PubMed | The Mintaka Foundation for Medical Research, Ghent University, Biogrammatics, Inc., LifeSci Partners LLC and 3 more.
Type: | Journal: Protein expression and purification | Year: 2016
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. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.
PubMed | University of Graz, Austrian Center of Industrial Biotechnology, Synthetic Genomics, Inc, University of Chicago and Biogrammatics, Inc.
Type: | Journal: Journal of biotechnology | Year: 2016
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. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris.
Austin R.J.,Institute for Systems Biology |
Kuestner R.E.,Institute for Systems Biology |
Chang D.K.,Institute for Systems Biology |
Madden K.R.,Seattle Cancer Care Alliance |
And 2 more authors.
Journal of Proteome Research | Year: 2011
The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation. © 2011 American Chemical Society.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 400.00K | Year: 2011
DESCRIPTION (provided by applicant): The goal of the proposed research is to identify and develop new basic tools for the expression of recombinant genes for the methylotrophic yeast Pichia pastoris. In recent years this yeast has become an important hostfor the production of biopharmaceuticals with over five thousand publications on its use and at least two human biopharmaceuticals (human serum albumin and ecallantide) on the market in the US and others at various stages in the clinical trial pipeline. This project will be done in part by and wholly for Biogrammatics, Inc. a small biotechnology company incorporated in January of 2008 whose business is to develop, utilize and sell improved tools for heterologous protein expression in this yeast. The business strategy of Biogrammatics involves two general objectives: 1) the sale of novel P. pastoris components and; 2) the construction, testing and improvement of custom recombinant strains of this yeast for customers. Three specific aims are outlined in thisapplication: 1) to conduct a genome wide search for new strong and/or well regulated promoters for expression of recombinant gene in P. pastoris; 2) to develop improved methods for preparation of competent P. pastoris cells for DNA-mediate transformationsand 3) to identify and develop of improved signal sequences to direct the secretion of recombinant proteins from this yeast The project, if funded through a two year Phase I SBIR-SHIFT award mechanism will result in products for Biogrammatics without theneed for a Phase II. At the initiation of this project, the head PI on the application, Dr. Cregg will shift from being a professor at the Keck Graduate Institute to an employee of Biogrammatics. ) PUBLIC HEALTH RELEVANCE: Proteins/peptides are the fastest growing sector in 900 billion dollar therapeutic agent market and hold the promise for true personalized medicine and stem cell therapies. Optimization of Pichia pastoris secretory promoters, transformation efficiency and signal sequences will significantly increase the success rate for recombinant protein production, leading to faster clinical trials and drug approvals in the rProtein therapeutic arena.
Biogrammatics, Inc. | Date: 2014-03-07
Isolated nucleic acids, expression methods, host cells, expression vectors, and DNA constructs for producing proteins, and proteins produced using the expression methods are disclosed. More specifically, nucleic acids isolated from Pichia pastoris having promoter activity and expression methods, host cells, expression vectors, and DNA constructs of using the Pichia pastoris promoters to produce different proteins and polypeptides are disclosed.
Category: Expression Vectors Initial Tab: Overview Pichia Expression Vectors. Recombinant protein expression in Pichia is most common from strains with expression vectors integrated into the Pichia genome ...
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 ...