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 the negative control, "NO DNA") contained 200 ng of genomic DNA. Clone 139 was b-gal and Cath A positive, clone WC7 - MOC control and "+"- positive control (10 ng, pPIC-9-b-Gal).

Figure 4

Co-transfection of the G115 pichia pastoris-Cath A strain with pPIC9-b-Gal and protein expression

A.  PCR amplification

B.  b-Gal enzyme assay

Enzyme activity in yeast homogenates after cultivation for 48, 72 and 84 h. Clone 139 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. Lanes 1 and 6- yeast homogenate of negative clone WC7, lanes 2 and 4-  homogenate of clone B1 (b-Gal positive), lanes 3 and 5- homogenate of double transfected clone 139 (b-Gal and Cath A) and lanes 7 and 9- yeast culture media of double- and monotransfected clones, correspondently after cultivation for 96 h. Lane 8- yeast culture medium of negative clone WC7 after cultivation for 96 h.

A

B

C