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Glycosylation of diverse recombinant proteins in glycoengineered E. coli. (a) Western blot analysis of (from left to right) MBP-GT with or without its native signal peptide, TOP7-GT with the DsbA signal peptide, GFP-GT with the TorA signal peptide, and the murine anti-digoxin IgG 26.10 with a PelB signal peptide on the light chain and a DsbA signal peptide on the heavy chain, which was also appended with the GT. Proteins were expressed in cells carrying pACYCpgl () or pACYCpglmut (). Blots were probed with anti-His (left) or hR6P (right) antibodies. Western blot analysis of wild-type Fc (wt Fc) and Fc DQNAT was conducted with anti-human antibodies (top) and SBA (bottom). (b) Fluorescence of cells expressing spTorA-GFP or spTorA-GFP-GT in pgl or pgl mut cells as indicated. Data are the averages of results from three replicate experiments, and the standard error was less than 5%. (c) ELISA signals for plates coated with BSA-digoxin conjugate (Dig) and probed with 26.10 IgG purified from pgl or pgl mut cells. Control wells without BSA-digoxin (Dig) were incubated with 26.10 IgG purified from pgl cells.
Source publication
The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor
proteins in bacteria. We developed a recombinant N-glycan acceptor pe...
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... the Top7 protein, a de novo-designed / protein that folds into a struc- ture not observed in nature (36). We modified Top7 with an N-terminal DsbA export signal and C-terminal GT. Following expression in glycosylation-competent cells, spDsbA-Top7-GT reacted with anti-His antibodies and was clearly glycosylated based on its reactivity toward hR6P (Fig. 3a). Next we exam- ined GFPmut2, a well-folded, fluorescence-activated cell sort- ing (FACS)-optimized variant of GFP (11). GFP is fluorescent only if it is allowed to fold in the cytoplasm of Gram-negative bacteria (15). Due to this peculiar property, export via the Tat pathway is the only way to localize fluorescent GFP to the periplasm ...
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... bacteria (15). Due to this peculiar property, export via the Tat pathway is the only way to localize fluorescent GFP to the periplasm (18, 46, 55). Thus, an spTorA-GFP-GT chimera was created and expressed in the presence of the functional pgl locus. Extracts from strains containing this construct elicited a strong signal with the hR6P antiserum (Fig. 3a). Finally, we examined a full-length mammalian antibody, the murine anti- digoxin 26-10 antibody (IgG 26.10) (41). Simmons and col- leagues showed previously that expression of aglycosylated IgGs is possible in E. coli via separate expression and targeting of the IgG heavy and light chains to the periplasm (50). In a similar fashion, we ...
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... and heavy chains of IgG 26.10 to the periplasm via a DsbA and a PelB export signal, respectively (41). Additionally, we amended the heavy chain for glycosylation via a C-terminal GT acceptor sequence. Fol- lowing expression and purification of this IgG, proteins reac- tive toward hR6P were detected only in the presence of the functional pgl locus (Fig. 3a), indicating the covalent attach- ment of bacterial ...
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... constructs following their expression in glycosylation-competent cells. In the case of spTorA-GFP-GT, the fluorescence activity of GFP was unaf- fected by the addition of C. jejuni N-glycans, although the addition of the GT sequence did cause a nearly 30% decrease in spTorA-GFP fluorescence relative to that of the version expressed without the GT (Fig. 3b). Thus, we concluded that while the tag itself moderately decreased the fluorescence of GFP, this was independent of the glycosylation process. We also examined the binding activity of E. coli-expressed IgG 26.10 against its cognate antigen digoxin. The activities of IgG 26.10 proteins produced in cells carrying the active or inactive ...
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... decreased the fluorescence of GFP, this was independent of the glycosylation process. We also examined the binding activity of E. coli-expressed IgG 26.10 against its cognate antigen digoxin. The activities of IgG 26.10 proteins produced in cells carrying the active or inactive pgl gene clusters were virtually identical, as measured by ELISA (Fig. 3c), which is similar to the case for GFP, indicat- ing that the process of glycosylation did not affect the antigen binding activity of IgG ...
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... tem. To address this, we cloned a mutant of the IgG1 Fc domain with triple mutations (Q295D Y296Q S298A) that encoded the preferred DQNAT sequon at N297 for PglB- mediated glycosylation (the Fc DQNAT mutant). Following ex- pression in E. coli cells carrying the pgl genes, the Fc DQNAT mutant but not wild-type Fc was effectively N glycosylated (Fig. 3a). It is noteworthy that despite this glycosylation at N297, the bacterially glycosylated Fc DQNAT was not observed to bind the FcRI receptor, as determined by ELISA, whereas an aglyco- sylated Fc variant (Fc E382V M428I ) that was engineered to bind FcRI (28) gave a strong ELISA signal (see Fig. S3 in the supplemental material). This ...
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... not wild-type Fc was effectively N glycosylated (Fig. 3a). It is noteworthy that despite this glycosylation at N297, the bacterially glycosylated Fc DQNAT was not observed to bind the FcRI receptor, as determined by ELISA, whereas an aglyco- sylated Fc variant (Fc E382V M428I ) that was engineered to bind FcRI (28) gave a strong ELISA signal (see Fig. S3 in the supplemental material). This indicates either that the bacterial N-glycan is insufficient for creating the "open" conformation needed for Fc receptor binding (35) or that the amino acids surrounding Asn297 (i.e., Q295, Y296, and S298) are needed ...
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... of naturally occurring oligosac- charides (38). Glycan diversity could be "genetically encoded" by expression of different glycosyltransferases to control the specific glycoform, and covalent transfer onto target proteins could be achieved using PglB, which is relatively promiscuous in its choice of both oligosaccharide (16) and protein (Fig. 3) substrates. Another potential use of the GT is in the develop- ment of therapeutic glycoprotein conjugates. For example, bacterial polysaccharides conjugated to proteins have proven effective as vaccines, as evidenced by the Haemophilus influen- zae type b conjugate vaccine (56). By expanding the spectrum of recombinant protein ...
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... For pJexpress401-ScADH-CGH (Table 1), pJexpress401-IFN was used as the source of the vector sequence. The source of the desired insert, the entire ScADH-CGH expression cassette, including the proposed glycan binding sitesglyco-tags (GT; 5 repeats of the D-Q-N-A-T motif; Fisher et al. (2011)) and His tag, plasmid pUC57-ScADH-CGH was obtained from Genscript. Both plasmids, pJexpress401-IFN and pUC57-ScADH-CGH, were digested with restriction endonucleases XbaI/SalI and fragments purified by preparative electrophoresis followed by isolation of the vector and insert sections obtained, were used as input reactants for the ligation reaction. ...
... It is also possible to utilize SRP-dependent co-translational transport for the periplasmic protein DsbA (ssDsbA). Fisher et al. (2011) compared ssDsbA, ssTorA, ssPelB and the signal peptide for the Sec pathway native to the MBP protein. Although they noted a different degree of distribution of individual glycosylated forms (mono-, di-, tri-, etc.), this does not change the fact that CjPglB was able to glycosylate proteins trans-ported by co-translational SRP as well as post-translational Sec and Tat mechanisms. ...
N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli . We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.
... E. coli does not favor the formation of disulfide bonds in the cytoplasm [44], a challenging characteristic for the expression of eukaryotic proteins, since most of them depend on these bridges for their proper structure [45]. Furthermore, the ability of E. coli to glycosylate proteins is exceptionally rare, a process fundamental to the structure and function of some proteins [46,47]. Therefore, bacteria are preferentially used for the expression of proteins devoid of PTMs or in circumstances where such modifications are not crucial for protein functionality [48]. ...
The global food production system faces several challenges, including significant environmental impacts due to traditional agricultural practices. The rising demands of consumers for food products that are safe, healthy, and have animal welfare standards have led to an increased interest in alternative proteins and the development of the cellular agriculture field. Within this innovative field, precision fermentation emerges as a promising technological solution to produce proteins with reduced ecological footprints. This review provides a summary of the environmental impacts related to the current global food production, and explore how precision fermentation can contribute to address these issues. Additionally, we will report on the main animal-derived proteins produced by precision fermentation, with a particular focus on those used in the food and nutraceutical industries. The general principles of precision fermentation will be explained, including strain and bioprocess optimization. Examples of efficient recombinant protein production by bacteria and yeasts, such as milk proteins, egg-white proteins, structural and flavoring proteins, will also be addressed, along with case examples of companies producing these recombinant proteins in a commercial scale. Through these examples, we will explore how precision fermentation supports sustainable food production and holds the potential for significant innovations in the sector.
... Each acceptor protein varied in its number of potential N-glycosylation sites. IdeS and NanA-constructs contain two N-glycosylation sequons [56] located on the N-and C-terminus, whilst ExoA contains two internal N-glycosylation sites and four N-and Cterminal sites (ExoA-10tag). ...
