In recent years, the field of structural biology has seen many advances in technology for the production of recombinant proteins, mainly led by the high-throughput techniques of the structural genomics community. These technologies have largely focussed on expression using
Glycoproteins represent a unique challenge to the structural biologist due to the size and heterogeneity of the oligosaccharide chains. Glycans can constitute from 1 % to over 80 % of the total protein mass  with variation in the type and number of sugars attached to a glycosylation site and also the occupancy of a site. The heterogeneity introduced by glycosylation can hinder crystallization of a glycoprotein  thus limiting the use of X-ray crystallography, one of the major techniques using in protein structure solution.
The following chapter reviews options for the production of glycoproteins from different types of expression system, both prokaryotic and eukaryotic, along with methods for addressing glycan heterogeneity in order to produce homogeneous glycoprotein samples which are amenable for structural studies. The use of such homogeneous glycoprotein samples in both crystallization and nuclear magnetic resonance (NMR) experiments are discussed.
2. Glycosylation in mammalian cells
In eukaryotes, glycans are added to the polypeptide chain in the endoplamic reticulum (ER) and the Golgi as the protein is secreted (see Figure 1). There are two types of glycosylation, one involves linkage of an oligosaccahride to an asparagine residue and the second involves linkage to a serine or threonine residue referred to as N- and O-glycosylation respectively.
N-glycosylation occurs co-translationally in the ER at the consensus sequon Asn-X-Ser/Thr/Cys-X where X is any amino acid except proline. The exact sequence of the sequon has a bearing on the occupancy of the glycosylation site. N-glycosylation is often essential for the expression and folding of a glycoprotein , with the initial glycan formed in the ER being further modified and decorated in the Golgi apparatus. This elaboration of the glycan core leads to a large number of possible structures which are classified as high mannose, complex or hybrid (Figure 2).
O-glycosylation (See Figure 3) usually occurs in regions containing large numbers of sequential serine, threonine and proline residues which are known as mucin domains. These regions show little secondary structure and are therefore often excluded from structural studies as proteins containing such disorder are unlikely to crystallize. O-glycosylation occurring outside mucin regions is difficult to predict accurately and so is usually only detected after production of a protein. As O-glycosylation occurs in the Golgi, it has little bearing on the early stages of protein folding and therefore sites found to be O-glycosylated can be engineered out of the protein by site-directed mutagenesis of the acceptor Ser/Thr residues.
3. Expression systems
For the correct production and folding of glycoproteins, the expression host needs to post-translationally modify the protein chain by adding sugars at the glycosylation sites. Therefore, production of glycoproteins is most often performed using eukaryotic cells, although the aglycosylated protein can be expressed as inclusion bodies in
In addition to finding an appropriate system for the over-expression of the target glycoprotein, the ability of this system to incorporate selenomethioine into the protein must be assessed. Selenomethioine labelling enables phasing of X-ray diffraction data by multi-wavelength anomalous diffraction (MAD) . Proteins expressed using
Another approach is to express the glycoprotein in the periplasm of
Recently an engineered eukaryotic protein glycosylation pathway has been inserted into
3.2. Insect cells
Glycoproteins produced by insect cells, such as
The glycoforms produced by insect cells can be trimmed by treatment with endoglycosidases, for example endoglycosidase (endo) H or endo F1 and endo D will remove oligomannose and paucimannose sugars respectively leaving one GlcNAc residue on N-glcyosylation sites. In addition, endo F2 can be used in combination with endo F3 to cleave oligomannose and biantennary complex sugars and core fucosylated bi and triantennary complex gycans. Using this de-glycosylation strategy Fan
Selenomethionine labelling in insect cell systems can be difficult due to the toxicity of selenomethionine and the long incubation times needed as late baculovirus promoters such as polyhedron or P10 are normally used . The levels of incorporation for secreted glycoproteins is higher than for intracellular proteins as unlabelled protein is removed during the media exchange process . For example, 85 % selenomethionine incorporation was achieved for envelope glycoprotein D from HSV1  and 76 % for palmitoyl protein thioesterase 1 (PDB entry 1EI9 and 1EH5) . In 2007, Cronin
3.3. Yeast cells
Yeast have been used for the production of human glycoproteins, with the most popular expression hosts being
There are examples in the literature where retaining the N-glycans on the glycosylated protein produced in yeast is important, such as the of human and mouse glutaminyl cyclases (QC), enzymes linked with Alzheimer’s disease, (PDB entry 3SI0) . The structure of human QC had already been solved using protein produced in
Production of selenomethionine labelled proteins is possible in yeast with incorporation levels of around 50 % routinely reported for both
3.4. Mammalian cells
Mammalian cells are a popular choice for the expression of glycoproteins, particularly for the production of human proteins. These cells have the correct machinery to fold and post-translationally modify glycoproteins. The two main cell lines used are human embryonic kidney (HEK) 293 cells and Chinese hamster ovary (CHO) cells, both of which are readily transfected with polythyleneimine (PEI), calcium phosphate or commercial lipids giving expression in 60-80 % of the cells [32-33]. There are two main variants of HEK 293 cells, (i) 293T which expresses the SV40 large-T antigen and (ii) 293E which expresses the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1). Plasmids containing the SV40 or EBV origins of replication are amplified within these variants, giving more copies of the plasmid per cell and therefore a higher levels of protein expression . An alternative method of introducing genes into mammalian cells is to use viral-mediated transduction such as the BacMam system , which has been shown to give milligram quantities of protein for structural studies .
Mammalian cells can be grown in either attached or suspension culture and automation of the culturing processes is possible in both cases [37-38]. Structural studies have been performed on proteins produced both by transient transfection, formation of a stable cell line, and using stable pools. Transient transfection is attractive due to the short timeframe between transfection and purified protein. Glycoprotein yields for non-antibodies of up to 40 mg/L have been obtained from attached HEK cells  and 36 mg/L from HEK cells in suspension , with yields of 27 mg/L from CHO cells in suspension .
Formation of a stable cell line producing a glycoprotein of interest often gives higher yields than utilizing transient transfection, however establishment of a stable cell line can take 2-6 months . Automation can give a three-fold increase in throughput, although the timeline is the same . An advantage of the system is that once the stable cell line has been established, production of the glycoprotein is fast and robust.
Use of stable transfection pools for expression are becoming increasingly popular as the protein yield is usually higher than for transient transfection but the timeline is shorter than for making a stable cell line. Post-transfection, the cells are sorted, often using a fluorescence marker, in order to enrich the pool for high producers. Using such a method, highly productive pools of cells can be obtained in 3 weeks, though yields may decline over time in culture as the transfection cells are not clonal. Using this method, expression levels of monoclonal antibodies from 100 mg/L to 1 g/L can be achieved in 2 months post transfection using CHO cells .
Selenomethionine labelling in mammalian cells is not as efficient as in
Unlike insect and fungi, mammalian cells produce glycoproteins with complex oligosaccharide chains (Figure 2) which are very heterogeneous and therefore not readily amenable to crystallization. Complete removal of N-linked glycans can be achieved by treatment of a purified glycoprotein with Peptide N-glycosidase (PNGase) F thus aiding crystallization . In practice, two problems are encountered which limit this approach. Firstly, incomplete removal of glycans leading to partial de-glycosylation of the product; and secondly, insolubility due to protein aggregation following removal of all the sugars. Alternatively, glycosylation can be completely blocked in cells by treatment with tunicamycin. However, if glycosylation is required for proper folding and/or solubility in situ, then this approach will compromise the synthesis of the product. Two methods have been developed to get round these problems both of which depend upon manipulating N-glycosylation during glycoprotein biosynthesis by blocking the action of processing enzymes using either chemical inhibitors or null mutant cell lines.
Three inhibitors have been used in the production of glycoproteins to manipulate the glycosylation pathway: N-butyldeoxynojirimycin (NB-DNJ), swainsonine and kifunensine. NB-DNJ inhibits α-glucosidase, thus blocking the early stages of N-glycan processing and giving products which contain high mannose or hybrid type sugars (Figure 4A). NB-DNJ has mainly being used in combination with the mutant CHO cell line, CHO Lec220.127.116.11 (Section 3.4.2), for instance in the crystallization of human costimulatory molecule B7-1 which is important in human immune response which gave crystals diffracting to 2.7 Å after treatment with endo H . Swainsonine blocks α-mannosidase II resulting in high mannose or hybid type sugars shown in Figure 4D. Kifunensine strongly inhibits α-mannosidase I activity resulting in sugars of the form Man9GlcNAc2 (Figure 4D) . Treatment of cells with the any of the above inhibitors results in relatively simple and chemically uniform glycoforms. Further, the high mannose and hybrid glycans resulting from the use of these drugs are cleavable using endoglycosidase (endo) H or endo F1 to leave one GlcNAc residue attached to the N-glycosylation site.
In practice, kifunensine is the most commonly used inhibitor in the production of glycoproteins for structural studies as the resulting glycans are the most homogeneous. In fact, the structures of glycoproteins can be solved following kifunensine treatment, without the use of endoglycosidase (for example, PDB entry 2WAH ), however more commonly the sugars are trimmed with endo H before crystallization studies. For example, Bishop
3.4.2. Mutant cell lines
CHO and HEK cell lines have been generated with mutations in their glycosylation pathways which disrupt the action of GlcNAc transferase I (GnTI) [50-51]. CHO Lec3.2.1 contains four mutations which are in the
The GnTI-deficient cell line, HEK 293 GnTI- (also known as HEK 293S) produces glycoproteins with high mannose glycans (Figure 4C) which are endo H and endo F1 sensitive. Use of HEK 293 GnTI- cells gives product containing a very uniform glycosylation pattern of the form Man5GlcNAc2 with only traces of other glycan patterns being detected [51, 55]. This cell line has proved popular, recently facilitating structure solution of NetrinG1 and NetrinG2 in complex with their respective ligands at 3.25 Å and 2.6 Å resolution respectively (PDB entries 3ZYJ and 3ZYJ) ; the human glutamate receptor GluR2 amino terminal domain at 1.8 Å resolution (PDB entries 2WJW and 2WJX) ; and the orphan domain of the membrane glycoprotein endoglin using small angle X-ray scattering (SAXS) .
