scholarly journals pOPIN-GG: A resource for modular assembly in protein expression vectors

2021 ◽  
Author(s):  
Adam R Bentham ◽  
Mark Youles ◽  
Melanie N Mendel ◽  
Freya A Varden ◽  
Juan Carlos De la Concepcion ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Protein Function ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Restriction Enzymes ◽  
Expression Vectors ◽  
Vector Systems ◽  
Protein Expression And Purification ◽  
Modular Cloning

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

scholarly journals Construction of gateway-compatible baculovirus expression vectors for high-throughput protein expression and in vivo microcrystal screening

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yanyang Tang ◽  
Justin Saul ◽  
Nirupa Nagaratnam ◽  
Jose M. Martin-Garcia ◽  
Petra Fromme ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Expression Vectors ◽  
Baculovirus Expression

scholarly journals Plasmodium sporozoites require the protein B9 to invade hepatocytes

2021 ◽  
Author(s):  
Priyanka Fernandes ◽  
Manon Loubens ◽  
Carine Marinach ◽  
Romain Coppee ◽  
Morgane Grand ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Protein A ◽  
Blood Feeding ◽  
Host Cells ◽  
Infected Mosquito ◽  
Mammalian Host ◽  
Genetic Tagging ◽  
Structural Levels

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite in the liver. This leads to the release of thousands of merozoites into the blood circulation and initiation of the pathogenic blood stages of malaria. Merozoite invasion of erythrocytes has been well characterized at the molecular and structural levels. In sharp contrast, the molecular mechanisms of sporozoite invasion of hepatocytes are poorly characterized. Here we report a new role during sporozoite entry for the B9 protein, a member of the 6-cysteine domain protein family. Using genetic tagging and gene deletion approaches in rodent malaria parasites, we show that B9 is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. Structural modelling indicates that the N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an invasion complex with Rh5 and RIPR in P. falciparum merozoites. We provide evidence that the beta-propeller domain of B9 is essential for protein function during sporozoite entry and interacts with P36 and P52, both also essential for productive invasion of hepatocytes. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

scholarly journals Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fernando Hinostroza ◽  
Alan Neely ◽  
Ingrid Araya-Duran ◽  
Vanessa Marabolí ◽  
Jonathan Canan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Function ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Coarse Grained ◽  
Cell Physiology ◽  
Missense Mutations ◽  
Small Change ◽  
Dynamics Simulations ◽  
Dynamin 2

Abstract High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.

scholarly journals Overview of Bacterial and Yeast Systems for Protein Expression

2021 ◽  
pp. 113-118
Author(s):  
Maheswara Reddy Mallu ◽  
Siva Reddy Golamari ◽  
Sree Rama Chandra Karthik Kotikalapudi ◽  
Renuka Vemparala
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Large Scale ◽  
Recombinant Protein Expression ◽  
Expression Vectors ◽  
Scale Production ◽  
Protein Coding ◽  
Recombinant Gene Expression ◽  
Vector Systems ◽  
Large Scale Production

Over the past decade the variety of hosts and vector systems for recombinant protein expression has increased dramatically. Researchers now select from among mammalian, insect, yeast, and prokaryotic hosts, and the number of vectors available for use in these organisms continues to grow. With the increased availability of cDNAs and protein coding sequencing information, it is certain that these and other, yet to be developed systems will be important in the future. Despite the development of eukaryotic systems, E. coli remains the most widely used host for recombinant protein expression. Optimization of recombinant protein expression in prokaryotic and eukaryotic host systems has been carried out by varying simple parameters such as expression vectors, host strains, media composition, and growth temperature. Recombinant gene expression in eukaryotic systems is often the only viable route to the large-scale production of authentic, post translationally modified proteins. It is becoming increasingly easy to find a suitable system to overexpress virtually any gene product, provided that it is properly engineered into an appropriate expression vector.

scholarly journals High-Throughput Protein Expression and Purification for Proteomics Research

2002 ◽  
Vol 2 ◽  
pp. 146-147
Author(s):  
Sharon A. Doyle ◽  
Michael Murphy ◽  
Jennifer Primus ◽  
Paul Richardson ◽  
Trevor Hawkins
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Expression And Purification ◽  
Protein Expression And Purification ◽  
Proteomics Research
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