Studies of recombinant protein expression :targeting signals, 3'untranslated regions and trans-acting factors

PhD ThesisAt present mammalian cell factories are being employed for recombinant protein production. However, the yields of proteins produced from such systems are often poor. This thesis describes experiments to study the effects of altering targeting signals (signal peptide) and 3’ untranslated regions (3’UTR) in an expression vector on protein expression. A variety of gene constructs containing Gaussia princeps luciferase as reporter were created using a seamless cloning method. In these constructs a variety of signal peptides, some with altered hydrophobicity, were combined with the Gaussia luciferase coding region and either the native Gaussia luciferase or human albumin 3’UTR. These were then transfected into CHO AA8 Tet-Off cells to measure how modification of the signal peptide/3’UTR affects protein expression. The results indicate that the Albumin 3’UTR, in conjunction with an appropriate signal peptide, boosts protein production by approximately 3 fold compared to the native Gaussia luciferase 3’UTR. Deletion analysis of the Albumin 3’UTR showed that deletion of regions 1-50, 1-100, 1-150, 101-150 significantly reduces protein production compared with deletion of regions 51-100, 51-150 and 1-50&101-150. Interestingly, mRNA abundance levels were significantly decreased for constructs containing deletions in regions 1-50, 1-150 and 1-50&101-150. UV Cross linking and electrophoretic mobility gel shift competition assays showed strong competition by RNA transcripts from the deletion construct 1-50, which was then used as bait for isolating bound protein/s from a CHO cell extract. Three proteins, including CUG-BP1 an RNA-binding protein involved in mRNA stability and translation were identified by mass spectrophotometry analysis. Knock down of CUG-BP1 expression using siRNA, led to impairment of complex formation between CHO cell protein extract and Albumin 3’UTR RNA transcripts, and in addition it led to an increase in the reporter activity and mRNA expression level in cells expressing the reporter gene with the full length Albumin 3’UTR and deletion variant 51-100. It is hypothesised that the differences in mRNA expression levels and secreted luciferase activity were due to CUG-BP1 binding to the Albumin 3’UTR. Further work is needed to explore the effects of CUG-BP1 on mRNA translation and stability.BBSRC, UniTargetingResearch AS,Berge

Allosteric modulation of zinc speciation by fatty acids

Background: Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another. Scope of review: Here we review the Zn2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn2 +, its effect upon cellular Zn2 + uptake and its importance in the diagnosis of myocardial ischemia are considered. Major conclusions: We previously identified the major binding site for Zn2 + on albumin. Furthermore, we revealed that Zn2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn2 +-binding to albumin in blood plasma. General significance: Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn2 + to albumin is important for the control of circulatory/cellular Zn2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin

Investigating native metal ion binding sites in mammalian histidine-rich glycoprotein

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Accepted Author Manuscript version arising. They acknowledge support by the Wellcome Trust (204821/Z/16/Z), the British Heart Foundation (PG/15/9/31270 and FS/15/42/31556), and the Leverhulme Trust (RPG-2018–397). J.L.W. acknowledges support by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1).Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion, and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterization. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterization of the ZnII binding sites of HRG is a challenge; their number and composition as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To the best of our knowledge, this is the first time that this suite of EPR techniques has been applied to count and characterize endogenous metal ion binding sites in a native mammalian protein of unknown structure.Publisher PDFPeer reviewe

Albumin-mediated extracellular zinc speciation drives cellular zinc uptake

This work was financially supported by the Leverhulme Trust (RPG-2017-214) and BBSRC (BB/J006467/1 and BB/V014684/1). We thank Prof. Andrew Riches (University of St. Andrews) for provision of materials, and Dr. Elizabeth Bolitho (University of Warwick) for assistance with cell culture experiments.The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux.Publisher PDFPeer reviewe

Investigating Native Metal Ion Binding Sites in Mammalian Histidine-Rich Glycoprotein

Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterisation. Binding of divalent metal ions, particularly zinc(II), to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterisation of the zinc(II) binding sites of HRG is a challenge; their number and composition, as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To our best knowledge this is the first time this suite of EPR techniques has been applied to count and characterise endogenous metal ion binding sites in a native mammalian protein of unknown structure

Crosstalk between zinc and fatty acids in plasma

This work was supported by the Leverhulme Trust (grant ref. RPG-2017-214), BBSRC (grant ref. BB/J006467/1) and the British Heart Foundation (grant refs. PG/15/9/31270 and FS/15/42/31556).In mammalian blood plasma, serum albumin acts as a transport protein for free fatty acids, other lipids and hydrophobic molecules including neurodegenerative peptides, and essential metal ions such as zinc to allow their systemic distribution. Importantly, binding of these chemically extremely diverse entities is not independent, but linked allosterically. One particularly intriguing allosteric link exists between free fatty acid and zinc binding. Albumin thus mediates crosstalk between energy status/metabolism and organismal zinc handling. In recognition of the fact that even small changes in extracellular zinc concentration and speciation modulate the function of many cell types, the albumin-mediated impact of free fatty acid concentration on zinc distribution may be significant for both normal physiological processes including energy metabolism, insulin activity, heparin neutralisation, blood coagulation, and zinc signalling, and a range of disease states, including metabolic syndrome, cardiovascular disease, myocardial ischemia, diabetes, and thrombosis.Publisher PDFPeer reviewe

Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins

The work described here was supported by NIH grants 1R01GM117325-01, 5U54GM094662-05 and R01GM053163, BBSRC grant BB/J006467/1 and British Heart Foundation grant PG/15/9/31270.Zinc is an essential nutrient in the body; it is required for the catalytic activity of many hundreds of human enzymes and virtually all biological processes, therefore its homeostasis and trafficking is of crucial interest. Serum albumin is the major carrier of Zn2+ in the blood and is required for its systemic distribution. Here we present the first crystal structures of human serum albumin (HSA) and equine serum albumin (ESA) in complex with Zn2+. The structures allow unambiguous identification of the major zinc binding site on these two albumins, as well as several further, weaker zinc binding sites. The major site in both HSA and ESA has tetrahedral geometry and comprises three protein ligands from the sidechains of His67, His247 and Asp249 and a water molecule. Isothermal titration calorimetric studies of a HSA H67A mutant confirm this to be the highest affinity Zn2+ site. Furthermore, analysis of Zn2+ binding to HSA and ESA proved the presence of secondary sites with 20-50-fold weaker affinities, which may become of importance under particular physiological conditions. Both calorimetry and crystallography suggest that ESA possesses an additional site compared to HSA, involving Glu153, His157 and His288. The His157 residue is replaced by Phe in HSA, incapable of metal coordination. Collectively, these findings are critical to our understanding of the role serum albumin plays in circulatory Zn2+ handling and cellular delivery.Publisher PDFPeer reviewe

Albumin-mediated alteration of plasma zinc speciation by fatty acids modulates blood clotting in type-2 diabetes

This work was supported by the British Heart Foundation (grant numbers PG/15/9/31270, FS/15/42/31556) and travel grants from the Commonwealth Scholarship Commission (grant number MWCN-2017-294) and the International Co-operation project of Qinghai Province (grant number 2021-HZ-806).Zn2+ is an essential regulator of coagulation and is released from activated platelets. In plasma, free Zn2+ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn2+ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn2+ in blood clot formation, we hypothesise that Zn2+ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn2+-binding ability of HSA. Amongst the NEFAs tested, palmitate (16:0) and stearate (18:0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16:1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn2+ alone. The effects of elevated Zn2+ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.Publisher PDFPeer reviewe

Plasma non-esterified fatty acids contribute to increased coagulability in type-2 diabetes through altered plasma zinc speciation

Zn2+ is an essential regulator of coagulation and its availability in plasma is fine-tuned through buffering by human serum albumin (HSA). Non-esterified fatty acids (NEFAs) transported by HSA reduce its ability to bind/buffer Zn2+. This is important as plasma NEFA levels are elevated in type-2 diabetes mellitus (T2DM) and other diseases with an increased risk of developing thrombotic complications. The presence of 5 mol. eq. of myristate, palmitate, stearate, palmitoleate and palmitelaidate reduced Zn2+ binding to HSA. Addition of myristate and Zn2+ increased thrombin-induced platelet aggregation in platelet-rich plasma and increased fibrin clot density and clot time in a purified protein system. The concentrations of key saturated (myristate, palmitate, stearate) and monounsaturated (oleate, vaccinate) NEFAs positively correlated with clot density in subjects with T2DM (and controls). Collectively, these data strongly support the concept that elevated NEFA levels contribute to an increased thrombotic risk in T2DM through dysregulation of plasma zinc speciation