypothesized that pEXT22 exhibits slower synthesis therefore allowing proper protein folding in a relaxed time frame without overwhelming the Tat system. However, the pET23/ptac seemed to attain better protein export.  Further experiments can be done to distinguish if the protein exported is in an active form by measuring its activity[24].

The tat null strain, has the tatABCDE genes responsible for the activity of the Tat system deleted [48]. No export of proteins with a TorA signal peptide are observed in this strain [48], therefore we can be certain proteins given a TorA signal peptide are exported exclusively by the Tat system (Figures 3-4).

In previous studies, an scFv is exported by the Tat pathway without prior disulfide bond formation and acquires its disulfide bonds in the periplasm[31]. In this study, ScFvM and various cysteine variants were expressed in cells alongside CyDisCo components, thus enabling disulfide bond formation in the cytoplasm prior to export to the periplasm. The results show that ScFvM is exported more efficiently in the presence of the CyDisCo components (Figures 5-6, ScFvM+ lanes). These results indicate that Tat proofreading mechanism more readily recognizes scFvM as correctly folded when in a less dynamic state i.e. when disulfide bond formation occurs prior to export.

Upon substituting the Cysteine residue position 162 in the VL domain with Serine, the disulfide bond in the VL domain is unable to form and the protein is not recognized by the Tat system for export even in the presence of CyDisCo components.

Upon substituting the same residue with Alanine at the same domain, the protein is expressed and exported efficiently to the periplasm by the Tat system, in the absence of a disulfide bond at that domain.

The difference might be in the properties exhibited by substituting amino acids. Although cysteine and serine differ by only one atom, the nucleophilic thiol of the Cysteine is much more reactive than the hydroxyl group of the Serine [49]. Cysteine plays a role as well in enhancing the stability of extracellular proteins by the formation of disulfide bonds in a non-reducing environment[49]. Both molecules are polar but the difference lies in the ability of the C162 domain to accommodate a Serine residue, more specifically the large Oxygen atom which could be distorting correct folding [50].

Upon substituting the same cysteine residue with alanine, the protein was efficiently exported by the Tat system and therefore deemed ‘’folded’’ by the proofreading mechanism. Alanine is a hydrophobic molecule, non-polar and smaller in size than Cysteine [51][52]. The VL domain was able to accommodate the C162A substitution with little effect on export efficiency. This could be due to the smaller size of alanine compared to Cysteine and its ability to fit in the inner hydrophobic region of the structure whilst still leaving room for the reduced cysteine residue [53].

Moreover, the substitution of a cysteine with a serine at position 97 in the VH domain had no major effect on the protein folding and export since the Tat system was still able to recognize the ScFv as folded and export it at a lower efficiency. This might be due to the structure and folding in the VH domain which is shown to be independent of the presence of a disulfide bond to be considered as fully folded.

The combination of both substitutions of cysteine to serine in both domains showed no export at all, verifying the requirement of a fully folded structure in the VL domain of the ScFv for its recognition by Tat.

Therefore, the folding of the protein structure in the VL domain of the ScFv is necessary for its recognition by the Tat system. However, the disulfide bond in the VH domain could be omitted and this will not affect the ScFv folding and its export by the Tat pathway.

All of these constructs were expressed in cells with CyDisCo components to test the proofreading ability of the Tat pathway in recognizing the different structural stabilities of the ScFv mutants. Amino acid substitutions of essential residues in the protein may hinder the structural properties and folding of the protein.

Disulfide-bonded proteins are not broken on an SDS-free gel due to the absence of the beta-mercaptoethanol reducing agent therefore they migrate faster. We can distinguish if the targeted protein has the disulfide bonds and is in its oxidized form or doesn’t and is in the reduced form. The ScFvM+ has acquired its disulfide bonds in the cytoplasm whereas the ScFvM- has acquired them in the periplasm. Thus, validating the function of CyDisCo components. This experiment has to be repeated for the uncertainty of the C97S+ since it was shown to be exported. However, its corresponding band is not present on the non-reducing gels for better interpretation.

For the assays of the BT6 protein maquettes, upon removing the histidine ligands, the heme-binding ability is reduced. The BT6h2 is able to bind 2 heme therefore it is folded tightly into a stable conformation which is exported efficiently by the Tat system. The BT6h1 is a more dynamic structure due to removal of histidine ligands and is able to only bind one heme. In a previous study, this protein is exported by the Tat system but not as efficiently as the BT6h2 (unpublished data). However, in this study no export could be seen in this specific protein maquette. This could be due to several reasons of which is the low competency of these plasmids since it took a couple of transformations to get colonies. Then the BT6h0 was tested for export but since it has no heme binding sites, it was very flexible and unable to attain a folded conformation. This maquette could not be exported by the Tat system in any of our experiments.

In the BL21 (DE3) strain, at 37°C, we can observe a cytoplasmic and periplasmic BT6h2 protein expression defining the efficient export of BT6h2 to the periplasm. Upon reducing the temperature to mild hypothermic condition (30°C), no protein expression could be seen for any of the sample fractions. Further trials of fractionation at this temperature are needed due to the oddness of the blot.

The W3110 strain was used in previous studies with the BT6 protein maquettes (data unpublished). In this study with W3110, we can observe that at 37°C only protein expression is seen in the periplasm for BT6h2. Therefore, BT6h2 is efficiently exported by the Tat system. Upon reducing the temperature to mild hypothermic condition (30°C), results were consistent and similar to the 37°C blot. No protein expression in BT6h1 is not really justifiable in this experiment due to its inconsistency with the previous study (unpublished).

Therefore, conformational stability of the protein is very important for its recognition by the Tat system and there is an extent to which the Tat system can accept flexible conformations.

All the bands observed at 15kDa and 10kDa correspond either to unspecific antibody binding or protein degradation as confirmed in previous studies [24].

5. Conclusion

Upon testing for the hGH, the pEX23 high copy vector seemed to confer higher protein expression with better export compared to the low copy pEXT22 vector. By using CyDisCo components, disulfide bond formation is enabled in the cytoplasm and results in more efficient export of the scFvM used in this study. In this study, minor differences the Tat system might tolerate and approve in structural changes of the proteins passing through it were identified. The effect of amino acid substitutions in heavy and light chain domains of ScFvM in the presence and absence of the CyDisCo components were studied to see how a single substitution of one amino acid can hinder export. Furthermore, how these amino acids might affect the internal conformation and structure of the protein and not allow it to fold properly and be recognized by the Tat system.

Moreover, the Tat system has been proven to accept conformational changes in the stabilities of the proteins passing through it. Taken as an example the BT6 protein maquettes where reduced protein folding capability is accompanied by a reduction in export and an unfolded protein structure is not subjected to export by the Tat pathway at all.

Further experimentation can be done to verify if the protein is being exported in an active form and to repeat the uncertainties obtained in this study. The proofreading mechanism of the Tat system seems to play a major role in the trafficking of proteins and further studies are needed to identify which features it accepts or rejects for a protein passing through it.

Acknowledgments

I express my sincere gratitude to our Professor Colin Robinson for giving me this opportunity to conduct experiments in his lab as part of the biosciences faculty in the University of Kent.

I would like to thank Alexander Jones and Daphne Mermans, alongside the other members of The Colin Robinson lab for their constant guidance and support while working on this project.

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