Although not currently in mainstream use for the production of glycoproteins, two other expression systems are worth mentioning, namely cell-based production in the protozoan
The structure of human Cu/Zn superoxide dismutase has been recently published and represents the first structure solved using
Cell-free synthesis systems have been used for structural studies for a number of years and are commonly based on lysates from
4. Variable occupancy of glycosylation sites
In addition to the types of oligosaccharide chain attached to a glycosylation site, i.e. the glycoform, heterogeneity occurs in the occupancy of a glycosylation site. The occupancy of an N-glycosylation site depends upon the recognition sequon along with the structure of the protein in proximity to the glycosylation site. Some N-glycosylation sites have very low occupancy as a result of the local sequence composition, secondary structure and also distance to the C-terminus [67-69]. Bioinformatics programmes such as NetNGlyc and NetOGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services /NetOGlyc/) [70-71] can predict occupancy of N-glycosylation and O-glycosylation sites respectively; however these are sequence based predictions and so need to be verified experimentally. After experimental identification of glycosylation site occupancy, variably occupied sites can be removed. Removal of variably occupied glycosylation sites should not affect the activity of a glycoprotein as these glycans are only present in a proportion of the sample and therefore must not be important for folding and solubility.
4.1. Identification methods
Analysis of glycosylation site occupancy is usually carried out using mass spectrometry. For a review of recent developments in glycoprotein analysis and glycomics see Zaia . In the most straightforward experiment, the occupancy of an N-glycosylation site can be determined using a combination of tryptic digest and PNGase F treatment, followed by analysis of the resulting peptide . The peptide contains asparagine at the proposed site if no glycan is present and, due to the action of PNGase F, an aspartic acid if sugars are attached. As glycopeptides are not readily ionized within a mass spectrometer, they can be enriched in the sample using hydrophilic interaction liquid chromatography (HILIC), thus increasing the likelihood of a glycosylation site-containing peptide being detected [74-75]. Mapping of glycoproteins, including both analysis of the glycosylation site and analysis of the glycans themselves, has been miniaturized and automated, such as methods using functionalized magnetic nanoparticles  and integrating chromatography steps  which allow for rapid acquisition of results.
Determination of O-glycosylation site occupancy is more difficult than for N-glycoslation sites as there is no endoglycosidase which universally releases O-linked glycans in the same way that PNGase F does for N-linked glycans. However, Halfinger
Variable occupancy of glycosylation sites has also been detected by mutation studies. Here all the potential sites are mutated and the resulting protein analysed for activity. For example, Garman
4.2. Removal of glycosylation sites
Glycosylation sites found to be non-essential are often removed using site directed mutagenesis of the asparagine codon to a glutamine codon as this amino acid is similar in size and charge, but is not an attachment site for a glycan chain.
Formation of an N174Q mutant version of human ephrinA2 was essential in obtaining crystals of the EphA4:ephrinA2 complex which resulted in the structure being solved to 2.3 Å resolution (PDB entry 2WO3) . In contrast, previous attempts at crystallization using wild type ephrinA2 with EphA4 were not successful. Although the N174 was shown to be invariably glycosylated, this site is not conserved across the ephrin family, (Nettleship, Bowden, unpublished results), and therefore was presumed to be non-essential.
In the case of human alpha-N-acetylgalactosaminidase, which has five N-glycosylation sites, mutation of N201 to glutamine led to crystals giving diffraction to 1.9 Å resolution (PDB entry 3H53) as opposed to the 8 Å resolution data collected using wild type glycoprotein crystals .
5. Structural studies
5.1. Crystallization and X-ray crystallography
Glycoproteins crystallize in a variety of conditions and trials are usually set up using the same screens as non-glycosylated proteins; such as those found in kits sold by Hampton Research, Molecular Dimensions and Emerald Biosystems. A number of reviews have addressed the problems connected with crystallization of glycoproteins such as the increase in surface entropy associated with large post-translational modifications and the microheterogenity of glycans [82-83]. However, it is to be noted that the presence of glycans can be an advantage for crystallization as they can form essential intermolecular contacts in crystal lattices . Indeed glycoprotein crystallization has a success rate of around 50 % which is comparable to that for non-glycosylated proteins . Methods around glycoprotein crystallization have developed to include automation and miniaturization using microscale crystallization techniques with as little as 65 μg of protein sample .
