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A Pichia Vector for Multicopy integration and Secreted Expression – pPIC9K expression vector

A Pichia Vector for Multicopy integration and Secreted Expression – pPIC9K expression vector. 미생물유전학 실험실 임 효 경. Pichia expression vector ; don’t have episomal plasmid (differ from S.cerevisiae expression vector) → must be integrated into chromosome → multicopy integration.

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A Pichia Vector for Multicopy integration and Secreted Expression – pPIC9K expression vector

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  1. A Pichia Vector for Multicopy integration and Secreted Expression – pPIC9K expression vector 미생물유전학 실험실 임 효 경

  2. Pichia expression vector ; don’t have episomal plasmid (differ from S.cerevisiae expression vector) → must be integrated into chromosome → multicopy integration

  3. ◎ pPIC9K expression vector system - isolation of multicopy inserts by in vivo method in order to test whether increasing the copy number of recombinant gene will lead to a subsequent increase in secreted protein expression - utilizes resistance to Geneticin(G418 sulfate) to screen for possible multicopy inserts ◎ Frequency of Multicopy Inserts - multiple plasmid integration in Pichia at a frequency between 1 and 10% of all His+ transformants

  4. ◎ Generation of Multicopy Inserts in vivo - kan gene confers resistance to Geneticin in Pichia -single copy of pPIC9K integrated into the Pichia genome ..Geneticin resistance level : ~0.25 ㎎/㎖ - multiple integrated copies of pPIC9K ..Geneticin resistance level : 0.5 ㎎/㎖(1~2 copies) ~4㎎/㎖(7~12 copies) → genetic linkage between the kanamycin gene and the “expression cassette” → kan gene on pPIC9K can be used as a tool to detect pPIC9K transformants

  5. Multiple insertion of expression cassette linked to the kan gene

  6. ◎ Procedure - Transformation into Pichia - His4+ initial selection - Geneticin selection - PCR - incubation in media - add MeOH (→ induction of vector) - secretion of desired protein into the media by signal sequence - purify

  7. ◎ pPIC9K vector - 9276 bp fusion vector - four unique restriction site for cloning (SnaBⅠ, EcoRⅠ, AvrⅡ, NotⅠ) - α-factor signal sequence (bases 949~1218) - HIS4 selection marker - 5’ AOX1, 3’ AOX1 - Amp pBR322 origin - kan (kanamycin resistance gene) - TT

  8. ◎ Promoter • ＊ AOX1 gene • regulation of the AOX1 gene is a two step process • ( repression/derepression mechamism + induction mechanism) • methanol is “inducer” • necessary for detectable levels of AOX1 expression

  9. ◎ PAOX1 and Multiple clonig site in pPIC9K

  10. ◎ Advantages of pPIC9K vector - Identifies the 1~10% of spontaneous His+ transformants that have multiple inserts - Average size of vector is similar to other Pichia expression vectors - Multiple inserts are located at a single locus - secretion of desired protein into the media by signal sequence

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