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Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence

Paper ID Volume ID Publish Year Pages File Format Full-Text
23350 43438 2013 7 PDF Available
Title
Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence
Abstract

The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6) – three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30 – obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6.The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments

► The insertion of Ala-Pro between MFα prepro sequence and protein of interest leads to an improved processing of the signal sequence. ► Expression of three variants of IL-6 reveals activity of an Ala-Pro specific endoprotease. ► Modeling of the transition confirmed better exposition of the Kex2p-cleavage site in dependency of Ala-Pro insertions.

Keywords
IL-6, interleukin-6; IFNα-2a, interferon-α 2a; IFNα-2b, interferon-α 2b; MFα, mating factor αHansenula polymorpha; Interleukin-6; Interferon-α 2a; Alpha mating factor; Secretion; Processing; Signal sequence; Kex2p
First Page Preview
Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 2, 20 August 2013, Pages 94–100
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering