Crystallization and low-resolution structure solution of the SALM3–PTPσ synaptic adhesion complex

Synaptic adhesion molecules are major organizers of the neuronal network and play a crucial role in the regulation of synapse development and maintenance in the brain. Synaptic adhesion-like molecules (SALMs) and leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-PTPs) are adhesion protein families with established synaptic function. Dysfunction of several synaptic adhesion molecules has been linked to cognitive disorders such as autism spectrum disorders and schizophrenia. A recent study of the binding and complex structure of SALM3 and PTPσ using small-angle X-ray scattering revealed a 2:2 complex similar to that observed for the interaction of human SALM5 and PTPδ. However, the molecular structure of the SALM3–PTPσ complex remains to be determined beyond the small-angle X-ray scattering model. Here, the expression, purification, crystallization and initial 6.5 Å resolution structure of the mouse SALM3–PTPσ complex are reported, which further verifies the formation of a 2:2 trans-heterotetrameric complex similar to the crystal structure of human SALM5–PTPδ and validates the architecture of the previously reported small-angle scattering-based solution structure of the SALM3–PTPσ complex. Details of the protein expression and purification, crystal optimization trials, and the initial structure solution and data analysis are provided.

Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells

Background Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods. Methods In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques. Discussion This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered ‘research-use only’, others will be recommended for scaling and application in the development of cell-based therapies.

Recombinant protein expression and purification of codon-optimized Pfu-Sso7d v2

This protocol has been optimized for the recombinant expression of a codon-optizimed Pfu-Sso7d DNA polymerase. This is a fusion protein composed of the Pfu enzyme from Pyrococcus furiosus for DNA amplification by PCR fused to a small 7 kDa protein from Sulfobulus solfataricus that binds to double-stranded DNA without any preference for specific sequences, thus enhancing polymerization processivity without affecting the catalytic activity or thermal stability of the enzyme. The goal of this protocol was to eliminate the use of large volumes for dyalisis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions. We also eliminated the use of DTT, which is often found in other similar protocols. The sequence plasmid encoding the codon-optimized Pfu-Sso7d enzyme used here can be found at https://benchling.com/s/seq-2TcUPjO2uMbDG5ufTQN4

Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications

The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.

Recombinant protein expression and purification of Taq DNA polymerase v1

This is a slightly modified and simplified version of a protocol by Thomas G.W. Graham et al, which is available at https://gitlab.com/tjian-darzacq-lab/bearmix and has been described in depth in the article 10.1371/journal.pone.0246647, for the recombinant expression of a E602D mutant of Taq DNA polymerase in pET-28a that is available in Addgene (Addgene plasmid # 166944 ; http://n2t.net/addgene:166944 ; RRID:Addgene_166944). The main goal of this protocol is to eliminate the use of large volumes for dialysis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions.

Characterization and computational simulation of human Syx, a RhoGEF implicated in glioblastoma

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic - Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.

The Penn State Protein Ladder system for inexpensive protein molecular weight markers

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.

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

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.