The characterization of the tetratricopeptide repeat protein 13 in mammals

2012 ◽  
Author(s):  
◽  
Stephen G. Shannon
Keyword(s):  
Protein Expression ◽  
Sequence Similarity ◽  
Amino Acid Level ◽  
Pathogen Resistance ◽  
Current Evidence ◽  
Tetratricopeptide Repeat ◽  
Resistance Proteins ◽  
Mammalian Cell Lines ◽  
Mouse Tissues ◽  
Immune Tissues

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Tetratricopeptide repeat domain protein 13 (TTC13) is a member of a large family of proteins known as tetratricopeptide repeat (TPR) proteins that has more than 5,000 members (Zeytuni 2012). One common feature of all TPR proteins is that they all contain TPR domains, which are thought to mediate protein-protein interactions and/or protein scaffolding. Interestingly, these TPR proteins are highly conserved between species. For example, sequence similarity between SUPPRESSOR of rps4 -RLD (SRFR1) and TTC13 was not only restricted to the TPR domain but also observed throughout the full length of both proteins. Extensive evidence suggested that SRFR1 was involved in pathogen resistance in plants by regulating resistance proteins in Arabidopsis thaliana (Kwon 2009). However, the mechanism by which SRFR1 regulated resistance proteins still remains unresolved. In contrast, nothing is known about the mammalian ortholog of SRFR1, TTC13. The goal of my project was to characterize TTC13 and to, ultimately, determine if TTC13 serves a similar role in mammals as SRFR1 in plants. Sequence analysis revealed that SRFR1 had 26% identity and 46% similarity at the amino acid level with TTC13. To begin addressing the role of TTC13 in mammals, I analyzed the expression of TTC13 mRNA and protein in mouse tissues. These analyses indicated that TTC13 mRNA was ubiquitously expressed in all examined mouse tissues. However, TTC13 protein expression varied in each tissue. For example, TTC13 protein was highly expressed in both kidney and liver, whereas TTC13 protein expression was not detectable in other tissues that were positive for TTC13 mRNA. Moreover, moderate protein expression was detected in the lung, heart and immune tissues (e.g. thymus, spleen, lymph nodes, T and B cells). To determine the cellular distribution of TTC13, I performed immunoflouresence and subcellular fractionation experiments. pFlag-TTC13 was localized to the cytoplasm in COS-1 cells by immunofluorescence. Cytoplasmic localization was confirmed by immunoblotting cytoplasmic and nuclear fractions for TTC13. Taken together, current evidence suggested that TTC13 was a cytoplasmic protein that was highly expressed in kidney and liver. Interestingly, TTC13 was also expressed in mammalian immune tissues in low to moderate levels, suggesting that TTC13 may have a role in the mammalian immune system. I conducted cell viability and cell cycle experiments in mammalian cell lines to investigate if TTC13 functions similarly to other TPR proteins.

Tissue-specific mismatch repair protein expression: MSH3 is higher than MSH6 in multiple mouse tissues

DNA Repair ◽  
2013 ◽  
Vol 12 (1) ◽  
pp. 46-52 ◽  
Author(s):  
Stéphanie Tomé ◽  
Jodie P. Simard ◽  
Meghan M. Slean ◽  
Ian Holt ◽  
Glenn E. Morris ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mismatch Repair ◽  
Tissue Specific ◽  
Repair Protein ◽  
Mouse Tissues ◽  
Mismatch Repair Protein

scholarly journals The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells

2020 ◽  
Vol 48 (4) ◽  
pp. 1607-1626 ◽  
Author(s):  
Pawel J Sikorski ◽  
Marcin Warminski ◽  
Dorota Kubacka ◽  
Tomasz Ratajczak ◽  
Dominika Nowis ◽  
...  
Keyword(s):  
Protein Expression ◽  
Methylation Status ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
In Vitro Susceptibility ◽  
Mammalian Cell Lines ◽  
Expression Characteristic

Abstract 7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m6A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

scholarly journals Fast and accurate HLA typing from short-read next-generation sequence data with xHLA

2017 ◽  
Vol 114 (30) ◽  
pp. 8059-8064 ◽  
Author(s):  
Chao Xie ◽  
Zhen Xuan Yeo ◽  
Marie Wong ◽  
Jason Piper ◽  
Tao Long ◽  
...  
Keyword(s):  
Sequence Data ◽  
Sequence Similarity ◽  
Amino Acid Level ◽  
Hla Typing ◽  
Sequencing Data ◽  
Desktop Computer ◽  
Short Read ◽  
Short Read Sequencing ◽  
Hla Genes ◽  
Human Chromosome 6

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99–100% four-digit typing accuracy for both class I and II HLA genes, taking only∼3 min to process a 30× whole-genome BAM file on a desktop computer.

Analysis of Protein Expression and Virus-like Particle Formation in Mammalian Cell Lines Stably Expressing HIV-1 gag and env Gene Products with or without Active HIV Proteinase

Virology ◽  
1993 ◽  
Vol 192 (2) ◽  
pp. 605-617 ◽  
Author(s):  
Hans Georg Kräusslich ◽  
Christina Ochsenbauer ◽  
Anke-Mareil Traenckner ◽  
Klaus Mergener ◽  
Michael Fäcke ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Particle Formation ◽  
Gene Products ◽  
Mammalian Cell Lines ◽  
Virus Like Particle ◽  
Hiv 1

scholarly journals High Efficiency Recombinant Protein Purification Using mCherry and YFP Nanobody Affinity Matrices

2021 ◽  
Author(s):  
Anh T.Q. Cong ◽  
Taylor L. Witter ◽  
Matthew J. Schellenberg
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Protein Purification ◽  
Real Time ◽  
High Efficiency ◽  
Expression System ◽  
Expression Systems ◽  
Protein Fusion ◽  
Real Time Monitoring ◽  
Mammalian Cell Lines

Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the proteins native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target protein using X-ray crystallography. From these analyses we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human Topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.

scholarly journals The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

2019 ◽  
Author(s):  
Pawel J. Sikorski ◽  
Marcin Warminski ◽  
Dorota Kubacka ◽  
Tomasz Ratajczak ◽  
Dominika Nowis ◽  
...  
Keyword(s):  
Protein Expression ◽  
Methylation Status ◽  
Structural Diversity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
In Vitro Susceptibility ◽  
Mammalian Cell Lines ◽  
Expression Characteristic

ABSTRACT7-Methylguanosine 5’-cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5’-terminal nucleotides and additional methylations (2’-O-methylation and m6A). Currently available 5’-capping methods do not address this diversity. We report trinucleotide 5’-cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (± 2’-O-methylation). HPLC-purified mRNAs carrying these 5’ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa, and JAWS II). In all cases the highest expression was achieved for mRNAs carrying 5’-terminal A and m6A, whereas the lowest was observed for G and Gm. The 2’-O-methylation of the first transcribed nucleotide (cap 1) significantly increased expression compared to cap 0 only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

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