Dataset Information

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression.

ABSTRACT: L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular region of CD62L. This resulted in excess of 20 mg/L yield of recombinant CD62L. In an attempt to understand the different expression levels among four similar CD62L constructs that differ primarily in signal sequences, we calculated the presence of potential RNA pseudoknots in their signal sequences. The results showed the presence of pseudoknots involving the start codon and between the signal sequence and gene in the mRNA of the non-expressing constructs, suggesting a potential inhibitory role of RNA pseudoknots in recombinant protein expression.

SUBMITTER: Spencer M

PROVIDER: S-EPMC5599116 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression.

Spencer Matthew M Max Nathan N Ireland Joanna J Zou Zhongcheng Z Wang Ruipeng R Sun Peter P

Protein expression and purification 20170824

L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular regio ...[more]

PMID: 28842197

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Project description:MotivationSearching genomes for non-coding RNAs (ncRNAs) by their secondary structure has become an important goal for bioinformatics. For pseudoknot-free structures, ncRNA search can be effective based on the covariance model and CYK-type dynamic programming. However, the computational difficulty in aligning an RNA sequence to a pseudoknot has prohibited fast and accurate search of arbitrary RNA structures. Our previous work introduced a graph model for RNA pseudoknots and proposed to solve the structure-sequence alignment by graph optimization. Given k candidate regions in the target sequence for each of the n stems in the structure, we could compute a best alignment in time O(k(t)n) based upon a tree width t decomposition of the structure graph. However, to implement this method to programs that can routinely perform fast yet accurate RNA pseudoknot searches, we need novel heuristics to ensure that, without degrading the accuracy, only a small number of stem candidates need to be examined and a tree decomposition of a small tree width can always be found for the structure graph.ResultsThe current work builds on the previous one with newly developed preprocessing algorithms to reduce the values for parameters k and t and to implement the search method into a practical program, called RNATOPS, for RNA pseudoknot search. In particular, we introduce techniques, based on probabilistic profiling and distance penalty functions, which can identify for every stem just a small number k (e.g. k <or= 10) of plausible regions in the target sequence to which the stem needs to align. We also devised a specialized tree decomposition algorithm that can yield tree decomposition of small tree width t (e.g. t <or= 4) for almost all RNA structure graphs. Our experiments show that with RNATOPS it is possible to routinely search prokaryotic and eukaryotic genomes for specific RNA structures of medium to large sizes, including pseudoknots, with high sensitivity and high specificity, and in a reasonable amount of time.

Project description:The diverse landscape of RNA conformational space includes many canyons and crevices that are distant from the lowest minimum free energy valley and remain unexplored by traditional RNA structure prediction methods. A complete description of the entire RNA folding landscape can facilitate identification of biologically important conformations. The Crumple algorithm rapidly enumerates all possible non-pseudoknotted structures for an RNA sequence without consideration of thermodynamics while filtering the output with experimental data. The Crumple algorithm provides an alternative approach to traditional free energy minimization programs for RNA secondary structure prediction. A complete computation of all non-pseudoknotted secondary structures can reveal structures that would not be predicted by methods that sample the RNA folding landscape based on thermodynamic predictions. The free energy minimization approach is often successful but is limited by not considering RNA tertiary and protein interactions and the possibility that kinetics rather than thermodynamics determines the functional RNA fold. Efficient parallel computing and filters based on experimental data make practical the complete enumeration of all non-pseudoknotted structures. Efficient parallel computing for Crumple is implemented in a ring graph approach. Filters for experimental data include constraints from chemical probing of solvent accessibility, enzymatic cleavage of paired or unpaired nucleotides, phylogenetic covariation, and the minimum number and lengths of helices determined from crystallography or cryo-electron microscopy. The minimum number and length of helices has a significant effect on reducing conformational space. Pairing constraints reduce conformational space more than single nucleotide constraints. Examples with Alfalfa Mosaic Virus RNA and Trypanosome brucei guide RNA demonstrate the importance of evaluating all possible structures when pseduoknots, RNA-protein interactions, and metastable structures are important for biological function. Crumple software is freely available at http://adenosine.chem.ou.edu/software.html.

Dataset Information

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression.

Publications

Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression.

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