Manfred Suckow

Identification of three residues in the basic regions of the bZIP proteins GCN4, C/EBP and TAF-1 that are involved in specific DNA binding.

The bZIP regions of the eukaryotic transcription factors GCN4 and C/EBP have similar protein sequences but they recognize different DNA sequences. In order to understand their specificity, a vector was constructed which permits overexpression in Escherichia coli of those domains of GCN4 that are necessary and sufficient for specific DNA binding i.e. the basic region and the leucine zipper. Specific DNA binding was monitored with gel shift experiments. The residues of the basic region of GCN4 were systematically replaced by those of C/EBP to transform GCN4 into C/EBP with respect to DNA binding. Residues −17, −16 and −14 were found to be responsible for switching GCN4 to C/EBP binding specificity (we define as residue +1 the first leucine of the first leucine heptad repeat of GCN4). We broadened the specificity of GCN4 to TAF‐1 by replacing residues −15 and −17 and we changed the specificity of C/EBP to TAF‐1 by swapping residue −17 of a particular hybrid. Thus residues positioned from −14 to −17 of the basic region play a key role in recognizing specific DNA sequences.

Strain and process development for the production of human cytokines in Hansenula polymorpha

The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.

Analysis of heat shock promoters inHansenula polymorpha: TheTPS1 promoter, a novel element for heterologous gene expression

The strength and regulatory characteristics of the heat-inducibleHSA1, HSA2 andTPS1 promoters were compared with those of the well-established, carbon source-regulatedFMD promoter in aHansenula polymorpha-based host systemin vivo. In addition, theSaccharomyces cerevisiae-derivedADH1 promoter was analysed. WhileADH1 promoter showed to be of poor activity in the foreign host, the strength of the heat shockTPS1 promoter was found to exceed that of theFMD promoter, which at present is considered to be the strongest promoter for driving heterologous gene expression inH. polymorpha.

Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha

Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to mainly contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition the cells essential functions are maintained under stress conditions like high salinity, heat or aridity stress. Hansenula polymorpha was engineered to catalyze the transformation of monomeric substrates to 5-hydroxyectoine. For this purpose four genes encoding the enzymes of the 5-hydroxyectoine biosynthesis pathway of Halomonas elongata, EctA, EctB, EctC, and EctD, were inserted into the genome of H. polymorpha. Subsequently the syntheses of ectoine and 5-hydroxyectoine were analyzed and optimized. We showed that H. polymorpha is a suitable system for recombinant 5-hydroxyectoine synthesis in gram per liter scale (2.8 g L⁻¹ culture supernatant, 365 μmol/g dcw) in which almost 100% conversion of ectoine to 5-hydroxyectoine without necessity of high salinity were achieved.

Mutant bZip-DNA complexes with four quasi-identical protein-DNA interfaces.

The complex between the yeast transcriptional activator GCN4 and the palindromic ATF/CREB site 5′‐ A4T3G2A1C0*G0′T1′C2′A3′T4′‐3′ shows dyad symmetry. The basic region of GCN4 contains a segment of 18 amino acids with a partially palindromic sequence: N‐LKRARNTEA*ARRSRARKL‐C. Symmetric residues are underlined. Apart from the ATF/CREB site, GCN4 also binds well to the symmetric variants with guanine in position 4 (5′‐G4T3G2A1C0*G0′T1′C2′A3′C4′‐3′) or thymine in position 0 (5′‐A4T3G2A1T0*A0′T1′C2′A3′T4′‐3′). The half‐sites of these sequences can be regarded as short pseudo‐palindromes with central guanine 2/cytosine 2′ base pairs. We investigated whether the geometry of the peptide of the basic region of GCN4 could be functionally related to the pseudo‐palindromic character of some target half‐sites. Since inspection of the X‐ray structures of GCN4‐DNA complexes reveals that several amino acid‐DNA interactions are symmetric within the wild‐type half‐complexes, we introduced mutations into a GCN4 bZip peptide that improve the symmetry of the peptide. We found that most of the constructs retain specific DNA recognition. For one mutant, we conclude that it is not only capable of forming DNA complexes showing the well‐known overall dyad symmetry, but that the protein‐DNA interface of each half‐complex can be divided further into two quasi‐identical, quasi‐symmetric substructures.

The use of highly expressed FTH1 as carrier protein for cytosolic targeting in Hansenula polymorpha

The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha.

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

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.

Promoter Assessment in Hansenula polymorpha Using a lacZ Reporter Gene

Analysis of the strength and regulatory characteristics of commonly used Hansenula polymorpha-detived promoter elements using LacZ as reporter gene.