Strep A is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydratte biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within E. coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by NMR spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.
... The protein recognition sequon for N-linked glycosylation is D/E-X-N-X-S/T, where X represents any amino acid except proline, and positive D/E and S/T amino acids are at the ±2 positions, pivotal in locating asparagine (N) as the acceptor amino acid 79 . Unlike eukaryotic glycosylation, bacterial N-linked glycosylation occurs after protein folding, therefore to be accessible to PglB engineered Glycotags of the D/E-X-N-X-S/T sequon can be added to the N-and C-termini of carrier proteins allowing enhanced glyco-modification in vaccine design 80,81 . ...
... However, such approaches may reduce overall polysaccharide attachment due to limiting the region of the chain which is activated and available for attachment 100 . This in some respects is similar to bioconjugation where protein carriers are engineered with glycotags at the N-and C-termini, which is more likely to preserve the protein's stability and structure, and therefore B and T cell epitopes 81 . Investigation of the use of bioconjugation for generation of a Strep A glycoconjugate would help lead the way to a cost-effective Strep A vaccine. ...
The Group A Carbohydrate (GAC) is a defining feature of Group A Streptococcus (Strep A) or Streptococcus pyogenes . It is a conserved and simple polysaccharide, comprising a rhamnose backbone and GlcNAc side chains, further decorated with glycerol phosphate on approximately 40% GlcNAc residues. Its conservation, surface exposure and antigenicity have made it an interesting focus on Strep A vaccine design. Glycoconjugates containing this conserved carbohydrate should be a key approach towards the successful mission to build a universal Strep A vaccine candidate. In this review, a brief introduction to GAC, the main carbohydrate component of Strep A bacteria, and a variety of published carrier proteins and conjugation technologies are discussed. Components and technologies should be chosen carefully for building affordable Strep A vaccine candidates, particularly for low- and middle-income countries (LMICs). Towards this, novel technologies are discussed, such as the prospective use of bioconjugation with PglB for rhamnose polymer conjugation and generalised modules for membrane antigens (GMMA), particularly as low-cost solutions to vaccine production. Rational design of “double-hit” conjugates encompassing species specific glycan and protein components would be beneficial and production of a conserved vaccine to target Strep A colonisation without invoking an autoimmune response would be ideal.
... It has been previously reported that recombinant YebF is secreted by laboratory strains of E. coli into the extracellular medium after first being translocated into the periplasm by the Sec-system (Zhang et al., 2006). A wide variety of proteins, including N-glycosylated protein domains, are readily secreted into the growth medium via fusion with YebF (Fisher et al., 2011;Haitjema et al., 2014). ...
... Tat can only transport fully folded proteins from the cytoplasm to the periplasm, so we hypothesized that a disulfide-containing protein that required disulfide formation to reach a native state, for example, one that was dependent on CyDisCo in our system, would be a good model protein to test the true capabilities of the Tat system. Based on its reported use as a transport mechanism for secretion to the medium (Fisher et al., 2011;Haitjema et al., 2014;Zhang et al., 2006), we initially chose a small disulfide bonded E. coli protein as a test protein-YebF. ...
High‐value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well‐studied TorA signal peptide that is Tat‐specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond‐containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level. Most studies related to Tat secretion have used the well‐studied Tat‐specific TorA signal peptide, but this signal peptide also tends to induce degradation of the protein of interest, resulting in poor yields. In this study, we show that YebF can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on the CyDisCo system for disulfide bond formation in the cytoplasm by two other Tat signal peptides: those of MdoD and AmiC.
... To enable conjugation of O-PS antigens to these carrier proteins, both were modified at their C termini with four tandem repeats of an optimized bacterial glycosylation motif, DQNAT (Chen et al., 2007), followed by a 6x-His tag to enable detection via Western blot analysis and purification by Ni-NTA chromatography. A signal peptide sequence derived from the E. coli DsbA protein was fused to the N-terminus to localize CRM 197 and PD to the periplasm in a manner that is compatible with N-linked glycosylation (Fisher et al., 2011). Each of the resulting plasmids, pTrc99A-CRM 197 4xDQNAT and pTrc99A-PD 4xDQNAT , were used to transform E. coli strain CLM24 carrying plasmid pMW07-O148 that encoded the O-PS biosynthetic enzymes and plasmid pMAF10 that encoded CjPglB (Feldman et al., 2005). ...
... CjPglB is well known for its remarkably relaxed oligosaccharide substrate specificity that allows transfer of diverse Und-PP-linked glycans including numerous different O-PS structures (Feldman et al., 2005;Wacker et al., 2006). The ability of CjPglB to site-specifically modify diverse acceptor proteins is aided by the introduction of a genetically encoded N-linked glycosylation tag that can be appended N-or C-terminally in single or multiple copies, or can be inserted at internal locations in the acceptor protein (Fisher et al., 2011). Here, the introduction of four tandemly repeated DQNAT motifs at the C-termini of CRM 197 and PD facilitated their use as acceptor protein substrates for CjPglB and significantly expanded the set of carrier proteins available for bioconjugation, which historically has focused on a narrow set of carriers that are not currently used in any licensed vaccines-most notably Pseudomonas aeruginosa exotoxin A (ExoA) (Kay et al., 2019). ...
Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler’s diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.
... Display of N-linked glycoproteins on ribosomes stands alongside a growing list of screening and selection tools that are amenable to glycoprotein engineering. Besides the glycophage display methods discussed above, several other high-throughput genetic assays for N-linked glycosylation have been described including enzyme-linked immunosorbent assay (ELISA)-based detection of periplasmic N-glycoproteins, 31 cell-surface display of N-glycans and N-glycoproteins, 5,8 and a colony replica blotting strategy called glycosylation of secreted N-linked acceptor proteins (glycoSNAP). 4,6 Collectively, these assays are enabling the creation and evaluation of an unprecedentedly large number of intact glycoproteins (>150 in one study alone 6 ) for which the structure−activity relationships associated with N-glycan installation can be systematically catalogued or technologically exploited. ...
Ribosome display is a powerful in vitro method for selection and directed evolution of proteins expressed from combinatorial libraries. However, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this gap, we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glycostructural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free N-glycoprotein synthesis. This integration enabled a first-in-kind method for ribosome stalling of target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the observation that encoding mRNAs remained stably attached to ribosomes provides evidence of a genotype-glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-glycoproteins with advantageous biological and biophysical properties.
... Native E. coli also rarely performs glycosylation, which can be indispensable for proper protein structure and function [25,26]. Even in strains modified to allow for this, glycosylation patterns differ from those in mammals [27]. In the absence of appropriate folding and modification machinery, overexpressed proteins can aggregate in intracellular inclusion bodies [28,29]. ...
This article presents a discussion of the process of precision fermentation (PF), describing the history of the space, the expected 70% growth over the next 5 years, various applications of precision fermented products, and the markets available to be disrupted by the technology. A range of prokaryotic and eukaryotic host organisms used for PF are described, with the advantages, disadvantages and applications of each. The process of setting up PF and strain engineering is described, as well as various ways that computational analysis and design techniques can be employed to assist PF engineering. The article then describes the design and implementation of a machine learning method, machine learning predictions having amplified secretion (MaLPHAS) to predict strain engineerings, which optimise the secretion of a recombinant protein. This approach showed an in silico cross‐validated R² accuracy on the training data of up to 46.6% and in an in vitro test on a Komagataella phaffii strain, identified one gene engineering out of five predicted, which was shown to double the secretion of a heterologous protein and outperform three of the best‐known edits from the literature for improving secretion in K. phaffii.
... era in glycoengineering with the potential to clone and express glycosylated proteins (and other glycostructures such as capsular polysaccharides and lipooligosaccharides) in E. coli cells. Subsequently, advances have been made in identifying the consensus sequence necessary for modification of a protein by CjPglB [9] and incorporating this short amino acid sequence, referred to as a 'Glyco tag' , into virtually any protein so that it would become a substrate for PglB-mediated glycosylation [10]. The coupling enzyme, PglB, has also been engineered for increased efficiency and broader substrate specificity, so that glycans with a galactose reducing end (the starting residue of a polymerized glycan) may be transferred in addition to the N-acetylated reducing end sugars preferred by the native C. jejuni PglB (CjPglB) [11]. ...
Background:
Glycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse.
Results:
In this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences.
Conclusions:
Amongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community.
... The discovery of the consensus sequon combined with the relaxed specificity of PglB towards different polysaccharide regions has contributed to burgeoning advancements in the field of glycoengineering. In 2011 Fisher et al., demonstrated that the addition of a sequence consensus 'glycotag' at either the N-or C-termini of a protein was sufficient for glycosylation [9]. This discovery meant that double-hit approach vaccines (where the protein and glycan are from the same pathogen) are a possibility, as any protein can become an acceptor. ...
Background
Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni.
Results
We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain.
Conclusions
We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.
