The oligonucleotide primers polyHis (CAT CAT CAT CAT CAT CAT) and GalAsense complimentary to GALcDNA sequence were used to amplify 243 b.p. cDNA fragment. Each sample (except negative control, "NO DNA") contained 50 ng of genomic DNA. Clones B1, B2, B4, B6, B9-B11 and B13-B18 were b-gal positive. Clones K1-K5 - MOC control and "+"- positive control (10 ng, pPIC-9-b-Gal.

Figure 2

Transformation of the G115 pichia pastoris strain with pPIC9-b-Gal and protein expression

A.  PCR amplification

B.  b-Gal enzyme assay

Enzyme activity in the cell culture medium after 36, 72 and 84 h cultivation. B1 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 partially purified enzyme preparation after ion-exchange chromatography (Mono Q) and gel-filtration (Superose 6). Lanes 1-5 corresponded to fraction numbers 18-22 after gel-filtration.

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

Cell culture mediumof b-Gal positive clone B1 obtained after 84 h cultivation was concentrated by ultrafiltration (PM 10, Amicon) was loaded onto the pET His*Tag column in the presence of 8M urea. The column was washed with binding buffer (20 mM Tris-HCl, 10 mM imidazol, 1M NaCl, pH 7.9) with (lane 2) end without (lane 3-5) urea and then the protein was eluted with elution buffer (20 mM Tris-HCl, 1 M imidazol, 500 mM NaCl, pH 7.9, line 6) and the column was stripped out from remained protein (line 7). 25 mkg of each fraction were tested with western blot

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