Darren Nesbeth

Step change in the efficiency of centrifugation through cell engineering: co-expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock.

Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well‐recognized. In this current article we demonstrate co‐expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto‐hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d50 = 0.5–0.6 µm). Critically, resultant step change improvements to the clarification performance under disc‐stack centrifugation conditions are shown. The cell‐engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale‐down mimics of continuous disc‐stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc‐stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co‐expression with a recombinant protein. Biotechnol. Bioeng. 2009; 104: 134–142

Novel detection of in vivo HLA–B27 conformations correlates with ankylosing spondylitis association

OBJECTIVE The class I major histocompatibility complex (MHC) molecule HLA-B27 exhibits a strong association with the autoimmune inflammatory arthritis disorder ankylosing spondylitis (AS) and with other related spondylarthropathies. In the absence of both a defined autoimmune response and a target autoantigen(s), the propensity of HLA-B27 to misfold has been hypothesized to be a major parameter in disease pathogenesis. We undertook this study to test the hypothesis that HLA-B27 misfolding is due to exposure of cysteine residues within the heavy chain to the oxidizing environment of the endoplasmic reticulum. METHODS A rapid acidification and alkylation modification method was used to examine cysteine residue exposure and accessibility within AS-associated and non-AS-associated HLA-B27 subtypes. RESULTS This novel approach to probing in vivo class I MHC structure revealed that the HLA-B27 heavy chain adopts conformations not previously described. Furthermore, amino acid residues specific to subtypes HLA-B*2706, B*2709, and B*2704 can have an impact on these novel conformations and on cysteine residue exposure. CONCLUSION HLA-B27 can adopt novel conformations, resulting in differential accessibility of cysteine residues, which can explain the propensity to misfold. Cysteine exposure in the HLA-B27 heavy chain is also affected by residues within the 114 and 116 regions, thereby providing a potential biochemical basis for the association of HLA-B27 subtypes with AS.

Endoplasmic Reticulum Degradation–Enhancing α‐Mannosidase–like Protein 1 Targets Misfolded HLA–B27 Dimers for Endoplasmic Reticulum–Associated Degradation

HLA–B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR‐induced ER‐associated degradation (ERAD) pathway in the disposal of HLA–B27 dimeric conformers.

Complex and unexpected dynamics in simple genetic regulatory networks

One aim of synthetic biology is to construct increasingly complex genetic networks from interconnected simpler ones to address challenges in medicine and biotechnology. However, as systems increase in size and complexity, emergent properties lead to unexpected and complex dynamics due to nonlinear and nonequilibrium properties from component interactions. We focus on four different studies of biological systems which exhibit complex and unexpected dynamics. Using simple synthetic genetic networks, small and large populations of phase-coupled quorum sensing repressilators, Goodwin oscillators, and bistable switches, we review how coupled and stochastic components can result in clustering, chaos, noise-induced coherence and speed-dependent decision making. A system of repressilators exhibits oscillations, limit cycles, steady states or chaos depending on the nature and strength of the coupling mechanism. In large repressilator networks, rich dynamics can also be exhibited, such as clustering and chaos. In populations of Goodwin oscillators, noise can induce coherent oscillations. In bistable systems, the speed with which incoming external signals reach steady state can bias the network towards particular attractors. These studies showcase the range of dynamical behavior that simple synthetic genetic networks can exhibit. In addition, they demonstrate the ability of mathematical modeling to analyze nonlinearity and inhomogeneity within these systems.

Synthetic biology routes to bio-artificial intelligence

The design of synthetic gene networks (SGNs) has advanced to the extent that novel genetic circuits are now being tested for their ability to recapitulate archetypal learning behaviours first defined in the fields of machine and animal learning. Here, we discuss the biological implementation of a perceptron algorithm for linear classification of input data. An expansion of this biological design that encompasses cellular ‘teachers’ and ‘students’ is also examined. We also discuss implementation of Pavlovian associative learning using SGNs and present an example of such a scheme and in silico simulation of its performance. In addition to designed SGNs, we also consider the option to establish conditions in which a population of SGNs can evolve diversity in order to better contend with complex input data. Finally, we compare recent ethical concerns in the field of artificial intelligence (AI) and the future challenges raised by bio-artificial intelligence (BI).

IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ∼8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140.

Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab’ fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab’ fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD‐A33 Fab’, to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac. We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab’ fragment expression. The Ptic promoter strain showed a 25−30% reduction in Fab’ expression relative to the original Ptac strain. Reduced Fab’ leakage and increased viability over the course of a fed‐batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab’ fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity.

Rapid acidification and alkylation: redox analysis of the MHC class I pathway.

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.

EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

HLA–B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR‐induced ER‐associated degradation (ERAD) pathway in the disposal of HLA–B27 dimeric conformers.

Open source approaches to establishing Roseobacter clade bacteria as synthetic biology chassis for biogeoengineering

Aim. The nascent field of bio-geoengineering stands to benefit from synthetic biologists’ efforts to standardise, and in so doing democratise, biomolecular research methods. Roseobacter clade bacteria comprise 15–20% of oceanic bacterio-plankton communities, making them a prime candidate for establishment of synthetic biology chassis for bio-geoengineering activities such as bioremediation of oceanic waste plastic. Developments such as the increasing affordability of DNA synthesis and laboratory automation continue to foster the establishment of a global ‘do-it-yourself’ research community alongside the more traditional arenas of academe and industry. As a collaborative group of citizen, student and professional scientists we sought to test the following hypotheses: (i) that an incubator capable of cultivating bacterial cells can be constructed entirely from non-laboratory items, (ii) that marine bacteria from the Roseobacter clade can be established as a genetically tractable synthetic biology chassis using plasmids conforming to the BioBrickTM standard and finally, (iii) that identifying and subcloning genes from a Roseobacter clade species can readily by achieved by citizen scientists using open source cloning and bioinformatic tools. Method. We cultivated three Roseobacter species, Roseobacter denitrificans, Oceanobulbus indolifexand Dinoroseobacter shibae. For each species we measured chloramphenicol sensitivity, viability over 11 weeks of glycerol-based cryopreservation and tested the effectiveness of a series of electroporation and heat shock protocols for transformation using a variety of plasmid types. We also attempted construction of an incubator-shaker device using only publicly available components. Finally, a subgroup comprising citizen scientists designed and attempted a procedure for isolating the cold resistance anf1 gene from Oceanobulbus indolifexcells and subcloning it into a BioBrickTM formatted plasmid. Results. All species were stable over 11 weeks of glycerol cryopreservation, sensitive to 17 µg/mL chloramphenicol and resistant to transformation using the conditions and plasmids tested. An incubator-shaker device, ‘UCLHack-12’ was assembled and used to cultivate sufficient quantity of Oceanobulbus indolifexcells to enable isolation of the anf1 gene and its subcloning into a plasmid to generate the BioBrickTM BBa_K729016. Conclusion.The process of ‘de-skilling’ biomolecular techniques, particularly for relatively under-investigated organisms, is still on-going. However, our successful cell growth and DNA manipulation experiments serve to indicate the types of capabilities that are now available to citizen scientists. Science democratised in this way can make a positive contribution to the debate around the use of bio-geoengineering to address oceanic pollution or climate change.