Native Disulfide Research Articles

Thiol/disulfide homeostasis in patients with chronic hepatitis B

Aim: The purpose of this study was to evaluate the relation between chronic hepatitis B (CHB) and the thiol/disulfide balance, used as a marker of oxidative stress. Materials and Methods: This study was conducted between May 2016 and July 2016 at the Erzurum Regional Training and Research Hospital Infectious Diseases Polyclinic. 63 subjects with CHB and 60 healthy volunteers without any known diseases were included in the study. In term of measuring dynamic thiol/disulfide homeostasis, we used the novel automated assay method developed by Erel and Neselioglu. Results: Native thiol (SH), total thiol (total SH) and disulfide (SS) levels were determined; measures such as SS/SH, SS/total SH, and SH/total SH were calculated. It is determined that CHB group’s SH levels (P=0.041), total SH levels (P=0.043) were lower than the control group’s. There is negative correlation between Anti-HBc total IgG and total SH, SH, SH/total SH, there is positive correlation between Anti-HBc total IgG and SS/SH, SS/total SH ratio. There is negative correlation between BMX index and total SH, SH, SH/total SH and positive correlation between SS/total SH, SS/SH. Positive correlation is determined between total protein, albumin and total SH, SH. Conclusions: In our study thiol levels as an antioxidant were found to be low in CHB patients. Thiol levels were again emphasized as a new marker in hepatitis B. Thiol levels in CHB are thought to shed light to slowing of disease course and improving new treatment efforts with more wide studies.

Thiol/disulfide homeostasis in patients with ocular-active and ocular-inactive Behçet disease.

To evaluate thiol/disulfide homeostasis in ocular-active (OA) and ocular-inactive (OI) Behçet disease (BD) patients and compare the data with healthy subjects. Twenty OABD patients, 20 OIBD patients and 20 healthy control subjects were included into the study. The BD ocular attack score 24 (BOS24) scoring system was used to assess the activity of disease in ocular BD patients. Systemic activity was also evaluated using BD current activity form (BDCAF). The native thiol (NT), total thiol (TT) and disulfide levels and NT/TT, disulfide/NT and disulfide/TT ratios were measured via using an innovative and automated method. BOS24 and BDCAF scores were 13.25 ± 2.32 and 4.18 ± 2.06 in OABD patients and 0.31 ± 0.47 and 2.14 ± 1.98 in OIBD patients, respectively. The NT, TT levels and NT/TT ratio were significantly reduced; in contrast, the disulfide levels, disulfide/NT and disulfide/TT ratios were significantly increased in OABD and OIBD patients compared to the healthy control subjects (p < 0.05). Moreover, while the levels of NT and TT were significantly reduced, the disulfide levels as well as disulfide/NT and disulfide/TT ratios were significantly elevated between OABD and OIBD patients (p < 0.05). However, the ratio of NT/TT did not significantly differ between OABD and OIBD patients (p = 0.449). The multiple regression model including BOS24 and BDCAF score statistically significantly predicted NT level, TT level and disulfide level (p < 0.001 for all). Thiol oxidation in BD patients resulted in a change of the thiol/disulfide balance. Therefore, thiol/disulfide homeostasis in BD patients can be used an innovative oxidative stress marker.

Evaluation of Thiol/Disulfide Homeostasis in Pediatric Patients with Diabetic Ketoacidosis.

This study aimed to investigate the value of Thiol/Disulfide homeostasis in pediatric diabetic ketoacidosis patients suffering from type 1 diabetes mellitus. This study featured children who were diagnosed with diabetic ketoacidosis and who were consecutively admitted to pediatric intensive care within one year of their diagnosis. Thiol/disulfide homeostasis was evaluated in 45 pediatric patients suffering from DKA, as well as 45 healthy controls of parallel gender and age. Thiol/disulfide homeostasis parameters were measured using a novel automated measurement method and the correlation between demographic data and parameters was measured. Pediatric patients were found to have low native thiols, total thiols and disulfide levels with type 1 diabetes after DKA (331.82±106.40, 362.71±113.31, 17.02±5.33 μmol/L, respectively) as compared to the control group (445.08±24.41, 481.21± 28.47, 18.06±5.12 μmol/L, respectively). Thiol/disulfide homeostasis was distorted in pediatric patients with DKA. Furthermore, it was found that they are not likely to return to normal, immediately after treatment.

