Human interferon-alpha 2b (IFN-alpha 2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-alpha 2b, Saccharomyces cerevisiae MF-alpha factor prepro sequence and a mutated alpha prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-alpha 2b into the culture medium of P. pastoris. The native secretion signal of IFN-alpha 2b did not secrete protein into the culture medium of P. pastoris. The alpha prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-alpha 2b (200 mg/1) into the culture medium, with the same amino acid sequence as that of the native IFN-alpha 2b secreted by human lymphocytes. The full alpha prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-alpha 2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full a prepro sequence was used for the secretion of IFN-alpha 2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal. (c) 2008 Elsevier Inc. All rights reserved.