Mechanisms and applications of disulfide bond formation

Project Leader: Prof. Lloyd Ruddock, Ph.D.
Faculty of Biochemistry and Molecular Medicine,
University of Oulu

Background and Significance

In 1982 the FDA approved the use of recombinant insulin, triggering a healthcare revolution with the introduction of recombinant protein based therapeutics. Since then life expectancy has increased by 7 years in Finland and by 8 years worldwide. However, this has been accompanied by a massive expansion in healthcare costs bringing increased inequality, increasing moral dilemmas regarding the value of life quality vs cost of treatment and threatening to undermine the fabric of healthcare in the developed world. Cheaper more efficient means of producing high quality protein and peptide therapeutics must be found.

The limitation in both the affordability of protein based therapeutics and in the expansion of protein based therapeutics and analytics is heavily linked to the complexity of large-scale production of active proteins with appropriate homogenous post-translational modifications (PTM), in particular disulfide bonds and N-glycans; the vast majority of protein based drugs include one or both PTMs. Both PTMs are naturally formed in the endoplasmic reticulum (ER), with the formation of the single native disulfide bonded state being essential to generate the biologically active conformation and disulfide bond formation often being the rate limiting step of protein folding in vivo and in vitro. N-glycosylation in contrast is usually heterogenous and is often not required to reach the native folded state. However, the N-glycan status can modulate biological activity, stability and pharmacokinetics of the protein. Hence N-glycan heterogeneity (which includes significant batch to batch variation) has major implications for therapeutic protein production and usage.

While the integration of multiple novel technologies in this project to generate Gen2Co is not trivial, the final product would be E.coli strains optimized for protein production with PTM. Such novel production strains would be simple to use and easily transfered from the laboratory to the market.

Recent Progress

Development of CyDisCo
Based on more than two decades of studies by the PI on understanding the mechanisms for disulfide bond formation we have developed systems which allow efficient disulfide bond formation in the cytoplasm of E. coli. Unlike previously published systems, our systems do not require disruption of the reducing pathways naturally found in the cytoplasm, and they work in any media and in any E.coli strain tested to date.

Our system, known as CyDisCo (cytoplasmic disulfide bond formation in E.coli), has a variety of formats, but those most widely used share the common feature of co- or pre-expression of a sulfhydryl oxidase and a protein disulfide isomerase i.e. catalysts of the two steps of native disulfide bond formation. The first patent has been granted and the system has been commercialized.

Studies with the CyDisCo system shows that the system is very successful and high yields of active, correctly folded, eukaryotic proteins can be obtained. CyDisCo can be combined with other technologies, such as systems that secrete folded proteins from the cytoplasm and N-glycosylation in the cytoplasm to generate Gen2Co, 2nd generation E.coli cell factories.

The patented variants of the system allow production of homogeneously folded human proteins with multiple disulfide bonds in E.coli grown in shake flasks with yields of up to 250 mg/litre culture. Recent results indicate that this can be extended into batch and fed-batch fermentation in defined minimal media even for proteins as complicated as full length human antibodies, with purified yields in excess of 1g/L of soluble folded protein being achieved for human growth hormone and interleukin 6 and up to 0.5g/L for human antibodies.

During this year we have gone back to redesign the fundamentals of the system and in parallel to introduce new factors into the system – again based on decades of fundamental studies on the mechanisms of protein folding. New variants that are more efficient in native disulfide bond formation as well as variants which aid other rate limiting steps in protein folding have been introduced. These new variants can increase the yield of folded protein obtained up to 4x.

Application of CyDisCo
The CyDisCo system was originally developed due to frustration with the inability to produce proteins involved in, or suspected to be involved in, protein folding and quality control in the ER in sufficient yields to undertake molecular characterization and structural studies.

This year one focus has been on increasing the breadth of proteins tested, both for academically interesting and industrially relevant proteins. Successful production of a range of scFv and Fab antibody fragments have been achieved in the cytoplasm, along with human N- and O-glycosyltransferases, gastrointestinal mucosa proteins, signalling molecules – including growth factors and hormones, proteins involved in development, angiogenesis and wound healing and a number of disulfide bond containing proteins involved in protein folding, quality control and ERAD - including high-yields of homogeneously folded human Ero1α/β.

As well as human Ero1α/β a range of other proteins involved in protein folding, quality control and ER-associated degradation (ERAD) have been made with the aim of increasing mechanistic understanding of the system and then the application of this knowledge to make our production systems more efficient.

Collaborative studies have commenced on several secreted proteins for functional and structural studies. During the past year three crystal structures have been solved for secreted proteins involved in angiogenesis and in wound healing and structural studies have commenced on a number of proteins including perlecan (a major structural protein in the extracellular matrix) as well as proteins playing a role in development, lung function and a novel chaperone family.

