Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1^102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences.

Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp B28 ) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.Administration of the two-chain 51-amino acid peptide hormone insulin is life sustaining for many of the 150 million diabetic patients in the world. Classical pancreatic extraction results in 10 -15 mg of insulin per pancreas and the number of porcine or bovine pancreases to produce current requirements of insulin (in excess of 7 metric tons) is simply not available. Consequently, a substantial fraction of insulin is today produced by eukaryotic secretory expression in the yeast Saccharomyces cerevisiae. Recently, second generation fast-acting genetically engineered insulin analogues have been engineered for improved diabetes therapy. This is exemplified by the clinically relevant fast-acting insulin, which features an amino acid substitution in position 28 of the B-chain from proline to aspartic acid and is used for treatment of diabetes mellitus (1).However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2, 3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4, 5)). Removal of the prepro-␣ factor by endoproteolytic cleavage by the Kex2 endoprotease in a late Golgi compartment generates a singlechain insulin precursor with self-association properties and structure essentially similar to that of human two-chain insulin (6, 7).Proteins exit the lumen of the endoplasmic reticulum (ER) 1 after folding, and frequently this is a rate-limiting step in secretion (8 -10). Herein we describe an improvement in eukaryotic secretion efficiency with application to insulin, using a strategy of structure-based rational engineering under the assumption that a more stably folded precursor molecule is ...

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The nucleotide sequence of the ␤glII A gene, encoding the extracellular ␤-1,3-glucanase II A (␤glII A ) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the ␤glII A enzyme has over 80% identity to the ␤glII isoenzyme, an endo-␤-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The ␤glII A enzyme lacks a glucan-or mannan-binding domain, such as those observed in ␤-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that ␤glII A has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

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