The strategy which is unique to the crystallization of glycoproteins is manipulation of the glycoform as described above. Such manipulations affect the propensity of a glycosylated protein to crystallize and also the quality of the resulting crystals. As shown in Figure 6, the type of oligosaccharide attached to the protein affects crystal morphology. Interestingly, in the case of human IgE-FcεRIα, the Man5GlcNAc2 glycoform gave better diffracting crystals than the shorter Man1GlcNAc2 (Figure 6E and 6D respectively) and therefore setting up crystallization trials with more than one glycoform can be advantageous.
X-ray crystal structures of glycoproteins often only show the initial GlcNAc residue even if more sugar residues are known to be attached to the protein because the glycan chains are flexible and so electron density for further sugar residues is not present. In rare cases, more of the oligosaccharide chain is resolved due to it either forming crystal contacts or interacting with the polypeptide chain. For example Figure 7 shows that the oligosaccharide chain attached to N297 of human IgG1-Fc could be fully resolved (PDB entry 2WAH) .
In solving a glycoprotein structure, it is important that the carbohydrate chains are built into the relevant electron density taking into account the restrictions on sugar conformation, including torsion angles, and linkages found in nature [87-88]. Recently, Crispin
5.2. NMR spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy can be used to study protein structure, but the technique faces some obstacles in terms of its use for glycoproteins, namely the 1H chemical shift overlap between carbohydrate and protein signal  and the difficulty of obtaining sufficient yield of labelled protein from eukaryotic systems. These challenges have led to the majority of glycoproteins structures solved by NMR being of the aglycosylated protein chains expressed in
Labelling with 15N or both 15N and 13C has been demonstrated in mammalian cells , insect cells  and yeast . Such eukaryotic expression systems have been used to solve a few recombinant glycoprotein structures containing glycans such as fragments of human fibronectin (PBD entry 1E8B), human thrombomadulin (PBD entry 1DQB) and the extracellular domain of human cytotoxic T lymphocylte-associated protein (CTLA)-4 (PDB entry 1AH1) [100-102].
The problem of signal overlap between proteins and carbohydrates has been tackled by Slynko
In NMR structures, the protein chains and attached glycans are in solution and so are able to move around. Unlike in crystal structures, solution NMR allows the flexibility of both the oligosaccharide chain and the protein chain around a glycosylation site to be observed. In the case of AcrA (PDB entry 2K32), the flexibility was seen to be in the α-helical loop region containing the N42 glycosylation site (Figure 8A), with the heptasaccharide glycan having a well defined rod-like structure . Human CTLA4 (PDB entry 1AH1) contains two N-glycosylation sites, N78 and N111 which are occupied with partially deglycosylated glycans of the form Man1GlcNAc2 (Figure 8B). The glycan attached to N78 interacts extensively with the side chains of a nearby β-sheet and is therefore ordered, whereas N111 had limited interaction with the protein chain and so only the initial GlcNAc residue is well defined .
6. Conclusions and future prospects
This chapter has reviewed some of the recent developments in the field of glycoprotein structural biology. Increasingly, the expression system of choice for the production of recombinant glycoproteins is the mammalian cell, and in particular HEK in the presence of kifunensine which modifies glycan processing. This enables treatment of the purified glycoprotein with endoglycosidase to reduce the sugar “load” to a single GlcNAc at each N-linked attachment site. Experience shows that by preparing glycoproteins with different glycoforms the chances of obtaining diffraction quality crystals are significantly increased. Combining this approach with the inclusion of novel additives in the crystallization experiment, for example “smart materials”, such as the molecularly imprinted polymers (MIPs) recently reported by Sarkidakis
Future developments in simpler low cost expression technology as exemplified by
The author would like to thank Ray Owens and Max Crispin for critical reading of the manuscript. The OPPF-UK is funded by the UK Medical Research Council and Biotechnology and Biological Sciences Research Council.
Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol. Nettleship J. E. Assenberg R. Diprose J. M. Rahman-Huq N. Owens R. J. 2010Oct; 172 1 55 65
On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. Apweiler R. Hermjakob H. Sharon N. 1999Dec 6; 1473 1 4 8
Computational analysis reveals abundance of potential glycoproteins in Archaea, Bacteria and Eukarya. Bioinformation. Zafar S. Nasir A. Bokhari H. 2011 6 9 352 5
Solutions to the Glycosylation Problem for Low- and High-Throughput Structural Glycoproteomics. In: Owens RJ, Nettleship JE, editors. Functional and Structural Proteomics of Glycoproteins Davis S. J. Crispin M. 2011 127 158
Historical Background and Overview. Varki A. Sharon N. 2009
Parodi AJ.Protein glucosylation and its role in protein folding. Annu Rev Biochem. 2000 69 69 93
An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat Chem Biol. JD Valderrama-Rincon Fisher. A. C. Merritt J. H. Fan Y. Y. CA Reading Chhiba. K. et al. 2012 8 5 434 6
Glycobiology. Guarino C. Delisa M. P. A. prokaryote-based cell-free. translation system. that efficiently. synthesizes glycoproteins. 2011Nov 8;8:8.
