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Current methods in structural proteomics and its applications in biological sciences

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.


Protein production. a Glycosylation: structure of a high mannose-type glycan. b Co-expression of a complex of four proteins with the pQLink system. M: marker, W: whole cellular protein, P: purified protein (Scheich et al. 2007). c Cell line development by recombinase-mediated cassette exchange (RMCE): cells are transfected with a vector containing a GFP gene flanked by recombination sites F3 and F and GFP-positive cells are isolated. Cassette exchange is initiated by co-transfecting a tagged cell line with an Flp recombinase expression vector and a targeting vector bearing the gene of interest (GOI). Flp recombinase exchanges the tagging gene cassette and a production cell line is obtained (Wilke et al. 2011)
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Fig3: Protein production. a Glycosylation: structure of a high mannose-type glycan. b Co-expression of a complex of four proteins with the pQLink system. M: marker, W: whole cellular protein, P: purified protein (Scheich et al. 2007). c Cell line development by recombinase-mediated cassette exchange (RMCE): cells are transfected with a vector containing a GFP gene flanked by recombination sites F3 and F and GFP-positive cells are isolated. Cassette exchange is initiated by co-transfecting a tagged cell line with an Flp recombinase expression vector and a targeting vector bearing the gene of interest (GOI). Flp recombinase exchanges the tagging gene cassette and a production cell line is obtained (Wilke et al. 2011)

Mentions: Animal cell lines are highly effective for the secretion of proteins with native glycosylation and disulfide bonds. Glycoproteins produced with the mammalian CHO or HEK293 cell lines carry heterogeneous, complex-type oligosaccharide chains attached to Ser/Thr (O-linked) or Asn (N-linked) side chains. Crystallization of glycoproteins is difficult because of the heterogeneity and flexible conformation of the bulky oligosaccharides, which can also mask possible sites of crystal contacts on the protein surface. Some glycosylation sites can be removed by mutagenesis. Regions with O-linked glycosylation are generally proline-rich and unfolded, and can be excluded from genetic constructs. However, many proteins require glycosylation for folding and transport through the secretory pathway. Enzymatic removal of N-linked glycans from the purified protein with endoglycosidase H or F leaves a single monosaccharide attached, which may increase the solubility of the deglycosylated protein. Enzymatic deglycosylation is efficient for oligosaccharides of the high-mannose type as obtained from the baculovirus system (Fig. 3a). Processing of N-linked glycans by mammalian cell lines results in complex-type oligosaccharides that are difficult to cleave enzymatically. Complex-type glycosylation can be prevented by chemical glycosylation inhibitors (Chang et al. 2007) or by mutating the host cells. The gene for the enzyme N-acetylglucosaminyl-transferase I (GnTI), which modifies high-mannose type oligosaccharides, has been mutated in the cell lines CHO Lec1, Lec3.2.8.1 (Stanley 1989) and HEK293S-GnTI(−) (Reeves et al. 2002). These cell lines and normal HEK293 cells treated with the glycosylation inhibitors kifunensine or swainsonine have enabled the production of many glycoproteins and their crystallization upon enzymatic deglycosylation (Aricescu et al. 2006; Chang et al. 2007; Davis et al. 1993; Standfuss et al. 2011). Optimized protocols and cell lines allow performing transient transfection of HEK293 at up to liter scale with inexpensive reagents (Aricescu et al. 2006). However, not all proteins can be produced in sufficient amounts by transient transfections. Stable cell lines allow the production of proteins more reproducibly and in much larger volumes in bioreactors. However, establishing lines with good performance requires considerable effort. Novel approaches of stable cell line development, based on preparative cell sorting and recombinase-mediated cassette exchange (RMCE Fig. 3c), combine faster development times with improved performance and have been used successfully for X-ray crystallography studies (Wilke et al. 2010, 2011).Fig. 3


Current methods in structural proteomics and its applications in biological sciences
Protein production. a Glycosylation: structure of a high mannose-type glycan. b Co-expression of a complex of four proteins with the pQLink system. M: marker, W: whole cellular protein, P: purified protein (Scheich et al. 2007). c Cell line development by recombinase-mediated cassette exchange (RMCE): cells are transfected with a vector containing a GFP gene flanked by recombination sites F3 and F and GFP-positive cells are isolated. Cassette exchange is initiated by co-transfecting a tagged cell line with an Flp recombinase expression vector and a targeting vector bearing the gene of interest (GOI). Flp recombinase exchanges the tagging gene cassette and a production cell line is obtained (Wilke et al. 2011)
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Related In: Results  -  Collection

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Fig3: Protein production. a Glycosylation: structure of a high mannose-type glycan. b Co-expression of a complex of four proteins with the pQLink system. M: marker, W: whole cellular protein, P: purified protein (Scheich et al. 2007). c Cell line development by recombinase-mediated cassette exchange (RMCE): cells are transfected with a vector containing a GFP gene flanked by recombination sites F3 and F and GFP-positive cells are isolated. Cassette exchange is initiated by co-transfecting a tagged cell line with an Flp recombinase expression vector and a targeting vector bearing the gene of interest (GOI). Flp recombinase exchanges the tagging gene cassette and a production cell line is obtained (Wilke et al. 2011)
Mentions: Animal cell lines are highly effective for the secretion of proteins with native glycosylation and disulfide bonds. Glycoproteins produced with the mammalian CHO or HEK293 cell lines carry heterogeneous, complex-type oligosaccharide chains attached to Ser/Thr (O-linked) or Asn (N-linked) side chains. Crystallization of glycoproteins is difficult because of the heterogeneity and flexible conformation of the bulky oligosaccharides, which can also mask possible sites of crystal contacts on the protein surface. Some glycosylation sites can be removed by mutagenesis. Regions with O-linked glycosylation are generally proline-rich and unfolded, and can be excluded from genetic constructs. However, many proteins require glycosylation for folding and transport through the secretory pathway. Enzymatic removal of N-linked glycans from the purified protein with endoglycosidase H or F leaves a single monosaccharide attached, which may increase the solubility of the deglycosylated protein. Enzymatic deglycosylation is efficient for oligosaccharides of the high-mannose type as obtained from the baculovirus system (Fig. 3a). Processing of N-linked glycans by mammalian cell lines results in complex-type oligosaccharides that are difficult to cleave enzymatically. Complex-type glycosylation can be prevented by chemical glycosylation inhibitors (Chang et al. 2007) or by mutating the host cells. The gene for the enzyme N-acetylglucosaminyl-transferase I (GnTI), which modifies high-mannose type oligosaccharides, has been mutated in the cell lines CHO Lec1, Lec3.2.8.1 (Stanley 1989) and HEK293S-GnTI(−) (Reeves et al. 2002). These cell lines and normal HEK293 cells treated with the glycosylation inhibitors kifunensine or swainsonine have enabled the production of many glycoproteins and their crystallization upon enzymatic deglycosylation (Aricescu et al. 2006; Chang et al. 2007; Davis et al. 1993; Standfuss et al. 2011). Optimized protocols and cell lines allow performing transient transfection of HEK293 at up to liter scale with inexpensive reagents (Aricescu et al. 2006). However, not all proteins can be produced in sufficient amounts by transient transfections. Stable cell lines allow the production of proteins more reproducibly and in much larger volumes in bioreactors. However, establishing lines with good performance requires considerable effort. Novel approaches of stable cell line development, based on preparative cell sorting and recombinase-mediated cassette exchange (RMCE Fig. 3c), combine faster development times with improved performance and have been used successfully for X-ray crystallography studies (Wilke et al. 2010, 2011).Fig. 3

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.