Expression and Characterization of Glycosylated and Catalytically Active Recombinant Human α-Galactosidase A Produced in Pichia pastoris

We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.

Section: Discussionsupporting

confidence: 92%

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

Hanquier

Sorlet

Desplancq

et al. 2003

“…Several groups, including our group, have produced recombinant ␣-GalA in various hosts. The amounts of recombinant ␣-GalA produced were as follows: 0.019 mg/ 10-cm culture dish under the expression system of COS-7 cells (8), and 4.5 mg/liter broth in P. pastoris (7). These results suggested that O. minuta has promising potential as a host for heterologous recombinant protein expression.…”

Section: Discussionmentioning

confidence: 88%

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Akeboshi

Chiba

Kasahara

et al. 2007

Human ␤-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of ␣-and ␤-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using ␣-1,6-mannosyltransferase-deficient (och1⌬) yeast as the host. Genes encoding the ␣-and ␤-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (␣␣) and HexB (␤␤). A total of 57 mg of ␤-hexosaminidase isozymes, of which 13 mg was HexA (␣␤), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the ␤-subunit. The purified HexA was treated with ␣-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ؎ 0.1 and 1.7 ؎ 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the ␤-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.

“…The optimum pH value for Pichia growth is approximately 5 (6), but the pH value is commonly reduced to counteract proteolysis (9,32) or to increase both the stability and productivity of heterologous proteins (5,29,56). However, a pH value of 4 or lower can compromise cells during the production process (27).…”

mentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...