Hendrickson WA, Horton JR, LeMaster DM.Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. Embo J. 1990May; 9 5 1665 72
Watson AA, Christou CM, O’Callaghan CA. Expression, purification and crystallization of the human UL16-binding protein ULBP1.Protein Expr Purif. 2011Sep; 79 1 44 8
Inflammation-Associated Nitrotyrosination Affects TCR Recognition through Reduced Stability and Alteration of the Molecular Surface of the MHC Complex. PLoS One. Madhurantakam C. Duru A. D. Sandalova T. Webb J. R. Achour A. Inflammation 2012e32805.
New pyrazolyl and thienyl aminohydantoins as potent BACE1 inhibitors: exploring the S2’ region. Bioorg Med Chem Lett. MS Malamas Erdei. J. Gunawan I. Barnes K. Hui Y. Johnson M. et al. 2011Sep 15; 21 18 5164 70
Structure. Lee H. J. Hota P. K. Chugha P. Guo H. Miao H. Zhang L. et al. N. M. R. structure of. a. heterodimeric S. A. M. S. A. M. complex characterization. manipulation of. Eph A. binding reveal. new cellular. functions of. S. H. I. P. 2012Jan 11; 20 1 41 55
The crystal structure of human alpha Meining W. Skerra A. 1microglobulin reveals a potential haem-binding site. Biochem J. 2012Apr 19;19:19.
Harrison RL, Jarvis DL.Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce "mammalianized" recombinant glycoproteins. Adv Virus Res. 2006 68 159 91
Structural basis for Fc gammaRIIa recognition of human IgG and formation of inflammatory signaling complexes. J Immunol. Ramsland P. A. Farrugia W. Bradford T. M. Sardjono C. T. Esparon S. Trist H. M. et al. 2011Sep 15; 187 6 3208 17
Fan QR, Hendrickson WA.Structure of human follicle-stimulating hormone in complex with its receptor. Nature. 2005Jan 20; 433 7023 269 77
Probability analysis of variational crystallization and its application to gp120, the exterior envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1). J Biol Chem. Kwong P. D. Wyatt R. Desjardins E. Robinson J. Culp J. S. BD Hellmig et. al 1999Feb 12; 274 7 4115 23
Structural basis of chemokine sequestration by a tick chemokine binding protein: the crystal structure of the complex between Evasin-1 and CCL3. PLoS One. Dias J. M. Losberger C. Deruaz M. CA Power Proudfoot. A. E. Shaw J. P. 2009e8514.
Selenium incorporation using recombinant techniques. Acta Crystallogr D Biol Crystallogr. Walden H. 2010Apr;66(Pt 4):352-7.
Crystallization and preliminary diffraction studies of the ectodomain of the envelope glycoprotein D from herpes simplex virus 1 alone and in complex with the ectodomain of the human receptor HveA. Acta Crystallogr D Biol Crystallogr. Carfi A. Gong H. Lou H. Willis S. H. Cohen G. H. Eisenberg R. J. et al. 2002May;58(Pt 5):836-8.
The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc Natl Acad Sci U S A. Bellizzi J. J. 3rd Widom J. Kemp C. Lu J. Y. Das A. K. Hofmann S. L. et al. 2000Apr 25; 97 9 4573 8
Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells. Protein Sci. Cronin C. N. Lim K. B. Rogers J. 2007Sep; 16 9 2023 9
Recombinant protein production in yeasts. Methods Mol Biol. Mattanovich D. Branduardi P. Dato L. Gasser B. Sauer M. Porro D. 2012 824 329 58
Damasceno LM, Huang CJ, Batt CA.Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol. 2012Jan; 93 1 31 9
Purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris. J Biochem. Noguchi S. Satow Y. 2006Dec; 140 6 799 804
Structural basis for receptor recognition of vitamin-B(12)-intrinsic factor complexes. Nature. Andersen C. B. Madsen M. Storm T. Moestrup S. K. Andersen G. R. 2010Mar 18; 464 7287 445 8
Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. Ruiz-Carrillo D. Koch B. Parthier C. Wermann M. Dambe T. Buchholz M. et al. 2011Jul 19; 50 28 6280 8
Huang KF, Liu YL, Cheng WJ, Ko TP, Wang AH.Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. Proc Natl Acad Sci U S A. 2005Sep 13; 102 37 13117 22
Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein. J Struct Biol. Laurila M. R. Salgado P. S. Makeyev E. V. Nettelship J. Stuart D. I. Grimes J. M. et al. 2005Jan; 149 1 111 5
Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing. Proc Natl Acad Sci U S A. Malkowski M. G. Quartley E. Friedman A. E. Babulski J. Kon Y. Wolfley J. et al. 2007Apr 17; 104 16 6678 83
Biologicals. Huh S. H. Do Lim H. J. Kim H. Y. Choi D. K. Song S. J. et H. al Optimization. of . Da k. linear polyethylenimine. for efficient. gene delivery. 2007Jun; 35 3 165 71
Efficiency of gene transfection reagents in NG108-15, SH-SY5Y and CHO-K1 cell lines. Methods Find Exp Clin Pharmacol. Martin-Montanez E. Lopez-Tellez J. F. MJ Acevedo Pavia. J. Khan Z. U. 2010Jun; 32 5 291 7
Episomal vectors for gene expression in mammalian cells. Eur J Biochem. Van Craenenbroeck K. Vanhoenacker P. Haegeman G. 2000Sep; 267 18 5665 78
Expression of a secreted protein in mammalian cells using baculovirus particles. Methods Mol Biol. BA Jardin Elias. C. B. Prakash S. 2012 801 41 63
BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Protein Expr Purif. Dukkipati A. Park H. H. Waghray D. Fischer S. Garcia K. C. Bac 2008Dec; 62 2 160 70
Automation of large scale transient protein expression in mammalian cells. J Struct Biol. Zhao Y. Bishop B. Clay J. E. Lu W. Jones M. Daenke S. et al. 2011Aug; 175 2 209 15
Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proc Natl Acad Sci U S A. Gonzalez R. Jennings L. L. Knuth M. Orth A. P. Klock H. E. Ou W. et al. 2010Feb 23; 107 8 3552 7
Acta Crystallogr D Biol Crystallogr. Aricescu A. R. Lu W. Jones E. Y. A. time cost-efficient system. for high-level. protein production. in mammalian. cells 2006Oct;62(Pt 10):1243-50.
Design of Experiment in CHO and HEK transient transfection condition optimization. Protein Expr Purif. Bollin F. Dechavanne V. Chevalet L. 2011Jul; 78 1 61 8
Wurm FM.Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004Nov; 22 11 1393 8
Automation of cell line development. Cytotechnology. Lindgren K. Salmen A. Lundgren M. Bylund L. Ebler A. Faldt E. et al. 2009Jan; 59 1 1 10
Rapid protein production using CHO stable transfection pools. Biotechnol Prog. Ye J. Alvin K. Latif H. Hsu A. Parikh V. Whitmer T. et al. 2010Sep-Oct; 26 5 1431 7
Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells. Protein Sci. Barton W. A. Tzvetkova-Robev D. Erdjument-Bromage H. Tempst P. Nikolov D. B. 2006Aug; 15 8 2008 13
Lee JE, Fusco ML, Saphire EO.An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat Protoc. 2009 4 4 592 604
Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B Davis S. J. Ikemizu S. Collins A. V. Fennelly J. A. Harlos K. Jones E. Y. et al. 7 1Acta Crystallogr D Biol Crystallogr. 2001Apr;57(Pt 4):605-8.
Carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions. J Mol Biol. Crispin M. Bowden T. A. Coles C. H. Harlos K. Aricescu A. R. Harvey D. J. et al. 2009Apr 17; 387 5 1061 6
Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat Struct Mol Biol. Bishop B. Aricescu A. R. Harlos K. CA O’Callaghan Jones. E. Y. Siebold C. 2009Jul; 16 7 698 703
Use of the alpha-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells. Acta Crystallogr Sect F Struct Biol Cryst Commun. Yu C. Crispin M. Sonnen A. F. Harvey D. J. Chang V. T. Evans E. J. et al. 2011Jul 1;67(Pt 7):785-9.
Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J Biol Chem. North S. J. Huang H. H. Sundaram S. Jang-Lee J. Etienne A. T. Trollope A. et al. 2010Feb 19; 285 8 5759 75
Structure. Chang V. T. Crispin M. Aricescu A. R. Harvey D. J. Nettleship J. E. Fennelly J. A. et al. Glycoprotein structural. genomics solving. the glycosylation. problem 2007Mar; 15 3 267 73
Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. PLoS One. Wilke S. Groebe L. Maffenbeier V. Jager V. Gossen M. Josewski J. et al. 2011e27829.
Fischer PB, Karlsson GB, Butters TD, Dwek RA, Platt FM.N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with changes in antibody recognition of the 1V2 region of gp120. J Virol. 1996Oct;70(10):7143-52.
