High‐throughput protein production – Lessons from scaling up from 10 to 288 recombinant proteins per week

Tegel, Hanna; Stéen, Johanna; Konrad, Anna; Nikdin, Hero; Pettersson, Katarina; Stenvall, Maria; Tourle, Samuel; Wrethagen, Ulla; Xu, LanLan; Yderland, Louise; Uhlén, Mathias; Hober, Sophia; Ottosson, Jenny

doi:10.1002/biot.200800183

Cited by 40 publications

(43 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Production of Antigen Standards for Absolute Quantification-The expression vector pAff8c containing a fragment coding for the quantification standard HisABPOneStrep was transformed into E. coli Rosetta DE3 cells (Novagen, Merck, Darmstadt, Germany), and HisABPOneStrep was expressed according to the standard protocol used within the Human Protein Atlas project (25). After purification using immobilized metal ion affinity chromatography and buffer exchange to 1ϫ PBS (10 mM NaP, 150 mM NaCl, pH 7.3) on a PD-10 desalting column (GE Healthcare, Uppsala, Sweden), a second purification step was performed using a StrepTrap™ HP column (GE Healthcare) on an Ä KTAexplorer system (GE Healthcare) according to the suggested protocol.…”

Section: Methodsmentioning

confidence: 99%

“…Heavy isotopelabeled ( 13 C and 15 N) arginine and lysine (Cambridge Isotope Laboratories, Tewksbury, MA) and light versions of the remaining 18 amino acids (Sigma-Aldrich) were added to the medium to a final concentration of 200 g/ml. After cultivation, the cells were lysed and the PrESTs were purified according to the standard Human Protein Atlas protocol (25).…”

Section: Methodsmentioning

confidence: 99%

“…For 127 of these, the corresponding recombinant PrESTs were available. The protein fragments had previously been expressed in E. coli and purified as described before (25). The 127 PrESTs were pooled together in equimolar amounts and used for trypsin cleavage using a standardized protocol (24).…”

Section: Fig 1 the Principle Of The Immuno-silac Methodmentioning

confidence: 99%

See 2 more Smart Citations

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Edfors

Boström

Zeiler

et al. 2014

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng Mass spectrometry-based proteomics is fast developing in the direction of clinical applications. Therefore, reliable quantification methods for absolute protein concentration determination are indispensible tools for future applications. So far, enzyme-linked immunosorbent assays and similar antibodybased methods excel in the sensitive detection of low levels of proteins in complex matrices, whereas mass spectrometry enables unbiased approaches and can provide unsurpassed specificity. The fact that most proteomes have a very high dynamic range between high and low abundant proteins, in particular for clinical samples, such as plasma and serum, often makes it necessary to use protein depletion of the most abundant proteins (1, 2) and/or elaborate fractionations (3-5) before running the mass spectrometry analysis. This has prompted several investigators to introduce a protein or peptide capture step using specific antibodies to allow for immunoaffinity enrichment prior to the MS analysis. In this way, a "sandwich" assay is obtained, but instead of having a readout in the analysis step based on a second antibody, the analysis step is performed using MS. In such an approach, either the intact protein is captured using an anti-protein antibody (6) or a peptide derived from the protein is captured using an antipeptide antibody that has been raised to the target peptide of interest (7)(8)(9)(10)(11). This is the principle behind stable isotope standards and capture by anti-peptide antibodies (SISCAPA), 1 developed by

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Edfors

Boström

Zeiler

et al. 2014

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…For a summary of the types of proteins found by proteomics in blood and blood cells see the recent review (Liumbruno et al, 2009). At present, there are few immunological or other reagents to measure and assay these many newly discovered proteins (Tegel et al, 2009). For now, only mass spectrometry is in a technical position to exploit many of the new discoveries made in human blood.…”

Section: A Importance Of Studying Blood Proteins and Peptides By Masmentioning

confidence: 99%

“…Where reagents are available, affinity chromatography has been shown to be at least as sensitive as multiple partition chromatography methods at or below the ng/mL range (Heidema et al, 2009). However, given the limited number and cost of affinity reagents (Tegel et al, 2009), there remains a need to fractionate and discover blood proteins by standard separation techniques.…”

Section: Affinity Chromatographymentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

View full text Add to dashboard Cite

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

show abstract

Strategies to Optimize Protein Expression in E. coli

2010

View full text Add to dashboard Cite

Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.

show abstract

High‐throughput protein production – Lessons from scaling up from 10 to 288 recombinant proteins per week

Cited by 40 publications

References 9 publications

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

Mass spectrometry of peptides and proteins from human blood

Strategies to Optimize Protein Expression in E. coli

Contact Info

Product

Resources

About