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8 Enzyme Engineering and Technology 193

tissues have been reported. The expressed proteins
are usually recovered in a bioactive, properly folded
form and secreted into the cell culture fluids.

Yeast

Yeast is a widely used expression system with many
commercial, medical, and basic research applica-
tions. The fact that yeast is the most intensively
studied eukaryote at the genetic/molecular level
makes it an extremely advantageous expression sys-
tem (Trueman 1995). Being a unicellular organism,
it retains the advantages of bacteria (low cultivation
cost, high doubling rate, ease of genetic manipula-

tion, ability to produce heterologous proteins in

large-scale quantities) combined with the advan-

tages of higher eukaryotic systems (posttranslational

modifications). The vast majority of yeast expres-

sion work has focused on the well-characterized

baker’s yeast Saccharomyces cerevisiae(Holz et al.

2003), but a growing number of non-Saccharomyces

yeasts are becoming available as hosts for recombi-

nant polypeptide production; these include Han-

senula polymorpha, Candida boidinii, Kluyvero-

myces lactis, Pichia pastoris(Fischer et al. 1999b,

Cregg et al. 2000, Cereghino et al. 2002), Schizo-

saccharomyces pombe(Giga-Hama and Kumagai

1999), Schwanniomyces occidentalis, and Yarrowia

Figure 8.12.Artificial chromosome cloning system. Initially, the circular vector is digested with restriction endonucle-
ases (RE) for linearization and then ligated with size-fractionated DNA (100 kb). The vector contains centromeric
and telomeric sequences, which assure chromosome-like propagation within the cell, as well as selection marker
sequences for stable maintenance.