The commercial primers AOX1 and AOX2 complimentary to  Pichia pastoris alcohol dehydrogenase gene (AOX1) sequence were used to amplify 1.9 kb cDNA fragment. Each sample (except negative control, "NO DNA") contained 200 ng of genomic DNA. Clones CA2-CA4, CA6-CA8 were Cath A positive. Clones CK1- CK5 were MOC control and "+"was positive control (10 ng, pPIC9k-Cath A. The upper band (2.1 kB) corresponded AOX1 and lower band (1.9 kB) was the gene of interest.

Figure 3

Transformation of the G115 pichia pastoris strain with pPIC9k- Cath A and protein expression

A.  PCR amplification

B.  Cath A enzyme assay

Enzyme activity in the cell culture medium was measured after 24 and 48 h cultivation. CA7 clone was grown in BMM (buffered methanol medium), pH 7.5 contained 1% casamino acids. Concentration of methanol was 0.5%

C.  Western blot analysis

Immunoblot of yeast culture media and homogenates. Lane 1 was Gibco BRL 10 kDa protein ladders, lanes 2-4- yeast culture media of Cath A negative CA1 after 24, 48 and 72 h cultivation lanes 5-7- homogenates of the same clones, lanes 8-10- yeast culture medium of Cath A positive CA7 clone after 24, 48 and 72 h cultivation and lane 11- yeast homogenate of the same clone after 24 h cultivation.

D.  Affinity chromatography in pET His*Tag system on polyHis       binding gel

Cell culture medium of Cath A positive clone CA7 obtained after 96 h cultivation was concentrated by ultrafiltration (PM 10, Amicon) and loaded onto the pET His*Tag column in the presence of 8M urea (flow through, lane 1). The column was washed with binding buffer (20 mM Tris-HCl, 10 mM imidazol, 1M NaCl, pH 7.9) with (lane 2) and without (lane 3) urea. Then the protein was eluted with elution buffer (20 mM Tris-HCl, 1 M imidazol, 500 mM NaCl, pH 7.9, line 4) and the column was stripped out from remained compounds. 15 mkg of each fraction were tested with western blot

A

B

C

D