Expression of hepatitis B S and preS2 proteins in methylotrophic yeasts

Claim 2

A process for the production of antigenic HBV particles consisting essentially of S and preS₂ comprising a) transforming a methylotrophic yeast with at least one expression cassette containing a structural gene for S, operably linked to a regulatory region and a 3′ termination sequence; and at least one expression cassette containing a structural gene for preS₂, operably linked to a regulatory region and a 3′ termination sequence; and thereafter b) culturing the resulting transformed yeast strain under suitable conditions to obtain the production of said HBV particles.

Claim 3

The process of claim 1 wherein said transformation is performed with at least one vector selected from the group consisting of plasmids or linear integrative site-specific vectors.

Claim 4

The process of claim 2 wherein the vectors are integrative.

Claim 5

The process of claim 3 wherein there is employed a) a first integrative site-specific vector contains the following serial arrangement: i) a first insertable DNA fragment, ii) a marker gene, and at least one expression cassette containing a structural gene for preS₂, operably linked to a regulatory region and a 3′ termination sequence, and iii) a second insertable DNA fragment; wherein the order of the marker gene and cassette of component (ii) may be interchanged; b) and a second integrative vector which contains the following: i) at least one insertable DNA fragment, ii) a marker gene, and at least one expression cassette containing a structural gene for S, operably linked to a regulatory region and a 3′ termination sequence, wherein the order of the marker gene and cassette of component (ii) may be interchanged.

Claim 6

The process of claim 4 wherein said insertable DNA fragment is derived from the DNA sequence of a gene isolated from Pichia pastoris and selected from the group consisting of AOX1 , p40, DHAS and HIS4 .

Claim 7

The process of claim 4 wherein at least one expression cassette comprises a) a regulatory region selected from the group consisting of AOX1 , p40, DHAS , isolated from Pichia pastoris , acid phosphatase, galactokinase, alcohol dehydrogenase, cytochrome c, alpha-mating factor and glyceraldehyde 3-phosphate dehydrogenase isolated from Saccharomyces cerevisiae operably linked to b) a structural gene for preS₂, operably linked to c) a 3′ termination sequence from Pichia pastoris selected from the group consisting of the 3′ termination sequences isolated from the AOX1 gene, p40 gene, DHAS gene and HIS4 gene.

Claim 8

The process of claim 4 wherein at least one expression cassette comprises a) a regulatory region selected from the group consisting of AOX1 , p40, DHAS , isolated from Pichia pastoris , acid phosphatase, galactokinase, alcohol dehydrogenase, cytochrome c, alpha-mating factor and glyceraldehyde 3-phosphate dehydrogenase isolated from Saccharomyces cerevisiae operably linked to b) a structural gene for S, operably linked to c) a 3′ termination sequence from Pichia pastoris selected from the group consisting of the 3′ termination sequences isolated from the AOX1 gene, p40 gene, DHAS and HIS4 gene.

Claim 9

The process of claim 4 wherein said marker gene is selected from the group consisting of HIS4 and ARG4 , isolated from Pichia pastoris , SUC2 isolated from Saccharomyces cerevisiae and neomycin gene of Tn903 and Tn601 .

Claim 10

The process of claim 8 wherein said marker genes are different and independently selected.

Claim 11

The process of claim 4 wherein said vector containing the preS₂ structural gene comprises a) a first insertable DNA fragment which is about one kilobase of the 5′ AOX1 regulatory region isolated from Pichia pastoris operably linked to b) a structural gene for preS₂, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is HIS4 isolated from Pichia pastoris ligated to e) a second insertable DNA fragment which is about 0.65 kilobases of the 3′ AOX1 termination sequence.

Claim 12

The process of claim 4 wherein said vector containing the S structural gene comprises a) an insertable DNA fragment which is about one kilobase of the 5′ AOX1 regulatory region isolated from Pichia pastoris operably linked to b) a structural gene for S, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is ARG4 isolated from Saccharomyces cerevisiae ligated to e) an insertable DNA fragment which is about 0.65 kilobases of the 3′ AOX1 termination sequence.

