Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin‐binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

Li, Yifeng; Franklin, Sarah; Zhang, Michael J.; Vondriska, Thomas M.

doi:10.1002/pro.546

Cited by 23 publications

(18 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To facilitate immunoprecipitation studies, we engineered an N-terminal streptavidin-binding peptide (SBP) tag [21] in frame with the Noxa coding sequence. The coding sequence of SBP-Noxa was subsequently cloned into the lentiviral expression vector pUB-eGFP-Thy1.1 [22] (generously provided by Dr. Zhibin Chen, the University of Miami, Miami, FL) by replacing eGFP with SBP-Noxa.…”

Section: Methodsmentioning

confidence: 99%

Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells

et al. 2016

View full text Add to dashboard Cite

Bortezomib, a clinical drug for multiple myeloma (MM) and mantle cell lymphoma, exhibits complex mechanisms of action, which vary depending on the cancer type and the critical genetic alterations of each cancer. Here we investigated the signaling mechanisms of bortezomib in mouse B lymphoma and human MM cells deficient in a new tumor suppressor gene, TRAF3. We found that bortezomib consistently induced up-regulation of the cell cycle inhibitor p21(WAF1) and the pro-apoptotic protein Noxa as well as cleavage of the anti-apoptotic protein Mcl-1. Interestingly, bortezomib induced the activation of NF-κB1 and the accumulation of the oncoprotein c-Myc, but inhibited the activation of NF-κB2. Furthermore, we demonstrated that oridonin (an inhibitor of NF-κB1 and NF-κB2) or AD 198 (a drug targeting c-Myc) drastically potentiated the anti-cancer effects of bortezomib in TRAF3-deficient malignant B cells. Taken together, our findings increase the understanding of the mechanisms of action of bortezomib, which would aid the design of novel bortezomib-based combination therapies. Our results also provide a rationale for clinical evaluation of the combinations of bortezomib and oridonin (or other inhibitors of NF-κB1/2) or AD 198 (or other drugs targeting c-Myc) in the treatment of lymphoma and MM, especially in patients containing TRAF3 deletions or relevant mutations.

show abstract

Section: Methodsmentioning

confidence: 99%

Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells

et al. 2016

View full text Add to dashboard Cite

show abstract

“…To facilitate immunoprecipitation experiments, we engineered an N-terminal FLAG tag or a C-terminal SBP-6×His tag [88] in frame with the MCC coding sequence, respectively. We subsequently generated two lentiviral expression vectors of tagged hMCC, including pUB-FLAG-hMCC and pUB-hMCC-SBP-6×His.…”

Section: Methodsmentioning

confidence: 99%

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

Edwards¹,

Baron²,

Moore³

et al. 2014

J Hematol Oncol

View full text Add to dashboard Cite

BackgroundIdentification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells.MethodsWe used microarray analysis to identify genes differentially expressed in TRAF3−/− mouse splenic B lymphomas. We employed lentiviral vector-mediated knockdown or overexpression to manipulate gene expression in human multiple myeloma (MM) cell lines. We analyzed cell apoptosis and proliferation using flow cytometry, and performed biochemical studies to investigate signaling mechanisms. To delineate protein-protein interactions, we applied affinity purification followed by mass spectrometry-based sequencing.ResultsWe identified mutated in colorectal cancer (MCC) as a gene strikingly up-regulated in TRAF3-deficient mouse B lymphomas and human MM cell lines. Aberrant up-regulation of MCC also occurs in a variety of primary human B cell malignancies, including non-Hodgkin lymphoma (NHL) and MM. In contrast, MCC expression was not detected in normal or premalignant TRAF3−/− B cells even after treatment with B cell stimuli, suggesting that aberrant up-regulation of MCC is specifically associated with malignant transformation of B cells. In elucidating the functional roles of MCC in malignant B cells, we found that lentiviral shRNA vector-mediated knockdown of MCC induced apoptosis and inhibited proliferation in human MM cells. Experiments of knockdown and overexpression of MCC allowed us to identify several downstream targets of MCC in human MM cells, including phospho-ERK, c-Myc, p27, cyclin B1, Mcl-1, caspases 8 and 3. Furthermore, we identified 365 proteins (including 326 novel MCC-interactors) in the MCC interactome, among which PARP1 and PHB2 were two hubs of MCC signaling pathways in human MM cells.ConclusionsOur results indicate that in sharp contrast to its tumor suppressive role in colorectal cancer, MCC functions as an oncogene in B cells. Our findings suggest that MCC may serve as a diagnostic marker and therapeutic target in B cell malignancies, including NHL and MM.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0056-6) contains supplementary material, which is available to authorized users.

show abstract

“…Because of the high affinity and specificity of the SBP tag to streptavidin, this system has been successfully applied for one-step affinity purification of proteins [5], [7], [26], for studying protein-protein interactions [26], [27] and for developing improved tandem tags in protein studies [6], [27], [28], [29]. To make this powerful technology to be cost effective, development of an idealized streptavidin mutein, SAVSBPM18, is essential.…”

Section: Discussionmentioning

confidence: 99%

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Wong

2013

PLoS ONE

View full text Add to dashboard Cite

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

show abstract

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin‐binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

Cited by 23 publications

References 35 publications

Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells

Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

Contact Info

Product

Resources

About