Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta.
The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His(6)-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni-nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P.angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.
Mack
Matthias
M
Institute for Technical Microbiology, Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim, Germany. m.mack@hs-mannheim.de
Wannemacher
Marion
M
Hobl
Birgit
B
Pietschmann
Patricia
P
Hock
Björn
B
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D018628:Gene Dosage; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
C066855:HGF protein, human; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D013925:Thromboplastin