In silico analysis of the different variable domain oriented single-chain variable fragment antibody-antigen complexes.

Single-chain variable fragment (scFv) antibodies hold great potential as diagnostic tools and therapeutic agents, especially for tumor cells. Since these applications require their production with improved properties, the design strategy of scFvs is crucial for their active, soluble, and high yield expression with high affinity towards their antigens. The order of VL and VH domains is one of the important parameters that affect the expression and binding affinity properties of scFvs. In addition, the optimum order of VL and VH domains could change for each scFv. In the present study, we used computer simulation tools to evaluate the effect of variable domain orientation on structure, stability, interacting residues of scFvs, and binding free energies of scFv-antigen complexes. We selected anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, and anti-IL-1ß scFv against IL-1ß which is an important inflammatory biomarker, as model scFvs. Molecular dynamics simulations of the scFv-antigen complexes for 100 ns resulted in stability and compactness for both scFv constructs. Interaction and binding free energies calculated by the Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) approach suggested that the relative binding energies of anti-HER2 scFv-VLVH and anti-HER2 scFv-VHVL constructs had similar binding affinity towards HER2, while a relatively more negative binding free energy obtained between anti-IL-1ß scFv-VHVL and IL-1ß pointed to a higher binding affinity. The in silico approach and the results obtained here could be applied as a guide for future experimental interaction studies for highly specific scFvs used as biotechnological tools.Communicated by Ramaswamy H. Sarma.

Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography.

A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (µ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 µm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. µ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by µ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4.

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 µg mL-1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.

The evaluation of disease awareness, caregiver burden, and workday loss in caregivers of COPD patients.

BACKGROUND: Our aim is to determine the caregiver burden of chronic obstructive lung disease (COPD) patient's caregivers, and to determine whether there is a workday loss. METHODS: 252 COPD patients and their caregivers were included. Disease information of the patients were recorded and a questionnaire was applied. Socio-demographic characteristics of the caregivers were recorded and a questionnaire consisting of 24 questions including COPD disease, treatment and loss of working days, and the Zarit Scale were used. RESULTS: 128(50.8%) of the patients according to GOLD were group-D, 97(38.5%) of the patient's relatives were working, 62(24.7%) were not able to go to work for 1-14 days, and 125(57.1%) spent outside the home from 1-14 nights, because those accompanied to patients. In univariate analysis were detected modified medical research council (mMRC) (p < 0.001), CAT (p < 0.001), the number of comorbidities of patients (p = 0.027), forced expiratory volume in 1 FEV1cc (p = 0.009), FEV1% (p < 0.001), the presence of long term oxygen therapy (LTOT), and the number of comorbidities of the patient's relatives (p = 0.06) increased the care load. In multiple linear regression analysis, age (p = 0.03), COPD assessment test (CAT) score (p = 0.001), FEV1% (<0.068) and the number of comorbidities of patients (p = 0.01) and the number of comorbidities of caregivers (p = 0.003) increased the caregiving burden. DISCUSSION: In COPD increases caregiving burden. This burden is greater in symptomatic patients and when comorbidities are present. Psychosocial and legal regulations should be investigated and solutions should be produced for the caregivers of COPD patients.

Investigation of the need for computed tomography pulmonary angiography in the decision to discontinue treatment for pulmonary thromboembolism.

BACKGROUND: Acute pulmonary thromboembolism (PTE) is an important cause of morbidity and mortality that can reduce quality of life due to long-term complications during and after treatment discontinuation. OBJECTIVES: The aim of this study was to evaluate patients for these complications before discontinuing treatment and determine the necessity of computed tomography pulmonary angiography (CTPA) imaging. METHODS: This retrospective study included 116 patients over the age of 18 who received anticoagulant treatment for at least 3 months and presented for treatment discontinuation to the Atatürk University Research Hospital Chest Diseases Outpatient Clinic between January 2015 and September 2019. RESULTS: CTPA performed at treatment discontinuation showed complete thrombus resolution with treatment in 73 patients (62.9%). High pulmonary artery obstruction index (PAOI) at diagnosis was statistically associated with findings of residual or chronic thrombus on CTPA at treatment discontinuation (p = 0.001). In the differentiation of patients with residual/chronic thrombus and those with thrombus resolution, D-dimer at a cut-off value of 474 µg/L had 60% sensitivity and 70% specificity. At a cut-off value of 35.5 mmHg, mean pulmonary artery pressure on echocardiography had sensitivity and specificity of 72% and 77%, respectively. At a cut-off of 23.75, PAOI had sensitivity and specificity of 93% and 69%, respectively. CONCLUSION: In addition to physical examination findings, D-dimer and echocardiography were guiding parameters in the evaluation of treatment discontinuation and thrombus resolution in patients presenting to the outpatient clinic for discontinuation of treatment for acute PTE. PAOI at diagnosis may be another important guiding parameter in addition to these examinations.