Claim 13

An integrative vector comprising a) at least one insertable DNA fragment which is an operable regulatory region of the 5′ AOX1 gene being about one kilobase in length isolated from Pichia pastoris operably linked to b) a structural gene for S, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is ARG4 isolated from Pichia pastoris ligated to e) a second insertable DNA fragment which is about 0.65 kilobases of the 3′ AOX1 termination sequence.

Claim 14

An integrative vector comprising a) at least one insertable DNA fragment which is an operable regulatory region of the 5′ AOX1 gene being about one kilobase in length isolated from Pichia pastoris operably linked to b) a structural gene for S, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is ARG4 isolated from Pichia pastoris ligated to e) a second insertable DNA fragment which is about 0.65 kilobases of the 3′ AOX1 termination sequence.

Claim 15

Methylotrophic yeast transformed with at least one expression cassette comprising a regulatory region operably linked to a structural gene for preS₂, operably linked to a 3′ termination sequence and at least one expression cassette comprising a regulatory region operably linked to a structural gene for S, operably linked to a 3′ termination seqeunce.

Claim 16

Methylotrophic yeast transformed with at least one expression cassette comprising a regulatory region operably linked to a structural gene for preS₂, operably linked to a 3′ termination sequence and at least one expression cassette comprising a regulatory region operably linked to a structural gene for S, operably linked to a 3′ termination seqeunce.

Claim 17

The methylotrophic yeast of claim 13 wherein the yeast is Pichia pastoris .

Claim 18

The methylotrophic yeast of claim 14 wherein the yeast is Pichia pastoris strain PPF1.

Claim 19

Pichia pastoris PPF1 of claim 15 wherein said PPF1 is transformed with (a) at least one linear integrative site specific vector which is a serial arrangement of i) a first insertable DNA fragment, ii) a marker gene and at least one Pichia -compatible expression cassette containing a structural gene for preS₂, operably linked to iii) a 3′ termination sequence selected from the group consisting of 3′ termination sequence isolated from AOX1 gene, p40 gene, DHAS gene, and HIS4 gene isolated from Pichia pastoris , iv) a second insertable DNA fragment, wherein the order of the marker gene and cassette of component (ii) may be interchanged; and (b) at least one integrative vector which contains the following: i) at least one insertable DNA fragment, ii) a marker gene and at least one Pichia compatible expression cassette containing a structural gene for S, operably linked to iii) a 3′ termination sequence selected from the group consisting of 3′ termination sequence isolated from AOX1 gene, p40 gene, DHAS gene, and HIS4 gene isolated from Pichia pastoris , wherein the order of the marker gene and cassette of component (ii) may be interchanged.

Claim 20

The linear site specific integrative vector containing the preS₂ structural gene of claim 16 wherein said vector comprises a) a first insertable DNA fragment which is about one kilobase of the 5′ AOX1 regulatory region isolated from Pichia pastoris operably linked to b) a structural gene for preS₂, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is HIS4 isolated from Pichia pastoris ligated to e) a second insertable DNA fragment which is about 0.65 kilobases of the 3′ AOX1 termination sequence.

Claim 21

The integrative vector containing the S structural gene of claim 16 wherein said vector comprises a) a first insertable DNA fragment which is about one kilobase of the 5′ AOX1 regulatory region isolated from Pichia pastoris operably linked to b) a structural gene for S, operably linked to c) the 3′ termination sequence of AOX1 isolated from Pichia pastoris ligated to d) a marker gene which is ARG4 isolated from Saccharomyces cerevisiae ligated to e) a second insertable DNA fragment which is about .65 kilobases of the 3′ AOX1 termination sequence.

Claim 22

Pichia pastoris PPF1/pTB012-1.

Claim 23

Pichia pastoris PPF1/pTB012-1.

Claim 24

Pichia pastoris PPF1 transformed as in claim 16 wherein said PPF1 is transformed with more than one copy of said linear integrative site-specific vector.

Claim 25

Pichia pastoris PPF1/pTB012-2.

Claim 26

Pichia pastoris PPF1/pTB012-2.