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Pichia Protein Purification Problem

Jonathan Kurtis, MD/PhD jkurtis at mail.med.upenn.edu
Thu Apr 8 10:56:20 EST 1999

I¹ve been having a tough time purifying a 6xhis tagged protein from Pichia
pastoris and was hoping someone could help.

The clone is a GS115 transformed with a pPICZb construct. This clone
expresses my protein (an 80 kDa actin binding protein) intracellularly to
what looks like well over 10 mg/10 ml of packed cells ( I tried and failed
to get it to secrete in pPICZalpha). After disrupting the cells in a Bead
Beater in 3x PBS, ph 8.0, 0.3% N-Laurl Sarkosine, I run the lysate over a
Ni-NTA superflow column. I wash with 3x PBS and elute with 50 mM Imidazole
in 3xPBS. The complete elution of my recombinant protein with such low
imidazole levels is consistent with the nature of this family of proteins
(the C terminus- where the 6x his tag is) is burried away from the
surface. I get excellent purification at this step (btw, largely due to
the 0.3% n-laurl sarkosine preventing most non-specific binding). Only 2
bands are visable by collodial coomassie, my 80 kDa band and another at 50
kDa. The eluate has a light yellow to deep tan (depending on the
concentration of protein) appearance. I have monitored the chromatography
with an FPLC setup and the tan color appears to be due to the 50 kDa
contaminant (my recombinant protein begins eluting before the co-elution
of the 80 and 50 kDa proteins).

I should be quite happy with this level of purity for a single step
process, but I have been unable to get rid of the 50 kDa contaminant by
either anion exchange (using Bio-Rad Macro Prep High Q resin, 15 cm x 1 cm
column) or hydroxyapatite (Bio-Rad, 10 cm x 1cm column). 

My plan is to make a batch of about 20 mg of partially purified product
with Ni-NTA and then run identical aliquats on the following resins: 1)
Cation exchange on Bio-Rad Macro Prep High S, 2) Hydrophobic interaction
chromatography on a Bio Rad resin.

I will try things like 20% ethanol or glycerol and 10 mM B-ME during the
second column step ( Don¹t want to mess with the Ni-NTA step, since it is
working so well).

Has anyone seen co-purification of a 50 kDa yellow/tan contaminant
before???  If so, how did you deal with it?

I understand that both flavins and AOX can copurify with recombinanat
proteins from Pichia, but their Mr are not near 50 kDa (Flavin??, AOX = 72

Thanks for your help,


Jonathan Kurtis, M.D./Ph.D.
Department of Pathology and Laboratory Medicine
6th Floor, Founders
Hospital of the University of Pennsylvania
3400 Spruce Street
Philadelphia, PA  19104-6523

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