Molecular and structural characterization of these continue to be a major focus in the coming year.

In parallel to these studies utilizing CyDisCo to understand the mechanisms of protein folding, we have also continued work on the mechanisms of action of key enzymes in oxidative protein folding such as PDI. It is known that PDI must be able to trigger conformational changes in bound non-native protein subtrates to allow access to buried thiols and disulfides. This is an area that has been poorly pursued due to the extreme difficulty in isolating/identifying intermediates and in studying the process. We have data from collaborative NMR studies that conformational exchange we had previously identified within PDI is linked to the ability to trigger change in the conformation of folding intermediate mimics (produced using CyDisCo). These studies feed into the development of the CyDisCo system.

Finally, we have used our knowledge of the mechanisms of oxidative folding in collaborative studies to elucidate the role of variants in a PDI-family members in Amyotrophic Lateral Sclerosis (ALS) and motor dysfunction.

Future Goals

The overall aim of the group is to provide a complete molecular description of the processes by which protein folding occurs within the ER and the application of this knowledge for the efficient production of disulfide bond containing proteins of scientific, medicinal or biotechnological importance.

With the final stages of development of CyDisCo in sight the primary foci of the group will switch towards:

  • The use of CyDisCo to obtain mechanistic understanding of the pathways and synergy of protein folding, quality control and ER-associated degradation.
  • The development of Gen2Co for efficient secretion of disulfide bonded proteins
  • The development of systems analogous to CyDisCo for the production of other PTMs in the cytoplasm of E.coli
  • Optimization of antibody folding and bispecific antibody production

Selected Publications

Gaciarz A, Veijola J, Uchida Y, Saaranen MJ, Wang C, Hörkkö S, Ruddock LW. Systematic screening of soluble expression of antibody fragments in the cytoplasm of E.coli. Micro Cell Factories 15:22, 2016.

Tejesvi MV, Picart P, Kajula M, Hautajärvi H, Ruddock LW, Kristensen HH et al. Identification of antibacterial peptides from endophytic microbiome. Appl Microbiol Biotechnol 100: 9283-9293, 2016.

Woehlbier U, Colombo A, Saaranen MJ, Perez V, Ojeda J et al (incl Ruddock LW). ALS-linked protein disulfide isomerase variants cause motor dysfunction. EMBO J 35:845-865, 2016.

Psioni GB, Ruddock LW, Bulleid N and Mollinari M. Division of labor among oxidoreductases: TMX1 acts preferentially on transmembrane polypeptides. Mol Biol Cell 26:3390-400, 2015.

Alanen HI, Walker KL, Lourdes Velez Suberbie M, Matos CF, Bönisch S, Freedman RB, et al. Efficient export of human growth hormone, interferon a2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochim Biophys Acta 1854:756-63, 2015.

Venkatesan R, Sah-Teli SK, Awoniyi LO, Jiang G, Prus P, Kastaniotis AJ, Hiltunen JK, Wierenga RK, Chen Z. insights into mitochondrial fatty acid synthesis from the structure of heterotetrameric 3-ketoacyl-ACP reductase/3R-hydroxyacyl-CoA dehydrogenase. Nat. Commun. 5:4805, 2014

Szambowska A, Tessmer I, Kursula P, Usskilat C, Prus P, Pospiech H, Grosse F. DNA binding properties of human Cdc45 suggest a function as a molecular wedge for DNA unwinding. Nucleic Acids Res. 42: 2308-2319, 2014

Matos CFRO, Alanen HI, Prus P, Uchida Y, Freedman RB, Keshavarz-Moore E, Robinson C and Ruddock LW. Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains Biotechnol Prog. 30:281-290, 2014

Kummu O, Turunen SP, Prus P, Lehtimäki J, Veneskoski M, Wang C, Hörkkö S. Human monoclonal Fab and human plasma antibodies to carbamyl epitopes cross-react with malondialdehyde-adducts. Immunology 141: 416-430. 2014

Irvine AG, Wallis AK, Sanghera N, Rowe ML, Ruddock LW, Howard MJ, Williamson RA, Blindauer CA, Freedman RB. Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway. PLoS One 9:e82511, 2014.

Saaranen MJ and Ruddock LW. Disulfide bond formation in the cytoplasm. Antioxid Redox Signal 19:46-53, 2013.

Hatahet F and Ruddock LW. Topological plasticity of enzymes involved in disulfide bond formation allows catalysis in either the periplasm or the cytoplasm. J Mol Biol 425:3268-3276, 2013.

Ruddock LW. Low-molecular-weight oxidants involved in disulfide bond formation. Antioxid Redox Signal 16:1129-1138, 2012.