Chiroptical Properties and Conformation of Four Lasiocepsin-Related Antimicrobial Peptides: Structural Role of Disulfide Bridges

We report an investigation of the role of disulfide bridges in the 27-residue antimicrobial peptide lasiocepsin (I) containing two disulfide groups (Cys8–Cys25, Cys17–Cys27) and three its analogs lacking one (II, III) or both (IV) native disulfides. Selective alternate incorporation of one or both disulfide bridges influences symmetry, conformation and biological properties of these peptides as demonstrated in their chiroptical (particularly Raman) properties. The effect of modifying the disulfide bridge pattern on the peptide secondary structure is investigated in water and in the presence of 2,2,2-trifluoroethanol and sodium dodecyl sulphate. A combination of experimental electronic and vibrational chiroptical data shows that both disulfide groups are necessary for stabilizing lasiocepsin secondary structure. While the Cys8–Cys25 disulfide group is important for sustaining lasiocepsin tertiary structure and maintaining its biological activity, the Cys17–Cys27 disulfide bridge has a supporting function consisting in reducing peptide flexibility.

Generation of Aggregates of α-Lactalbumin by UV-B Light Exposure.

Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of α-lactalbumin (α-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by α-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation. This process mediated efficient aggregation with up to 98% monomer conversion into aggregates through formation of intermolecular disulfide bonds, while only minor levels of nonreducible cross-links were observed. SDS-PAGE analysis revealed that illumination led to formation of dimeric, trimeric, and oligomeric forms of α-LA. LC-MS/MS analysis showed that all of the four native disulfide bonds in α-LA were cleaved by UV illumination but to different extents, and the extent of cleavage was found to be higher in the absence of calcium. Seventeen different non-native disulfides were formed after 24 h of UV illumination. Two dityrosine bonds were identified (Tyr103-Tyr103 and Tyr36-Tyr103) alongside ditryptophan (Trp118-Trp118) and tyrosine-tryptophan (Tyr50-Trp60) cross-links. In addition, Trp60, Trp118, Cys73, Cys91, Cys120, Phe80, Met90, His68, and His107 were found to be oxidized up to 12% as compared to a nonilluminated control. Our work illustrates that light exposure can be used for generation of α-LA aggregates, but optimization of the illumination conditions is required to reduce oxidative damage to Trp, Cys, Phe, Met, and His residues.

The thiol/disulfide balance in ketone positive and ketone negative pregnant women with nausea and vomiting - a prospective study in a tertiary center.

We aimed to investigate the thiol/disulfide balance in ketone positive (hyperemesis gravidarum) and ketone negative pregnant women with nausea and vomiting. A total of 60 patients under the 14th week of pregnancy were included in this study, and they were divided into two groups. Group 1 included 30 pregnant women with ketone positive, group 2 included 30 ketone negative pregnant women with nausea, and vomiting. The native thiol, disulfide, and total thiol concentrations were measured using an automated method and compared among the two groups. There were also three indexes that are derived from disulfide, native and total thiol (Index 1 = 100 × disulfide/native thiol); (Index 2 = 100 × disulfide/total thiol); (Index 3 = 100 × native thiol/total thiol). When compared with Group 1 and Group 2, total thiol was high, native thiol was low but not statistically significant. Disulphide (p = 0.046), index 1 (p = 0.036) and index 3 (p = 0.034) were statistically significant. Patients with ketone positive are shifting to OS direction due to lack of nutrients and electrolytes. This study emphasizes the therapeutic potential of antioxidant supplementation, which is becoming an increasingly used approach in treating the symptoms of women with ketone positive.

A Chemical Biology Approach to Probing the Folding Pathways of the Inhibitory Cystine Knot (ICK) Peptide ProTx-II.

Peptide toxins that adopt the inhibitory cystine knot (ICK) scaffold have very stable three-dimensional structures as a result of the conformational constraints imposed by the configuration of the three disulfide bonds that are the hallmark of this fold. Understanding the oxidative folding pathways of these complex peptides, many of which are important therapeutic leads, is important in order to devise reliable synthetic routes to correctly folded, biologically active peptides. Previous research on the ICK peptide ProTx-II has shown that in the absence of an equilibrating redox buffer, misfolded intermediates form that prevent the formation of the native disulfide bond configuration. In this paper, we used tandem mass spectrometry to examine these misfolded peptides, and identified two non-native singly bridged peptides, one with a Cys(III)-Cys(IV) linkage and one with a Cys(V)-Cys(VI) linkage. Based on these results, we propose that the C-terminus of ProTx-II has an important role in initiating the folding of this peptide. To test this hypothesis, we have also studied the folding pathways of analogs of ProTx-II containing the disulfide-bond directing group penicillamine (Pen) under the same conditions. We find that placing Pen residues at the C-terminus of the ProTx-II analogs directs the folding pathway away from the singly bridged misfolded intermediates that represent a kinetic trap for the native sequence, and allows a fully oxidized final product to be formed with three disulfide bridges. However, multiple two-disulfide peptides were also produced, indicating that further study is required to fully control the folding pathways of this modified scaffold.

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories.

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

The Role of Thiol–Disulfide and Ischemia-Modified Albumin in the Differential Diagnosis of Ovarian Pathologies in Children

Introduction: The aim of this study was to evaluate the association between ovarian pathologies and oxidative stress in children via the new method of thiol/disulfide homeostasis. Materials and Methods: The study was conducted in our clinic and included 24 cases of ovarian cysts (OCs) followed by us and 23 cases of operated OC or torsion and monitored by pediatric surgical intensive care unit. The control group consisted of 24 girls who admitted to the pediatric surgery outpatient clinic because of not-incarcerated inguinal hernia. Serum native thiol, total thiol, dynamic disulfide, albumin, and ischemia-modified albumin (IMA) levels of the patients and healthy volunteers were evaluated. Results: Native thiol (P = 0.41), total thiol (P = 0.57), dynamic disulfide (P = 0.98), albumin (P = 0.54), and IMA (P = 0.98) levels of the patients with OC were found similar with the ovarian torsion (OT) group. However, there were statistically significant differences in native thiol (P

Thiol/disulphide balance in patients with psoriasis.

IntroductionPsoriasis is a common chronic inflammatory skin disease with unknown etiopathogenesis.AimTo examine the thiol/disulfide balance in psoriasis patients and to compare the results with a healthy control group.Material and methodsEighty patients with psoriasis and 80 healthy individuals who were age- and gender-matched with these patients were included in this study. Serum native thiol, disulfide and total thiol levels were measured by a new automated method developed by Erel and Neselioglu. Disulphide/total thiol, disulphide/native thiol and native thiol/total thiol were calculated.ResultsWhile there was no statistically significant difference in terms of disulphide levels (SS) between the patient and control groups (p > 0.05), there were significant differences in terms of total thiol and native thiol (SH) levels, SS/SH, SS/total thiol and SH/total thiol ratios between the patient and control groups (p < 0.05). There was a statistically significant relationship between duration of the disease and the disulfide/native thiol ratio (p > 0.05).ConclusionsIn recent years, there have been few studies on the role of oxidative stress in the etiopathogenesis of psoriasis. In this study, we investigated in psoriasis patients, thiol/disulfide balance as a new oxidative stress marker. The results were compared with a healthy control group. Our results showed that thiol/disulphide balance shifted towards disulphide in psoriasis patients. This is important as a finding that supports the role of oxidative stress in the pathogenesis of psoriasis.

Dynamic thiol / disulfide homeostasis in metabolically healthy obese adolescents

Aim: This study aim to discuss the possible role of oxidant-antioxidant balance in providing metabolic health in obese adolescents by determining the oxidant-antioxidant status. Material and Methods: 98 metabolically healthy obese and 75 normal-weight adolescents participated in the study. Obese individuals were grouped according to the severity of obesity. Biochemical parameters including thiol/ disulfide homeostasis were analyzed. Serum native thiol (SH), total thiol (SH+SS) and disulfide (SS) concentrations were determined using a novel automated measurement method. Disulfide to native thiol ratio (SS/SH), disulfide to total thiol ratio(SS/(SH+SS)), and native thiol to total thiol ratio (SH/(SH + SS)) were calculated and were presented as percentage. Results: Native and total thiol levels-as an antioxidative parameter-was significantly higher in obese adolescents compared to healthy-weight adolescents (p0.001). Native thiol/total thiol ratio was slightly higher in the obese group, but not statistically significant (p=0.4). Disulfide levels, disulfide/native thiol and disulfide/total thiol ratios were not different between the groups. We found that the levels of the antioxidant parameters, native thiol, and total thiol increased significantly along with the severity of the obesity, while other parameters remained comparable. We also found a significant increase in antioxidant parameters correlated with the severity of obesity (p0.001). Conclusion: This study concluded that the increased antioxidant status and activity in obese adolescents may be related to the metabolic health.

Evaluation of thiol-disulfide homeostasis in pseudoexfoliation glaucoma and primary open-angle glaucoma.