Evaluation of 3-month follow-up of patients with postacute COVID-19 syndrome.

In addition to the highly variable clinical presentation of acute COVID-19 infection, it can also cause various postacute signs and symptoms. This study aimed to evaluate patients with postacute COVID-19 over 12 weeks of follow-up. The study included 151 patients who were diagnosed with COVID-19 by real-time polymerase chain reaction of a nasopharyngeal swab 1 month earlier, had radiologic findings consistent with COVID-19 pneumonia, and presented to the post-COVID-19 outpatient clinic between May and August 2021. The patients were divided into three groups based on COVID-19 severity: nonsevere pneumonia (Group 1), severe pneumonia (Group 2), and severe pneumonia requiring intensive care (Group 3). Evaluation of laboratory parameters at 4 and 12 weeks showed that Group 3 had a higher lactose dehydrogenase (LDH) level and a lower mean platelet volume than the other groups at both time points (p = 0.001 for all). Group 3 also had lower percent predicted forced vital capacity (FVC%), percent predicted forced expiration volume in 1 s (FEV1%), and percent predicted diffusion capacity of the lungs for carbon monoxide divided by alveolar volume (DLCO/VA%) compared to Groups 1 and 2 at Week 4 (p = 0.001, 0.004, 0.001, respectively) and compared to Group 1 at 12 weeks (p = 0.002, 0.03, 0.001, respectively). Patients with persistent dyspnea at 12 weeks had significantly lower FEV1%, FVC%, DLCO/VA%, and saturation levels in room air and significantly higher LDH, pro-BNP, D-dimer, and heart rate compared to those without dyspnea (p = 0.001 for all). Although the lungs are most commonly affected after COVID-19 infection, vascular and endothelial damage also causes multisystem involvement. Our study indicates that laboratory values, radiological signs, and pulmonary functional capacity improved in most patients after 12 weeks of follow-up.

In vitro selection of DNA aptamers against human osteosarcoma.

BACKGROUND: Osteosarcoma is a highly malignant bone tumor, most frequently occurring in the rapid bone growth phase. Effective treatment of this disease is hindered by the lack of specific probes for early diagnosis and the fast cancer widespread. METHODS: To find such probes, the cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) methodology was implemented against the human osteosarcoma MG-63 cell line towards the selection of new specific aptamers. After 10 rounds of selection, the aptamer DNA pool was Sanger sequenced and the sequences were subjected to a bioinformatic analysis that included sequence alignment, phylogenetic relationship, and secondary structure prediction. RESULTS: A DNA aptamer (OS-7.9), with a dissociation constant (Kd) value in the nanomolar range (12.8 ± 0.9 nM), revealed high affinity against the target cells at the physiological temperature. Furthermore, the selected aptamer also recognized lung carcinoma and colon colorectal adenocarcinoma cell lines, which are reported as common metastasis sites of osteosarcoma. CONCLUSIONS: These results suggest that OS-7.9 could recognize a common protein expressed in these cancer cells, possibly becoming a potential molecular probe for early diagnosis and targeted therapies for metastatic disease. Moreover, to the best of our knowledge, this was the first attempt to generate a DNA aptamer (OS-7.9 aptamer) against the MG-63-cell line by cell-SELEX.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage compounds. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most valuable biopolymers because of its superior mechanical properties. Here, we developed a bioprocess utilizing recombinant Bacillus megaterium strain for PHBV over-production from glucose, without any precursor addition. PHA production was performed in a controlled bioreactor by batch and fed-batch modes using wild-type B. megaterium and rec-B. megaterium cells overexpressing the native phaC gene. The effect of oxygen transfer rate on biomass formation and PHA accumulation was also investigated, under different dissolved oxygen levels. Structural and thermal properties of PHA were characterized by GC-FID, 1H-NMR, TGA and DSC analyses. Significantly, the copolymer produced from glucose as the carbon source in rec-B. megaterium was composed of 58 mol% of 3-hydroxyvalerate monomers. After 66 h, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ0 = 0.1 h-1 produced the highest CDW (7.7 g L-1) and PHA concentration (6.1 g L-1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.8 g g-1.

Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm.

Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

Microfluidic immobilized metal affinity chromatography based on Ti(IV)-decorated silica microspheres for purification of phosphoproteins.

A silica-based immobilized metal affinity chromatography (IMAC) sorbent with the morphological properties suitable for purification of large phosphorylated biomolecules was synthesized. The sorbent was designed in the form of monodisperse-porous silica microspheres, 5.3 µm in size, having bimodal pore size distribution with a large median pore size (40 nm) and high surface area (163 m2/g) decorated with Ti(IV) cations (i.e. Ti(IV)@THSPMP@SiO2 microspheres). The decoration of silica microspheres with Ti(IV) cations was made by using 3-(trihydroxysilyl)propyl methylphosphonate (THSPMP) as a bifunctiontional linker, by preserving their bimodal pore size distribution. The mesopores provided a large surface area for parking of adsorbed phosphoproteins as large phosphorylated biomolecules while the intraparticular transport of phosphoproteins was facilitated by the macropores providing a large median pore size. High equilibrium adsorption capacity and high desorption yield in the purification of phosphoproteins were obtained using Ti(IV)@THSPMP@SiO2 microspheres as the sorbent in batch- and microfluidic-IMAC systems. The phosphoproteins, α-casein and ß-casein were isolated from milk and human serum with almost quantitative yields and high purity in the batch IMAC system. The appropriate microcolumn permeability (3.66 × 10-14 m2) originating from its appropriate average diameter (5.3 µm), high porosity (0.948 cm3/g) and high surface area (163 m2/g) of Ti(IV)@THSPMP@SiO2 microspheres makes the synthesized sorbent a promising stationary phase for dynamic chromatography. Hence, a new phosphoprotein enrichment format, a microfluidic IMAC system was constructed and successfully operated for highly selective purification of phosphoproteins from non-fat milk as a complex sample. The microfluidic-IMAC system is a promising tool particularly for phosphoproteomic applications performed using samples in microliter or nanoliter scale, also involving an on-line connection of purification unit to LC-MS for the identification of large phosphorylated biomolecules enriched.

Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal affinity chromatography-based colorimetric determination of histidine-tagged proteins.

Monodisperse porous silica microspheres were functionalized with the iminodiacetic acid/copper(II) complex and then evaluated as a group-specific peroxidase-mimicking nanozyme for colorimetric determination of histidine-tagged (His-tagged) proteins. The green fluorescent protein (GFP) was selected as a typical His-tagged protein. The specificity for GFP and the peroxidase-like activity for the selected substrate were obtained by immobilizing the complex on the porous microspheres. The modified microspheres were also evaluated as a group specific immobilized metal affinity chromatography (IMAC) sorbent for the purification of GFP from Escherichia coli extract. The peroxidase-like activity of the microspheres was inhibited by the GFP adsorbed onto the microspheres due to the interaction of His-tagged protein with the immobilized Cu(II) complex. Ortho-phenylenediamine is used as a substrate for the enzyme mimic. The photometric response (measured at 416 nm) is linear in the 9.0-92 µg·mL-1 GFP concentration range in E. coli lysate. The limit of detection is 6.9 µg·mL-1. Graphical abstractSchematic representation of metal affinity chromatography-based colorimetric determination of histidine-tagged proteins using silica microspheres functionalized with iminodiacteic acid/copper (II) complex as a peroxidase mimic.

Ni(II)-decorated porous titania microspheres as a stationary phase for column chromatography applications: Highly selective purification of hemoglobin from human blood.

Titania (TiO2)-based monodisperse-porous stationary phase/sorbent was synthesized by decoration of Ni(II) ions onto TiO2 microspheres 4.2 µm in size, obtained by a staged-shape template hydrolysis and condensation protocol. Ni(II) ions were attached onto iminodiacetic acid-3-glycidoxypropyltrimethoxysilane (IDA-GPTMS) bound-titania microspheres by metal-chelate complex formation. The appropriate mean size, sufficiently high surface area and high porosity providing an appropriate column permeability make Ni(II)-decorated TiO2 microspheres a good sorbent/stationary phase for batch/continuous-column chromatography applications. Ni(II)-decorated TiO2 microspheres were investigated as a sorbent for purification of a typical histidine-rich protein, hemoglobin (Hb) via immobilized metal affinity chromatography (IMAC) in batch fashion, by including bovine serum albumin (BSA) as reference. The saturation capacities of batch adsorption runs performed with bovine Hb and BSA were determined as 137 ±â€¯9 and 45 ±â€¯3 mg/g, respectively. Human Hb with the purity of > 95% was recovered from whole blood by IMAC conducted in batch-fashion. Ni(II)-decorated microspheres were also evaluated as a stationary phase in a microfluidic-IMAC system, in which, human Hb was recovered from whole blood with a purity of 85%. The microfluidic-IMAC system constructed here, based on monodisperse-porous TiO2 microspheres, is a promising tool for genomics/proteomics applications involving isolation of valuable biomolecules from low-volume samples.

Established and Upcoming Yeast Expression Systems.

Yeast was the first microorganism used by mankind for biotransformation of feedstock that laid the foundations of industrial biotechnology. Long historical use, vast amount of data, and experience paved the way for Saccharomyces cerevisiae as a first yeast cell factory, and still it is an important expression platform as being the production host for several large volume products. Continuing special needs of each targeted product and different requirements of bioprocess operations have led to identification of different yeast expression systems. Modern bioprocess engineering and advances in omics technology, i.e., genomics, transcriptomics, proteomics, secretomics, and interactomics, allow the design of novel genetic tools with fine-tuned characteristics to be used for research and industrial applications. This chapter focuses on established and upcoming yeast expression platforms that have exceptional characteristics, such as the ability to utilize a broad range of carbon sources or remarkable resistance to various stress conditions. Besides the conventional yeast S. cerevisiae, established yeast expression systems including the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha, the dimorphic yeasts Arxula adeninivorans and Yarrowia lipolytica, the lactose-utilizing yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and upcoming yeast platforms, namely, Kluyveromyces marxianus, Candida utilis, and Zygosaccharomyces bailii, are compiled with special emphasis on their genetic toolbox for recombinant protein production.

Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems.

Monodisperse-porous silica microspheres 5.5 µm in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, ß-nicotinamide adenine dinucleotide (ß-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and ß-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and ß-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 µm in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and ß-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and ß-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.

Protein A and protein A/G coupled magnetic SiO2 microspheres for affinity purification of immunoglobulin G.

Protein A carrying magnetic, monodisperse SiO2 microspheres [Mag(SiO2)] with bimodal pore size distribution including both mesoporous and macroporous compartments were proposed as an affinity sorbent for IgG purification. Protein A was tightly bound onto the aldehyde functionalized-Mag(SiO2) microspheres. The mesoporous compartment provided high surface area for protein A binding and IgG adsorption while the macropores made easier the intraparticular diffusion of protein A and IgG. The selection of relatively larger microspheres with high saturation magnetization allowed faster magnetic separation of affinity sorbent from the IgG isolation medium, less than 1min. With these properties, the proposed sorbent is an alternative to the common sorbents in the form of core-shell type, magnetic silica nanoparticles with more limited surface area and slower magnetic response. By using protein A attached-Mag(SiO2) microspheres with the concentrations lower than 50mg/mL, IgG isolation from rabbit serum was performed with a purity higher than 95%, with an isolation yield comparable to commercial magnetic resins, and in shorter isolation periods. IgG could be also quantitatively isolated from rabbit serum with the sorbent concentrations higher than 50mg/mL. Successive IgG isolation runs indicated that no significant protein A leaching occurred from the magnetic matrix.

Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions.

Glycan microarrays have become a powerful platform to investigate the interactions of carbohydrates with a variety of biomolecules. However, the number and diversity of glycans available for use in such arrays represent a key bottleneck in glycan array fabrication. To address this challenge, we describe a novel glycan array platform based on surface patterning of engineered glycophages that display unique carbohydrate epitopes. Specifically, we show that glycophages are compatible with surface immobilization procedures and that phage-displayed oligosaccharides retain the ability to be recognized by different glycan-binding proteins (e.g. antibodies and lectins) after immobilization. A key advantage of glycophage arrays is that large quantities of glycophages can be produced biosynthetically from recombinant bacteria and isolated directly from bacterial supernatants without laborious purification steps. Taken together, the glycophage array technology described here should help to expand the diversity of glycan libraries and provide a complement to the existing toolkit for high-throughput analysis of glycan-protein interactions.

Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation.

Glycosylation is the most prevalent post-translational modification found on proteins, occurring in all domains of life. Ever since the discovery of asparagine-linked (N-linked) protein glycosylation pathways in bacteria, major efforts have been made to harness these systems for the creation of novel therapeutics, vaccines, and diagnostics. Recent advances such as the ability to produce designer glycans in bacteria, some containing unnatural sugars, and techniques for evolving glycosylation enzymes have spawned an entirely new discipline known as bacterial glycoengineering. In addition to their biotechnological and therapeutic potential, bacteria equipped with recombinant N-linked glycosylation pathways are improving our understanding of the N-glycosylation process. This review discusses the key role played by microorganisms in glycosciences, particularly in the context of N-linked glycosylation.

Production of recombinant proteins by yeast cells.

Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.

Production of secretory and extracellular N-linked glycoproteins in Escherichia coli.

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 peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

A filamentous phage display system for N-linked glycoproteins.

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X(1)-N-X(2)-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10(5) per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the -2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.