Quality and authenticity of heterologous proteins synthesized in yeast.

Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments. There has been recent progress in characterizing and controlling the factors involved in determining authenticity.

O-linked oligosaccharide on the 75-kDa neurotrophin receptor.

Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O-glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O-glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of disialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)(1-2-) Gal beta 1-3GalNAc. Modification of the O-linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.

The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) domain has been predicted from sequence comparisons to represent a trypsin-like serine protease. By expressing wild-type and mutant HCV-1 cDNAs in transfected mammalian cells, we have identified putative nonstructural proteins 3 (72 kDa), 4 (10 kDa and 27 kDa) and 5 (58 kDa) and have shown that their processing from the viral polyprotein precursor is dependent on Ser1165 located in the proposed protease catalytic site. Data obtained from in vitro RNA translations indicate that unlike the processing of the NS2/NS3 junction, NS3/NS4 processing is dependent on Ser1165. In contrast to the situation for the related flaviviral NS3 proteases, the HCV NS3-mediated cleavage of the NS3/NS4 junction does not require the upstream NS2 domain and may not occur at dibasic sites.

The effects of using recombinant vaccinia viruses expressing either large or small HDAg to protect woodchuck hepadnavirus carriers from HDV superinfection.

Live rVVs expressing either p24 delta or p27 delta were produced and used to immunize woodchuck hepadnavirus carriers. Upon challenge with infectious HDV, circulating HDV RNA levels appeared to be similar in both controls and vaccinees. Although extended follow-up studies of these animals is necessary before making firm conclusions, including an analysis of circulating HDAg levels, these preliminary results provide no evidence for a protective immunity conferred by the rVVs. In contrast, we have shown in other studies that repeated immunization of woodchucks with purified, recombinant p24 delta subunit does confer significant protection against HDV challenge in some of the vaccinees (A. Ponzetto, et al., this volume). The underlying immunological mechanisms responsible for the different outcome of these varied vaccination regimens remain to be elucidated.

Rabbit-derived anti-HD antibodies for HDAg immunoblotting.

We studied the sensitivity and specificity of two rabbit-derived antibodies (W2 and W3) raised against two synthetic peptides of the HDAg: the C-terminus of the p24 protein (W2) and the C-terminus of the p27 protein (W3) (Chiron Co., Emeryville, CA, U.S.A.). The results were compared with those obtained with a human polyclonal anti-HD (W1). We have tested W2 and W3 against blotted serum samples from 25 patients (20 HBsAg positive anti-HD positive, three HBsAg positive anti-HD negative and two HBsAg negative anti-HD negative) and liver extracts from five HBsAg seropositive patients, two anti-HD positive and three anti-HD negative. In serum samples W3 sporadically reacted with a p31 protein present in both anti-HD positive and negative sera; W2 identified the p24 protein in 7/10 W1 positive samples but in none of 10 W1 negative; the p27 protein was captured by W2 only in one highly viremic serum. In every sample of liver extract W3 recognized a 48-kDa band. W1 and W2 stained 5 bands at 45, 27, 24, 16 and 12 kDa. The p45, p27 and p24 proteins were peculiar of HDV-infected livers; p16 and p12 were also detected in HDV free livers. W2 identified an additional 54-kDa protein in 4/5 liver extracts. For diagnostic purposes human polyclonal anti-HD represents the most sensitive probe for HDAg immunoblotting, with a specificity similar to that of rabbit-derived antibodies. The affinity of W2 and W3 antibodies for serum and liver HDAg appears to be lower than for HDAg recombinant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha.

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.