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.         Export assays of BT6 protein maquettes expressed in BL21 (DE3) strain at 37°C and 30°C. ………………………………………………….26
   8.         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.