High‐Throughput Cloning, Expression and Purification Technologies

Kreusch, Andreas; Lesley, Scott A.

doi:10.1002/0470012536.ch7

Cited by 3 publications

(4 citation statements)

References 29 publications

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“…Host cells used for the biosynthesis of recombinant proteins are relatively complex systems extending from bacteria (e.g., Escherichia coli), [1][2][3][4][5] to yeasts (e.g., Pichia pastoris) [6][7][8][9][10] and to eukaryotic cells such as chinese hamster ovarian cells (CHO). 11 During culture, these cells secrete a very large number of their own proteins that can easily contaminate the recombinant DNA product. [12][13][14][15][16] Even after sophisticated purification steps, significant levels of host cell proteins (HCP) may still remain present in the final purified biopharmaceutical.…”

Section: Introductionmentioning

confidence: 99%

“…Both are complex starting materials full of thousands of proteins that are potential impurities for the final product that may, in rare cases, cause adverse events in the patient ranging from a slight fever to long-term immunogenicity to toxic and, in even rarer cases, fatal events. Host cells used for the biosynthesis of recombinant proteins are relatively complex systems extending from bacteria (e.g., Escherichia coli ), − to yeasts (e.g., Pichia pastoris ) − and to eukaryotic cells such as chinese hamster ovarian cells (CHO) . During culture, these cells secrete a very large number of their own proteins that can easily contaminate the recombinant DNA product. − Even after sophisticated purification steps, significant levels of host cell proteins (HCP) may still remain present in the final purified biopharmaceutical. − Although host cell impurities are mostly innocuous to the receiving patient, regulatory agencies require demonstrations that HCPs are not only minimized but also analyzed with the most sensitive available methods.…”

Section: Introductionmentioning

confidence: 99%

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A New Approach for the Detection and Identification of Protein Impurities Using Combinatorial Solid Phase Ligand Libraries

et al. 2006

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We propose a novel method for detection of protein impurities present in plasma-derived and recombinant purified injectable biopharmaceuticals by enhancing the concentration of protein impurities, in essence "amplifying" their presence to detectable levels. The method is based on the capture of proteins using a combinatorial solid-phase hexapeptides ligand library previously described for the reduction of protein concentration difference in biological fluids. Three proteins have been investigated: Staphylococcus aureus Protein A, expressed in Escherichia coli and supplied as 99% pure, recombinant human albumin, expressed in Pichia pastoris and certified as 95% pure, and therapeutic albumin supplied as 96-98% pure injectable solution. In all cases, after treatment with the ligand libraries, a number of additional polypeptide chains, not visible in the control, could be detected and obtained in sufficient amounts for MS analysis. In the cases of the two recombinant proteins, it could be demonstrated that a number of these polypeptide chains were host cell proteins still present in the purified product. In addition, a substantial number of these spots were found to be cleavage products of the original recombinant DNA species. Such cleavage products were particularly abundant in the recombinant human albumin preparation. From pure injectable serum albumin, a number of human plasma protein impurities were also identified by LC-MS/MS analysis. Treatment with ligand libraries of purified proteins is thus seen as a very powerful method of capture and concentration of host proteins and cleaved products for further analysis to control better the quality of industrial biotechnology products.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A New Approach for the Detection and Identification of Protein Impurities Using Combinatorial Solid Phase Ligand Libraries

et al. 2006

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“…EDC and NHS (Lambert and Buckle, 2006) large-scale expression and purification of 96 proteins in parallel using recombinant expression systems (Kreusch and Lesley, 2004). Such methods for the elucidation of tertiary structures of proteins have potentially important applications in protein biotechnological products, such as comparison of batch to batch variability, particularly for proteins refolded from inclusion bodies, as well as comparison of tertiary structures of upcoming generic or "follow-on" protein products.…”

Section: Protein Function Cross-linking Reagents Usedmentioning

confidence: 99%

Three Dimensional Structures of Proteins and Protein Complexes from Chemical Cross-Linking and Mass Spectrometry: A Biochemical and Computational Overview

Chakravarti¹,

Lewis²,

Chakravarti³

et al. 2006

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In recent years, protein cross-linking technology using appropriate chemical cross-linking reagents and identification of cross-linked peptides and proteins by mass spectrometry has been used successfully to elucidate the intra-and inter-molecular interactions of protein molecules. Identification of the cross-linking sites within a single polypeptide chain or across polypeptide chains of a protein can provide insight into spatial distance constraints within the molecular structure and hence aid in the prediction of the low resolution three dimensional structure of the protein. In addition, finding cross-linked peptides in a multimeric protein complex is useful for deducing the composition, kinetics, nature of interaction and contact sites of the participating protein molecules in the complex. Here, we describe: (i) commonly used chemical reagents for cross-linking specific amino acid residues in protein molecules, (ii) application of mass spectrometry following digestion of cross-linked proteins with appropriate enzymes of known specificity, (iii) computer software tools for mass spectral data analysis to locate possible sites of intra-and/or inter-molecular cross-linking of protein molecules, and (iv) computational methods for predicting low resolution structure with the help of experimentally identified cross-links. We also discuss various specific examples of the application of the cross-linking technology to the identification of intramolecular and inter-molecular interactions of protein molecules.

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“…Presently, there is a movie of a large-scale fermenter in action [35], as well as narrated demonstrations of technical advances in crystallography [36–38] and NMR [7]. …”

Section: Introductionmentioning

confidence: 99%

The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: a structural biology web resource

Gifford

Carter

Gabanyi

et al. 2012

J Struct Funct Genomics

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The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB (http://sbkb.org), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a Functional Sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.

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High‐Throughput Cloning, Expression and Purification Technologies

Cited by 3 publications

References 29 publications

A New Approach for the Detection and Identification of Protein Impurities Using Combinatorial Solid Phase Ligand Libraries

A New Approach for the Detection and Identification of Protein Impurities Using Combinatorial Solid Phase Ligand Libraries

Three Dimensional Structures of Proteins and Protein Complexes from Chemical Cross-Linking and Mass Spectrometry: A Biochemical and Computational Overview

The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: a structural biology web resource

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