This study aimed to compare the serum thiol-disulfide homeostasis, total antioxidant status (TAS), and total oxidant status (TOS) in patients with pseudoexfoliation glaucoma (PEG) patients, primary open-angle glaucoma (POAG) patients, and healthy individuals (control). Ninety subjects were included in this study. Three groups were separated as PEG, POAG, and control. All groups were chosen to be similar in terms of age and gender. Blood samples were obtained following an overnight fasting state and were collected on the ice at 4°C. The serum samples were separated from the cells by centrifugation at 3000 rpm for 15 min and were stored at -80°C. Serum samples analyzed for TAS and TOS, native thiol, total thiol, disulfide, and native thiol/disulfide ratio. TAS and TOS levels of PEG patients were 1.2892 ± 0.0905 mmol/L; 5.0191 ± 2.7722 μmol/L, respectively. TAS and TOS levels of POAG patients were 1.2741 ± 0.1252 mmol/L; 4.1674 ± 1.7723 μmol/L, respectively. TAS and TOS levels of the control group were 2.3414 ± 0.1409 mmol/L; 4.0931 ± 0.1107 μmol/L, respectively. The TAS level was significantly lower in PEG and POAG groups compared to control. TOS level showed no significant differ ¬ ence between PEG, POAG, and control groups (P > 0.05). The mean serum total thiol and native thiol levels were significantly lower in patients with PEG compared to POAG and control group; there was no significant difference between the POAG and control group (P > 0.05). The mean serum disulfide level was significantly lower in patients with PEG compared to POAG (P = 0.018). Low levels of TAS were observed in patients with glaucoma, which was likely a response to the increased oxidative stress observed in these patients. While total thiol and native thiol levels were higher in the PEG group, the disulfide level was higher in the POAG group. TAS and TOS levels showed no significant difference between POAG and PEG groups.

Changes in thiol/disulfide homeostasis in patients with chronic kidney disease

Thiol/disulfide homeostasis (TDH) is a new marker of oxidative stress. In this study, we would like to determine the changes in TDH in hemodialysis (HD) patients with chronic kidney disease (CKD). This cross-sectional study was conducted in the Nephrology Clinic of Konya Training and Research Hospital. A total of 197 individuals including 75 HD patients, 41 end stage renal disease (ESRD) patients (having stage 3-5 CKD but not receiving hemodialysis), and 81 healthy controls were enrolled in the study. Serum native thiol, total thiol, and disulfide levels were measured with a new method developed by Erel and Neselioglu. It was determined that there was a statistically significant difference in the mean age, body mass index (BMI), modification of diet in renal disease (MDRD), and creatinine level between the three groups (p

Investigation of thiol-disulfide balance in children with pneumonia

Aim: We aimed to investigate the thiol-disulfide balance, role of oxidative stress in pneumonia and role of measurement of thiol as a new biological marker for the diagnosis of pneumonia in children with pneumonia who admitted to pediatric emergency service and general pediatric polyclinic.Material and Methods: In our study, patients between 6 months and 4 years of age, who admitted to the Pediatric Emergency Department and Pediatric Policlinic of between January 2017 and February 2018 with a diagnosis of community-acquired pneumonia, were evaluated prospectively. A total of 90 patients were included in the study, including 45 patients and 45 healthy children in the same age group. These patients were examined for age, gender, symptoms and physical examination findings, native thiol, total thiol and disulfide values. Results: There was no significant difference between the patient and control groups in terms of gender. Statistically significant difference was observed between native thiol, total thiol and disulfide values between patient and control group (p0.05). In the patient group, the levels of native thiol and total thiol were low and disulfide levels were found to be high. There was a significant difference between patient and control group in terms of disulfide / total thiol, disulfide / native thiol and native thiol / total thiol parameters (p0.05). Disulfide / total thiol and disulfide / native thiol parameters were high in the patient group. Nativethiol / total thiol ratio was found to be high in the control group. Conclusion: In children with pneumonia; native thiol, total thiol, disulfide and their ratios were found to be significantly correlated. It was found that oxidative stress has an important role in the pathogenesis of pneumonia. The use of thiols in the diagnosis of pneumonia may guide clinicians.

Assessment of Plasma Thiol-disulfide Balance in Pseudoexfoliation Syndrome and Pseudoexfoliation Glaucoma.

