We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD 65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD 65 at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD 65 was purified to greater than 90% purity.

Thrombopoietin (TPO) is a hematopoietic factor involved in the regulation of megakaryocytopoiesis. Full length recombinant human TPO (332 residues) has been expressed in BHK cells and purified to homogeneity using conventional means. Peptide, disulfide, and glycosylation mapping of human TPO from residues 1 to 246 has been carried out using liquid chromatography-electrospray mass spectrometry (LC-ESMS). A modification of the ramped orifice method of Carr and co-workers [Carr et al. (1993) Protein Sci. 2, 183-196] is employed, providing additional information for assignment of the LC-ESMS chromatograms. With the modification, b- and y-series peptide ions are produced via front-end CID which confirms the mass-based assignments. The results of our analysis of TPO indicate that the amino acid sequence of TPO 1-246 is as expected from the transfected cDNA with complete cleavage of the signal peptide. Two unique disulfides are formed between the four cysteines in the cytokine domain of TPO: Cys7-Cys151 and Cys29-Cys85. The glycosylation map indicates the position, occupancy, and structures of the N- and O-glycans in TPO 1-246. In addition, site specific structural characterization of the PNGase F-liberated N-glycans has been performed following purification by high-pH anionic exchange chromatography with pulsed amperometric detection (HPAEC-PAD); the results corroborate the LC-ESMS data. The N-glycans are of the complex type with the core-fucosylated disialylated biantennary and trisialylated triantennary structures predominating. The O-glycans are of the mucin type with the monosialylated and disialylated GalGalNAc-S/T structures predominating. Furthermore, we propose that the C-terminal domain of TPO be further divided into two domains on the basis of sequence homology among the cloned sequences and glycosylation/structural features: an N-glycan domain (154-246) and an O-glycan domain (247-332).

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Cross-link formation, within the duplex, by oligodeoxynucleotides containing a 5-[omega-(omega-haloacylamido)alkyl]-2'-deoxyuridine to a complementary oligodeoxynucleotide was investigated under conditions approximating the physiologic environment. The site and extent of crosslinking to the target strand were determined for several electrophilic haloacylamidoalkyl structures. The regiospecificity of alkylation was primarily determined by the length of the electrophilic haloacylamidoalkyl group, while the extent of reaction was dependent upon both the structure of the acylamidoalkyl group and the reactivity of the electrophile. Cross-linking was additionally modulated by the sequence of the duplex in the vicinity of the alkylation site. The exact placement of the electrophile adjacent to the targeted nucleophile, an N-7 group on a specific guanine base in the target strand, was the most important factor in determining the rate of reaction. With the optimal haloacylamidoalkyl structure and duplex sequence, the most rapid rate obtained was t1/2 = 1.3 h at 37 degrees C.

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