Journal ArticleDOI
High-yield secretion of recombinant gelatins by Pichia pastoris.
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TLDR
Recombinant non‐hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae α‐mating factor prepro signal to represent the highest level of heterologous protein secretion reported to date for P. pastoris.Abstract:
Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.read more
Citations
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Journal ArticleDOI
Heterologous protein production using the Pichia pastoris expression system.
TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed.
Journal ArticleDOI
Recombinant protein expression in Pichia pastoris.
TL;DR: Major advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe’s value and power in commercial and research labs alike.
Journal ArticleDOI
Production of recombinant proteins by microbes and higher organisms.
Arnold L. Demain,Preeti Vaishnav +1 more
TL;DR: The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells while transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.
Journal ArticleDOI
Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.
TL;DR: This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains.
Journal ArticleDOI
Microbial Enzymes: Tools for Biotechnological Processes
José L. Adrio,Arnold L. Demain +1 more
TL;DR: Microbial diversity and modern molecular techniques are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution.
References
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Journal ArticleDOI
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Journal Article
Cleavage of structural proteins during the assemble of the head of bacterio-phage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Journal ArticleDOI
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
TL;DR: In this paper, complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends, and these fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the three' extension of the complementary strand.
Book ChapterDOI
High-efficiency transformation of yeast by electroporation.
TL;DR: This chapter presents the procedure for high-efficiency transformation of yeast by electroporation, designed by adapting the principles of bacterial electroporarion to transformarion of Saccharomyces cerevisiae, taking care, in addition, to provide continuous osmotic support of the electrically compromised cells.
Journal ArticleDOI
Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-α-factor
TL;DR: Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase, and cloned DNA restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo.