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Custom Pichia pastoris Expression Strain Construction/Expression Testing
Strain construction and testing has been performed at BioGrammatics for companies and academic labs of all sizes (from Big Pharma to a single grad-student). Services include creating expression strains from scratch with all de novo reagents and synthetic DNA, as well as projects with existing components strains and/or vectors, such as a strain improvement project to increase expression levels of a specific recombinant protein with an existing Pichia strain.
No project is too small, and all projects are unique.
Please contact info@bioGrammatics to discuss a project and a quote.
Following are points for consideration in an expression project, as well as more details on what BioGrammatics includes in a strain construction/testing service.
1) Project goals and application for the protein/system.
2) Prior protein expression data and other information on the protein of interest (native source, known functions, structural data).
3) Protein assay. What methods of detection are available (Antibodies for a Western, enzymatic assay).
4) Requirement for an epitope or purification tag (i.e. 6his).
6) Ultimate scale of production and cost requirements.
Stain Construction and Testing Example.
Without any prior indication of how an ORF is expressed in Pichia, an initial expression test can focus a work-plan to define the scope and scale of a project to meet the project goals. An example work-plan for an initial strain development and expression testing service contract is outlined below.
Strain Construction and Expression testing, includes:
1) System design. Includes an analysis of the client goals and what expression tools will best help meet these goals, as well as, the evaluation of prior examples similar to a project.
2) Gene/ORF design. Includes a recommendation for the specific nucleotide sequence to be used for the clients desired gene products. BioGrammatics design algorithm considers: 1) codon usage from highly expressed genes and nearest neighbor-pair analysis from Pichia genome sequence data, 2) secondary structure and GC content, 3) Pichia specific RNA and protein processing, 4) N-linked glycosylation sites, 5) unique restriction enzyme site requirements and 6) other gene specific requirements. BioGrammatics will also evaluate Third Party gene design sequences. [Note, BioGrammatics will coordinate the gene synthesis from a reputable Third Party provider under a confidentiality agreement, however, the costs of gene synthesis are not included unless stated in the contract (currently $0.30 – 0.80/base)].
3) Cloning gene(s) into expression vectors and sequence verification of entire ORF and cloning junctions (performed under a confidentiality agreement with a reputable Third Party provider). BioGrammatics expression vectors utilize a seamless cloning scheme to generate precise, defined junctions (Fig/link).
4) Preparation of Expression vector DNA and Transformation into BioGrammatics Pichia pastoris strain(s). For example, most expression vectors are linearized for targeted integration at a specific site in the Pichia genome. Clean concentrated DNA is electroporated into a specific Pichia strain to create stable clones with the expression cassette integrated in the Pichia genome.
5) P. pastoris Clone Selection and Isolation/Purification.
6) Clone characterization. Marker testing, PCR analysis of select clones to confirm expected integration, and growth analysis.
7) Expression testing multiple strains/gene: generate cell biomass, induce and harvest (e.g. 48 – 72hr post MeOH induction). Prepare and store supernatant or cell samples.
8) Assay supernatants or cell extracts for expression of recombinant proteins. Standard analysis includes basic SDS-PAGE/Coomasie or Silver Staining, Western analysis if antibodies are available as well as protein specific activity assays. Assay development can also include plate assays (Yeasterns) and selections for the best expressing clones.
9) Repeat Expression testing on best clones (i.e. for verification, comparison, alternate time-points, pH or other grow/induction conditions, and/or for larger sample volumes).
10) Report and Recommendations.
Expression plasmids, select P. pastoris strains and any supernatant or cell fractions requested by Client.
Report (summary of work performed and recommendations).
Timeline estimate: for up to 10 ORF’s performed in parallel, project completion is estimated to be 4-8 weeks from time template DNA is received at BioGrammatics.
BioGrammatics will enter into a confidentiality agreement to discuss a project. The following 2-way confidentiality agreement is available for review, and/or as a template (link).
Master Service Agreement.
BioGrammatics offers the following Contract Service Agreement as an example/template for contact services.