Mark A. Payton

The Candida albicans PKC1 gene encodes a protein kinase C homolog necessary for cellular integrity but not dimorphism.

Using a DNA fragment derived from the Saccharomyces cerevisiae protein kinase C gene (PKC1) as a probe to screen an ordered array library of genomic DNA from the dimorphic pathogenic fungus Candida albicans, the C. albicans PKC1 gene (CaPKC1) was isolated. The CaPKC1 gene is predicted to encode a protein of 1079 amino acids with 51% sequence identity over the entire length with the S. cerevisiae Pkc1 protein and is capable of functionally complementing the growth defects of a S. cerevisiae pkc1Δ mutant strain on hypo‐osmotic medium. Deletion of both endogenous copies of the CaPKC1 gene in diploid C. albicans cells resulted in an osmotically remedial cell lysis defect of both the budding and the hyphal growth form and morphologically aberrant cells of the budding form. Despite these abnormalities, the transition between the two growth forms of C. albicans occurred normally in pkc1/pkc1 double disruptants. Capkc1p was modified at its C‐terminus with two repeats of the Staphylococcus aureus protein A IgG‐binding fragment (ZZ‐sequence tag) and partially purified by chromatography on DEAE–Sepharose and IgG–Sepharose. In vitro, Capkc1p preferably phosphorylated the S. cerevisiae Pkc1p pseudosubstrate peptide and myelin basic protein, but not histones, protamine or dephosphorylated casein, and failed to respond to cofactors known to activate several mammalian PKC isozymes.

Production of ethanol by thermophilic bacteria

Abstract Doubts about the viability of using classical yeast fermentations to produce ethanol have led in recent years to a search for alternative microbes capable of ethanol production. Among the front-runners for consideration are the thermophilic bacteria and this article shows how close these extraordinary microbes have come to replacing yeast in ethanol production.

PURIFICATION AND CHARACTERIZATION OF FUNGAL AND MAMMALIAN PHOSPHOMANNOSE ISOMERASES

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, andCandida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, andpH dependences. Surprisingly, in view of the large differences betweenPseudomonas aerugenosa andSaccharomyces cerevisiae PMI, the human andC. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.

The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation.

We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37°C, to growth at 37°C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.

Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli. The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E. coli B, a natural lon- prototroph. A simple purification method is described which takes advantage of the basic nature of the protein. The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein. The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.

Fermentation and Growth of Escherichia coli for Optimal Protein Production

Large‐scale production of recombinant proteins in Escherichia coli requires growth of cells in fermentors. This unit lists E. coli strains appropriate for use in fermentors, and also discusses important characteristics of fermentation equipment. Production of recombinant proteins in batch fermentations is described, as are variations of fermentation systems that enable continuous growth and protein production in high‐cell‐density, fed‐batch cultures and that permit labeling of recombinant proteins with heavy atom derivatives such as selenomethionine or with stable isotopes such as 2H, 13C, and 15N. Production of labeled proteins facilitates structural studies by X‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. The protocols in this unit are designed for expression systems directing intracellular or periplasmic localization of recombinant proteins; however, in the case of extracellular secretion of the desired protein, the culture medium itself, rather than pelleted cells, would be saved, concentrated, and subjected to purification processes. Fermentation experiments require careful monitoring of cell growth and assurance of preinoculation sterility, both which are described here.

Protein Expression in the Baculovirus System

Insect cell‐recombinant baculovirus co‐cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post‐translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large‐scale production of recombinant proteins in the baculovirus expression system. A method for large‐scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end‐point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small‐scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS‐PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed‐batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

Selection of Escherichia coli Expression Systems

This unit lists the most useful expression strains of E. coli for fermentation processes. Standard procedures are provided for several expression systems, namely, temperature induction via the pL promoter and chemical induction via the trp promoter, lac or tac promoters, and the T7 promoter. These protocols require that the gene encoding the protein of interest has been identified and cloned into an appropriate expression vector using standard molecular biology techniques. Transformation of a suitable host strain (e.g., by electroporation) is also described and is a prerequisite. Protocols for the analysis of plasmid stability and subsequent storage are provided. Support protocols describe how to prepare samples for electrophoresis, how to analyze the solubility of the expressed proteins, and how to make samples of periplasmic extracts and extracellular media (using TCA precipitation). Many of the support protocols are small‐scale analysis procedures that are used to guide subsequent purification strategies and determine the suitability of the expression system for further development and scale‐up.