Effects of N-butyldeoxynojirimycin and the Lec18.104.22.168 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization. Protein Sci. Butters T. D. Sparks L. M. Harlos K. Ikemizu S. Stuart D. I. Jones E. Y. et al. 1999Aug; 8 8 1696 701
Glycobiology. Crispin M. Harvey D. J. Chang V. T. Yu C. Aricescu A. R. Jones E. Y. et al. Inhibition of. hybrid complex-type glycosylation. reveals the. presence of. the Glc. N. Ac transferase. I-independent fucosylation. pathway 2006Aug; 16 8 748 56
Structural basis for cell surface patterning through NetrinG-NGL interactions. Embo J. Seiradake E. Coles C. H. Perestenko P. V. Harlos K. Mc Ilhinney R. A. Aricescu A. R. et al. 2011Nov 2; 30 21 4479 88
Crystal structure of the GluR2 amino-terminal domain provides insights into the architecture and assembly of ionotropic glutamate receptors. J Mol Biol. Clayton A. Siebold C. Gilbert R. J. Sutton G. C. Harlos K. Mc Ilhinney R. A. et al. 2009Oct 9; 392 5 1125 32
Structural and functional insights into endoglin ligand recognition and binding. PLoS One. Alt A. Miguel-Romero L. Donderis J. Aristorena M. Blanco F. J. Round A. et al. 2012e29948.
Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae. Acta Crystallogr Sect F Struct Biol Cryst Commun. Gazdag E. M. Cirstea I. C. Breitling R. Lukes J. Blankenfeldt W. Alexandrov K. 2010Aug 1;66(Pt 8):871-7.
Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif. Breitling R. Klingner S. Callewaert N. Pietrucha R. Geyer A. Ehrlich G. et al. 2002Jul; 25 2 209 18
Trends Biotechnol. Katzen F. Chang G. Kudlicki W. The past. present future of. cell-free protein. synthesis 2005Mar; 23 3 150 6
Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. Walter P. Blobel G. 1983 96 84 93
Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment. Appl Microbiol Biotechnol. Tarui H. Murata M. Tani I. Imanishi S. Nishikawa S. Hara T. 2001May; 55 4 446 53
J Biotechnol. Mikami S. Kobayashi T. Yokoyama S. Imataka H. A. hybridoma-based in. vitro translation. system that. efficiently synthesizes. glycoproteins 2006Dec 15; 127 1 65 78
Preparation of a cell-free translation system from PC12 cell. Neurochem Res. Shibutani M. Kim E. Lazarovici P. Oshima M. Guroff G. 1996Jul; 21 7 801 7
Curr Pharm Biotechnol. Ohashi H. Kanamori T. Shimizu Y. Ueda T. A. highly controllable. reconstituted cell-free. system--a breakthrough. in protein. synthesis research. 2010Apr; 11 3 267 71
Do N-glycoproteins have preference for specific sequons? Bioinformation. Rao R. S. Bernd W. 2010 5 5 208 12
Petrescu AJ, Milac AL, Petrescu SM, Dwek RA, Wormald MR.Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology. 2004Feb; 14 2 103 14
N-glycosylation efficiency is determined by the distance to the C-terminus and the amino acid preceding an Asn-Ser-Thr sequon. Protein Sci. Bano-Polo M. Baldin F. Tamborero S. MA Marti-Renom Mingarro. I. 2011Jan; 20 1 179 86
Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput. Gupta R. Brunak S. 2002 2002 310 22
Glycobiology. Julenius K. Molgaard A. Gupta R. Brunak S. Prediction conservation. analysis structural characterization. of mammalian. mucin-type O-glycosylation. sites 2005Feb; 15 2 153 64
Mass spectrometry and glycomics. Omics. Zaia J. 2010Aug; 14 4 401 18
Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry. Anal Biochem. Nettleship J. E. Aplin R. Aricescu A. R. Evans E. J. Davis S. J. Crispin M. et al. 2007Feb 1; 361 1 149 51
Nettleship JE.Hydrophilic Interaction Liquid Chromatography in the Characterization of Glycoproteins. In: Wang PG, He W, editors. Hydrophilic Interaction Liquid Chromatography (HILIC) and Advanced Applications: CRC Press; 2011 523 550
J, Elortza F, Jensen ON, Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J Proteome Res. Hagglund P. Bunkenborg J. Elortza F. Jensen O. N. Roepstorff P. A. new strategy. for identification. of N-glycosylated. proteins unambiguous assignment. of their. glycosylation sites. using H. I. L. I. C. enrichment partial deglycosylation. 2004May-Jun; 3 3 556 66
Kuo CW, Wu IL, Hsiao HH, Khoo KH.Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles. Anal Bioanal Chem. 2012Jan 29;29:29.
Integrated sample pretreatment system for N-linked glycosylation site profiling with combination of hydrophilic interaction chromatography and PNGase F immobilized enzymatic reactor via a strong cation exchange precolumn. Anal Chem. Qu Y. Xia S. Yuan H. Wu Q. Li M. Zou L. et al. 2011Oct 1; 83 19 7457 63
Evaluation of non-reductive beta-elimination/Michael addition for glycosylation site determination in mucin-like O-glycopeptides. Electrophoresis. Halfinger B. Sarg B. Lindner H. H. 2011Dec; 32 24 3546 53
Garman SC, Wurzburg BA, Tarchevskaya SS, Kinet JP, Jardetzky TS.Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha. Nature. 2000Jul 20; 406 6793 259 66
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling. Structure. Bowden T. A. Aricescu A. R. Nettleship J. E. Siebold C. Rahman-Huq N. Owens R. J. et al. 2009Oct 14; 17 10 1386 97
Clark NE, Garman SC. The 1.9 a structure of human alpha-N-acetylgalactosaminidase: The molecular basis of Schindler and Kanzaki diseases.J Mol Biol. 2009Oct 23; 393 2 435 47
- 82. Mesters JR, Hilgenfeld R. Protein Glycosylation, Sweet to Crystal Growth? Cryst Growth Des. 2007;7(11):2251-3.
Stura EA, Nemerow GR, Wilson IA.Strategies in the crystallization of glycoproteins and protein complexes. J Cryst Growth. 1992
Proc Natl Acad Sci U S A. Lin D. Y. Tanaka Y. Iwasaki M. Gittis A. G. Su H. P. Mikami B. et al. The P. D- -L P. D. complex resembles. the antigen-binding. Fv domains. of antibodies. cell T. receptors 2008Feb 26; 105 8 3011 6
Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcvarepsilonRI. Nat Struct Mol Biol. MD Holdom Davies. A. M. Nettleship J. E. Bagby S. C. Dhaliwal B. Girardi E. et al. 2011May; 18 5 571 6
Schrödinger LLC. The PyMOL molecular graphics system.ed.
Building meaningful models of glycoproteins. Nat Struct Mol Biol. Crispin M. Stuart D. I. Jones E. Y. 2007May;14(5):354; discussion-5.
Structure and Biosynthesis of Glycoprotein Carbohydrates. In: Moo-Young M, editor. Comprehensive Biotechnology. 2 ed: Elsevier; Crispin M. Scanlan C. N. Bowden T. A. 2011 73 90
Bioinformatics Databases and Applications Available for Glycobiology and Glycomics. In: Owens RJ, Nettleship JE, editors. Functional and Structural Proteomics of Glycoproteins Ranzinger R. Maaβ K. Lütteke T. 2011 59 90
GlycomeDB--a unified database for carbohydrate structures. Nucleic Acids Res. Ranzinger R. Herget S. von der Lieth. C. W. Frank M. Glycome D. 2011Jan;39(Database D373-6 373 6
SWEET- WWW-basedrapid 3D construction of oligo- and polysaccharides. Bioinformatics. Bohne A. Lang E. von der Lieth. C. W. S. W. E. E. 1999Sep; 15 9 767 8
Woods Group. GLYCAM Web.Complex Carbohydrate Research Center, University of Georgia, Athens, GA 2005 2012
GlyProt: in silico glycosylation of proteins. Nucleic Acids Res. Bohne-Lang A. von der Lieth. C. W. Gly 2005Jul 1;33(Web Server W214-9 214 9
pdb-care (PDB carbohydrate residue check): a program to support annotation of complex carbohydrate structures in PDB files. BMC Bioinformatics. Lutteke T. von der Lieth. C. W. 2004Jun 4;5(69):69.
Structure. Read R. J. Adams P. D. Arendall W. B. 3rd Brunger A. T. Emsley P. Joosten R. P. et al. A. new generation. of crystallographic. validation tools. for the. protein data. bank 2011Oct 12; 19 10 1395 412
J Am Chem Soc. Slynko V. Schubert M. Numao S. Kowarik M. Aebi M. Allain F. H. N. M. R. structure determination. of a. segmentally labeled. glycoprotein using. in vitro. glycosylation 2009Jan 28; 131 3 1274 81
Egorova-Zachernyuk TA, Bosman GJ, Degrip WJ.Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium. Appl Microbiol Biotechnol. 2011Jan; 89 2 397 406
Isotope labeling in insect cells. Methods Mol Biol. Saxena K. Dutta A. Klein-Seetharaman J. Schwalbe H. 2012 831 37 54
Pickford AR, O’Leary JM.Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris. Methods Mol Biol. 2004 278 17 33
The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding. Embo J. Pickford A. R. Smith S. P. Staunton D. Boyd J. Campbell I. D. 2001Apr 2; 20 7 1519 29
Wood MJ, Sampoli Benitez BA, Komives EA.Solution structure of the smallest cofactor-active fragment of thrombomodulin. Nat Struct Biol. 2000Mar; 7 3 200 4
Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28. Nat Struct Biol. Metzler W. J. Bajorath J. Fenderson W. Shaw S. Y. Constantine K. L. Naemura J. et al. 1997Jul; 4 7 527 31
N-linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase. Science. Kowarik M. Numao S. Feldman M. F. Schulz B. L. Callewaert N. Kiermaier E. et al. 2006Nov 17; 314 5802 1148 50
Protein crystallization facilitated by molecularly imprinted polymers. Proc Natl Acad Sci U S A. Saridakis E. Khurshid S. Govada L. Phan Q. Hawkins D. Crichlow G. V. et al. 2011Jul 5; 108 27 11081 6