Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris.

Background: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P, P) was first conducted in chemostat cultures.

Refined Pichia pastoris reference genome sequence.

Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments.

Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse(®) mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement.

Expression in the yeast Pichia pastoris.

The yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth. This chapter assumes the reader is proficient in molecular biology and details the more yeast specific procedures involved in utilizing the P.

Posttransformational vector amplification in the yeast Pichia pastoris.

Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones.

Classical genetics.

A significant advantage of Pichia pastoris as an experimental system is the ability to readily bring to bear both classical and molecular genetic approaches to a research problem. Although the advent of yeast molecular genetics has introduced new and exciting capabilities, classical genetics remains the approach of choice in many instances. These include the generation of mutations in previously unidentified genes (mutagenesis), the removal of unwanted secondary mutations (backcrossing), the assignment of mutations to specific genes (complementation analysis), and the construction of strains with new combinations of mutant alleles.

Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris.

Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate.