The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase.

Key features

• We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d.

• Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance.

• Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers.

• The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCR applications.

The study of translation is important to the understanding of gene expression. While genome-wide measurements of translation efficiency (TE) rely upon ribosome profiling, classical approaches to address translation of individual genes of interest rely on biochemical methods, such as polysome fractionation and immunoprecipitation (IP) of ribosomal components, or on reporter constructs, such as luciferase reporters. Methods to investigate translation have been developed that, however, require considerable research effort, including addition of numerous features to mRNA regions, genomic integration of reporters, and complex data analysis. Here, we describe a simple biochemical reporter assay to study TE of mRNAs expressed from a transiently transfected plasmid, which we term Nascent Chain Immunoprecipitation (NC IP). The assay is based on a plasmid expressing an N-terminally Flag-tagged protein and relies on the IP of Flag-tagged nascent chains from elongating ribosomes, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) quantification of eluted mRNA. We report that elution of mRNA following IP can be achieved by treatment with puromycin, which releases ribosome-mRNA complexes, or with purified Flag peptide, which instead releases nascent chain-ribosome-mRNA complexes. In the example described in this protocol, untranslated regions (UTRs) of a gene of interest were used to flank a FlagVenus coding sequence, with the method allowing to infer UTR-dependent regulation of TE. Importantly, our method enables discrimination of translating from non-translating mRNAs. Additionally, it requires simple procedures and standard laboratory equipment. Our method can be used to test the effect of regulators, such as microRNAs or therapeutic drugs or of various genetic backgrounds, on translation of any user-selected mRNA.

Key features

• The novel NC IP protocol builds upon a previously published method for detection of mRNA-binding proteins (Williams et al., 2022).

• The NC IP protocol is adapted for detecting mRNA actively undergoing translation.

• The method uses mammalian cell culture but could be adapted to multiple organisms, including budding yeast (S. cerevisiae).

Graphical overview

Design of the Nascent Chain Immunoprecipitation (NC IP) reporter and assay. Left. The construct carries a 3× Flag tag at the N-terminal end of Venus protein (FlagVenus). In this example, the reporter is adapted to study untranslated regions (UTR)-dependent expression by flanking FlagVenus coding sequence with UTRs of Aurora kinase A (AURKA) mRNA (depicted reporters refer to Cacioppo et al., 2023, Figure 3). The depicted reporters carry mutations in the proximal (p) or distal (d) polyadenylation signal (PAS). Right. Following reporter transfection, ribosomes are locked onto reporter mRNA by treating cells with cycloheximide (CHX), which prevents ribosome run-off and additional rounds of elongation, before cell lysis and immunoprecipitation (IP) of FlagVenus nascent chains via anti-Flag beads. Reporter mRNAs are then eluted, isolated, and quantified by RT-qPCR.

Recombinant proteins of high quality are crucial starting materials for all downstream applications, but the inherent complexities of proteins and their expression and purification create significant challenges. The Pichia pastoris yeast is a highly useful eukaryotic protein expression system. Pichia’s low cost, genetic tractability, rapid gene expression, and scalability make it an ideal expression system for foreign proteins. Here, we developed a protocol that has optimized the expression and isolation of a non-mammalian secreted metalloprotease, where we can routinely generate recombinant proteins that are pure and proteolytically active. We maximized growth and protein production by altering the feeding regime, through implementation of a non-fermentable and non-repressing carbon source during the methanol-induction phase. This approach increased biomass production and yielded milligrams of recombinant protein. Downstream applications involving active, recombinant fungal proteases, such as conjugation to nanoparticles and structure-related studies, are greatly facilitated with this improved, standardized approach.

Graphical abstract

Far-western blotting, derived from the western blot, has been used to detect interactions between proteins in vitro, such as receptor–ligand interactions. The insulin signaling pathway plays a critical role in the regulation of both metabolism and cell growth. The binding of the insulin receptor substrate (IRS) to the insulin receptor is essential for the propagation of downstream signaling after the activation of the insulin receptor by insulin. Here, we describe a step-by-step far-western blotting protocol for determining the binding of IRS to the insulin receptor.

Sclerotinia sclerotiorum causes white mold, leading to substantial losses on a wide variety of hosts around the world. Many genes encoding effector proteins play important roles in the pathogenesis of S. sclerotiorum. Therefore, establishment of a transformation system for the exploration of gene function is necessarily significant. Here, we introduce a modified protocol to acquire protoplasts for transformation and generate knockout strains, which completements the transformation system of S. sclerotiorum.