Objectives:The thiol-disulfide balance is very important in cellular events such as apoptosis and oxidative stress. This study is a comparison of plasma thiol-disulfide homeostasis in patients with pseudoexfoliation syndrome (PEXS) and pseudoexfoliation glaucoma (PEXG).Methods:Thirty-one patients with PEXS, 43 patients with PEXG, and 38 healthy controls were included in this prospective study. The plasma level of native thiol and disulfide were measured using a spectrophotometric assay and the native thiol/disulfide ratio was analyzed.Results:The demographic characteristics of the 3 groups were similar (p>0.05). Statistically significant differences were observed in the plasma disulfide levels (21.6±7.3 µmol/L vs. 17.4±6.8 µmol/L) and the native thiol/disulfide ratio (22.9±9.1 vs. 29.9±14.7) between the PEXG group and the controls (p=0.03, p=0.02, respectively).Conclusion:Significant differences in the plasma levels of disulfide and the native thiol/disulfide ratio in PEXG patients indicated a breakdown of the thiol-disulfide circuits.

Level of Certain Oxidants and Antioxidants in Patients with Uterine Fibroids

Objective: The mechanism of the leiomyoma formation process is still unknown. However, the menstrual cycle is associated with hypoxia, and ongoing hypoxia is associated with cellular events leading to the conversion of myometrial cells into uterine fibroids. After the hypoxic environment, the balance of cellular cytokines and growth factors changes. It was planned to evaluate the balance of oxidative agents and the role of oxidative stress in the formation of uterine fibroids.Study Design: The study was conducted in patients of gynecology outpatient clinic of Ankara Oncology Training and Research Hospital. Those volunteers with fibroids formed the study group, and the healthy ones were identified as the control group. A total of 64 subjects, 32 in both groups were included. The levels of catalase, ceruloplasmin, myeloperoxidase, native thiol, disulfide, and total thiol levels were investigated in groups.Results: Antioxidant parameters such as native thiol, disulfide, and total thiol levels were significantly decreased in uterine fibroid group. Catalase, ceruloplasmin, and myeloperoxidase levels were not statistically different between groups.Conclusion: Decrease in these antioxidant parameters showed that the hypoxia and the balance of oxidant and antioxidant substances changes may probably have associated with the pathophysiology of uterine fibroids.

Ischemia-modified albumin (IMA) and dynamic thiol-disulfide homeostasis in patients with postherpetic neuralgia

Abstract Background Ischemia-modified albumin (IMA) is an isotype of albumin that increases under oxidative stress, and plasma thiols are main defense mechanisms against oxidative stress. The objective of this study was to investigate thiol-disulfide homeostasis and serum IMA levels in postherpetic neuralgia (PHN) patients. Methods A total of 29 PHN patients and 30 healthy controls were included in the study. Serum total and native thiol concentrations and serum disulfide concentration were measured using the method described by Erel and Neselioglu. The albumin cobalt binding test was used to measure serum IMA levels. Results Serum IMA levels were 1.21 ± 0.58 AU and 0.75 ± 0.09 AU in the PHN and control groups, respectively (p &lt; 0.001). Serum total thiol concentrations were found to be 421.62 ± 90.28 μmol/L and 598.36 ± 73.63 μmol/L in the PHN and control groups, respectively (p &lt; 0.001). Serum native thiol concentrations were found to be 365.75 ± 92.07 μmol/L and 531.90 ± 72.9 μmol/L in the PHN and control groups, respectively (p &lt; 0.001). Serum disulfide concentrations were found to be 33.23 ± 5.33 μmol/L and 27.93 ± 7.81 μmol/L in the PHN and control groups, respectively (p = 0.003). The native thiol/total thiol ratio was significantly lower, and the disulfide/total thiol and disulfide/native thiol ratios were significantly higher in the PHN group compared to the controls. Conclusions IMA levels are high and dynamic thiol/disulfide homeostasis is disrupted in PHN patients.

Bridged Cysteine Conjugations.

Preparation of antibody-drug conjugates (ADCs) with a highly homogeneous drug loading in general requires site-selective conjugation of a cytotoxic payload. Typically, functionality utilized for attachment of the payload is achieved through engineering of suitable chemical handles or by enzymatic modification of the antibody. Relatively few methods to produce ADCs with homogeneous drug loading via endogenous amino acid conjugation have been developed. Herein we describe a robust method for the conjugation of antibodies using a cysteine rebridging approach to produce ADCs with highly homogeneous drug-to-antibody ratios (DAR) at the native interchain disulfides, called ThioBridge®. The process described relies upon an elegant cascade of addition-elimination reactions carried out under mild aqueous conditions that can be readily applied to wild-type antibodies without the need for prior modification via recombinant or enzymatic means. Using this method, conversions to a conserved DAR ADC are typically in the range of 70-95% and overall process yields of >70% are readily achieved.

Applications of catalyzed cytoplasmic disulfide bond formation.

Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.