During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

http://www.biotechlab.ch/UserFiles/File/787339_Ref_CererghinoCregg2000.pdf

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

Vestweber, Dietmar, and James E. Blanks. Mechanisms That Regulate the Function of the Selectins and Their Ligands. Physiol. Rev. 79: 181–213, 1999. — Selectins are a family of three cell adhesion m...

Mechanisms That Regulate the Function of the Selectins and Their Ligands

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant 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.

Recombinant protein expression in Pichia pastoris.

Understanding the molecular basis of lymphocyte homing to lymphoid organs was originally a problem of concern only to immunologists. With the discovery of L-selectin and its ligands, interested scientists have expanded to include glycobiologists, immunopathologists, cancer biologists, and developmental biologists. Going beyond its first discovered role in homing to lymph nodes, the L-selectin system is implicated in such diverse processes as inflammatory leukocyte trafficking in both acute and chronic settings, hematogenous metastasis of carcinoma cells, effector mechanisms for inflammatory demyelination of axons, and implantation of the early mammalian embryo. This review focuses on the ligands for L-selectin that are found on vascular endothelium, leukocytes, carcinoma cells, and at various extravascular sites. The discovery of selectins and their ligands has validated the long-predicted hypothesis that carbohydrate-directed cell adhesion is relevant in eukaryotic systems. Emphasis will be given to the carbohydrate and sulfation modifications of the ligands, which enable recognition by L-selectin. The rapid "homing" of labeled cells into the lymph nodes presumably had its basis in the special affinity of small lymphocytes for the endothelium of the postcapillary venules.

Ligands for L-selectin: homing, inflammation, and beyond.

In this chapter, we aim to present a consistent description of the structure and functions of caseins and the casein micelle. Function, it must be stressed, is interpreted here to mean both the biological functions of casein in the mammary gland and milk and the function in dairy foods. Thus, we tread a variation of a well-worn path in biology by exploring a structure-function trichotomy.

Casein Micelle Structure, Functions and Interactions

Disulphide-linked caseins and casein micelles

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858. After the final reverse-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial peptide map was established, and degenerate primers were designed and used for amplification of fragments of the gene encoding the activator. Inverse PCR was subsequently used for obtaining the full-length gene. The S. uberis plasminogen activator gene (skc) encodes a protein consisting of 286 amino acids including a signal peptide of 25 amino acids. In an amino acid sequence comparison the cloned activator showed an identity of approximately 26% to the streptokinases isolated from Streptococcus equisimilis and Streptococcus pyogenes. Interestingly, the activator from S. uberis was found to lack the C-terminal domain possessed by the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis revealed that 9 of these were highly similar to strain NCTC 3858. Sequencing of the skc gene from three of these strains indicated that the amino acid sequence of the protein is highly conserved within the species.

Purification and Cloning of a Streptokinase from Streptococcus uberis

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene.

/pdf/characterization-of-three-types-of-human-alpha-s1-casein-4hlskjothk.pdf

Laust B. Johnsen

Papers

Disulphide-linked caseins and casein micelles

Purification and Cloning of a Streptokinase from Streptococcus uberis

Characterization of three types of human alpha s1-casein mRNA transcripts.

Characterization of a bovine mammary gland pp3 cdna reveals homology with mouse and rat adhesion molecule glycam-1

Structural characterization of the fibroblast growth factor-binding protein purified from bovine prepartum mammary gland secretion.