Nguyen VD, Hatahet F, Salo KE, Enlund E, Zhang C, Ruddock LW. Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli. Micro Cell Fact 10:1, 2011.

Nguyen VD, Saaranen MJ, Karala AR, Lappi AK, Wang L, Raykhel IB, Alanen HI, Salo KE, Wang CC, Ruddock LW. Two endoplasmic reticulum PDI-peroxidases increase the efficiency of the use of peroxide during disulfide bond formation. J Mol Biol 406:503-515, 2011.

Lappi AK and Ruddock LW. Re-examination of the role of interplay between glutathione and protein disulphide isomerase. J Mol Biol 409:238-249, 2011.

Alanen HI, Raykhel IB, Luukas MJ, Salo KEH, Ruddock LW. Beyond KDEL: The role of positions 5 and 6 in determining ER localization. J Mol Biol 409:291-297, 2011.

Karala AR, Lappi AK, Ruddock LW. Modulation of an active-site cysteine pK a allows PDI to act as a catalyst of both disulfide bond formation and isomerization. J Mol Biol 396:883-892, 2010.

Karala AR and Ruddock LW. Bacitracin is not a specific inhibitor of protein disulfide isomerase. FEBS J 277:2454-2462, 2010.

Hatahet F, Nguyen VD, Salo KEH, Ruddock LW. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E.coli. Micro Cell Fact 9:67, 2010.

Saaranen MJ, Karala AR, Lappi AK, Ruddock LW. The role of dehydroascorbate in disulfide bond formation. Antioxid Redox Signal 12:15-25, 2010.

Karala AR, Lappi AK, Saaranen M, Ruddock LW. Efficient peroxide mediated oxidative refolding of a protein at physiological pH and implications for oxidative folding in the endoplasmic reticulum. Antioxid Redox Signal 11:963-970, 2009.

Rowe M, Ruddock LW, Kelly G, Schmidt J, Williamson R, Howard M. Solution structure and dynamics of ERp18, a small ER resident oxidoreductase. Biochemistry 48:4596-4606, 2009.

Hatahet F and Ruddock LW. Modulating proteostasis: peptidomimetic inhibitors and activators of protein folding. Curr Pharm Des 15:2488-2507, 2009.

Saaranen MJ, Salo KEH, Latva-Ranta MK, Kinnula VL, Ruddock LW. The C-terminal active site cysteine of Escherichia coli glutaredoxin 1 determines the glutathione specificity of the second step of peptide deglutathionylation. Antioxid Redox Signal 11:1819-1828, 2009.

Karala A, Lappi AK, Ruddock LW. The role of conserved arginine of PDI in catalysis. FEBS J 276:143-144, 2009.

Hatahet F and Ruddock LW. Protein disulfide isomerase: A critical evaluation of its function in disulfide bond formation. Antioxid Redox Signal 11:2807-2850, 2009.

Byrne LJ, Sidhu A, Wallis AK, Ruddock LW, Freedman RB, Howard MJ, Williamson RA. Mapping of the ligand binding site on the b’ domain of human PDI: interaction with peptide ligands. Biochem J 423:209-217, 2009.

Wallis AK, Sidhu A, Byrne LJ, Howard MJ, Ruddock LW, Williamson RA, Freedman RB. The ligand-binding b’ domain of human protein disulphide-isomerase mediates homo-dimerization. Protein Sci 18:2569-2577, 2009.

Patents 2016

US9238817B2 Method for producing natively folded proteins in a prokaryotic host (Lloyd Ruddock, January 19, 2016)

US9416388B2 Method for producing disulfide bond containing proteins in a prokaryotic cytoplasm (Lloyd Ruddock & Feras Hatahet, August 16 2016)

Research Group Members

Project Leader:
Lloyd Ruddock, Ph.D., Professor

Senior and Post-doctoral Investigators:
Ekaterina Biterova (Academy of Finland)
Mirva Saaranen, Ph.D. (Academy of Finland and University of Oulu)
Johanna Veijola (Biocenter Oulu)
Jiro Ogura (Uehara Memorial Foundation)

Ph.D. Students:
Zhang Chi, M.Sc. (Academy of Finland and Biocenter Oulu)
Anna Gaciarz, M.Sc. (Biocenter Oulu)
Kati Korhonen, M.Sc. (ISB, Academy of Finland)
Antti Moilanen (Academy of Finland)
Lisette Van Tassel, M.Sc. (Biocenter Oulu)

Laboratory Technicians, 2 (Sigrid Jusélius Foundation and University of Oulu)

Main source of salary in brackets.

Foreign Scientists, 7

National and International Activities

Visiting Researchers in 2016 (over two weeks)
Dr Jiro Ogura

Last updated: 10.4.2017