Yeast production of hepatitis B surface antigen

Claim 2

An essentially pure culture of a yeast capable of growth on methanol as carbon and energy source wherein said yeast is of the genus Pichia transformed with an expression cassette comprising (a) a regulatory region isolated from Pichia pastoris and selected from the group consisting of AOX1, p40, and DAS operably linked to (b) a polypeptide coding region coding for a hepatitis B surface antigen operably linked to (c) a Pichia transcription termination fragment.

Claim 3

An essentially pure culture of a strain of a strain of Pichia pastoris capable of growth on methanol as a carbon and energy source transformed with a plasmid selected from the group consisting of: pBSAG5, pBSAG5I, and pBSAGI5I.

Claim 4

An essentially pure culture of a strain of a strain of Pichia pastoris capable of growth on methanol as a carbon and energy source transformed with a plasmid selected from the group consisting of: pBSAG5, pBSAG5I, and pBSAGI5I.

Claim 5

An isolated DNA fragment comprising: (a) a regulatory region isolated from Pichia pastoris and selected from the group consisting of AOX1, p40 and DAS, said regulatory region being capable of controlling the transcription of messenger RNA when positioned at the 5' end of the polypeptide encoding region; wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host organism containing said DNA fragment is in contact; and (b) a polypeptide coding region operably linked to said regulatory region and coding for heptatitis B surface antigen.

Claim 6

An isolated DNA fragment comprising: (a) a regulatory region isolated from Pichia pastoris and selected from the group consisting of AOX1, p40 and DAS, said regulatory region being capable of controlling the transcription of messenger RNA when positioned at the 5' end of the polypeptide encoding region; wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host organism containing said DNA fragment is in contact; and (b) a polypeptide coding region operably linked to said regulatory region and coding for heptatitis B surface antigen.

Claim 7

A DNA fragment in accordance with claim 3 wherein said regulatory regions characterized as shown by the restriction map in FIG. 2 of the drawings.

Claim 8

A DNA fragment in accordance with claim 3 further comprising a 3' sequence of DNA downstream of the polypeptide coding region; wherein said 3' sequence of DNA is capable of controlling the polyadenylation and termination of transcription of messenger RNA coded for by said polypeptide coding region.

Claim 9

A DNA fragment in accordance with claim 3 wherein said DNA fragment further comprises one or more additional DNA sequences selected from the group consisting of bacterial plasmid DNA, bacteriophage DNA, yeast plasmid DNA, and yeast chromosomal DNA.

Claim 10

A DNA fragment in accordance with claim 6 wherein said yeast chromosal DNA comprises an autonomously replicating DNA sequence and a marker gene.

Claim 11

A DNA fragment in accordance with claim 3 wherein said DNA fragment further comprises one or more additional DNA sequences selected from the group consisting of bacterial plasmid DNA, bacteriophage DNA, yeast plasmid DNA, and yeast chromosomal DNA.

Claim 12

A DNA fragment is accordance with claim 8 wherein said yeast chromosomal DNA comprises an autonomously replicating DNA sequence and a marker gene.

Claim 13

A DNA fragment in accordance with claim 3 further comprising serially arranged DNA which comprises: a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment; wherein said first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous with portions of the genomic DNA of species of the genus Pichia; wherein said DNA fragment and said marker gene are positioned between the 3' end of said first insertable DNA fragment and the 5' end of said second insertable DNA fragment; and wherein said first and second insertable DNA fragments are oriented with respect to one another as they are so oriented in the genome of Pichia.

Claim 14

A DNA fragment in accordance with claim 3 wherein said polypeptide coding region consists essentially of the approximately 700 base pair EcoRI-StuI fragment depicted below: __________________________________________________________________________ 5'-GAATTCATGG AGAACATCAC ATCAGGATTC CTAGGACCCC TGCTCGTGTT ACAGGCGGGG TTTTTCTTGT TGACAAGAAT CCTCACAATA CCGCAGAGTC TAGACTCGTG GTGGACTTCT CTCAATTTTC TAGGGGGATC TCCCGTGTGT CTTGGCCAAA ATTCGCAGTC CCCAACCTCC AATCACTCAC CAACCTCCTG TCCTCCAATT TGTCCTGGTT ATCGCTGGAT GTGTCTGCGG CGTTTTATCA TATTCCTCTT CATCCTGCTG CTATGCCTCA TCTTCTTATT GGTTCTTCTG GATTATCAAG GTATGTTGCC CGTTTGTCCT CTAATTCCAG GATCAACAAC AACCAGTACG GGACCATGCA AAACCTGCAC GACTCCTGCT CAAGGCAACT CTATGTTTCC CTCATGTTGC TGTACAAAAC CTACGGATGG AAATTGCACC TGTATTCCCA TCCCATCGTC CTGGGCTTTC GCAAAATACC TATGGGAGTG GGCCTCAGTC CGTTTCTCTT GGCTCAGTTT ACTAGTGCCA TTTGTTCAGT GGTTCGTAGG GCTTTCCCCC ACTGTTTGGC TTTCAGCTAT ATGGATGATG TGGTATTGGG GGCCAAGTCT GTACAGCATC GTGAGTCCCT TTATACCGCT GTTACCAATT TTCTTTTGTC TCTGGGTATA CATTTAAGGC CT-3' __________________________________________________________________________

Claim 15

A plasmid comprising: the DNA fragment of claim 3, bacterial plasmid DNA, a selectable yeast marker gene, and a yeast autonomous replication sequence.

Claim 16

A plasmid in accordance with claim 12 wherein said plasmid is pBSAG5.

Claim 17

A plasmid in accordance with claim 12 wherein said plasmid is pBSAG5I, as depicted in FIG. 11.

Claim 18

A plasmid in accordance with claim 12 wherein said plasmid is pBSAGI5I, as depicted in FIG. 11.

Claim 19

An essentially pure culture of a strain of the Pichia pastoris transformed with the DNA fragment of claim 5.

Claim 20

A process for preparing hepatitis B surface antigen which comprises cultivating Pichia pastoris transformed with the plasmid of claim 12 in a nutrient medium which comprises methanol.

Claim 21

A process in accordance with claim 17 further comprising isolating and purifying said hepatitis B surface antigen.

Claim 22

A DNA fragment in accordance with claim 3 wherein said regulatory region is characterized as shown by the restriction map in FIG. 1(b) of the drawings.

Claim 23

A DNA fragment in accordance with claim 3 wherein said regulatory region is characterized as shown by the restriction map in FIG. 3 of the drawings.

Claim 24

A process for the production of hepatitis B surface antigen comprising: (a) transforming Pichia pastoris capable of growth on methanol as a carbon and energy source with an expression cassette containing a polypeptide coding region for a hepatitis B surface antigen operably linked to a regulatory region derived from Pichia pastoris and selected from the group consisting of AOX1, p40 and DAS and a transcription termination fragment; and thereafter (b) culturing the resulting transformed Pichia pastoris on methanol as a carbon and energy source under suitable conditions to obtain the production of hepatitis B surface antigen.

Claim 25

A process for the production of hepatitis B surface antigen comprising: (a) transforming Pichia pastoris capable of growth on methanol as a carbon and energy source with an expression cassette containing a polypeptide coding region for a hepatitis B surface antigen operably linked to a regulatory region derived from Pichia pastoris and selected from the group consisting of AOX1, p40 and DAS and a transcription termination fragment; and thereafter (b) culturing the resulting transformed Pichia pastoris on methanol as a carbon and energy source under suitable conditions to obtain the production of hepatitis B surface antigen.

Claim 26

The process of claim 21 wherein said expression cassette is contained within a vector which is selected from the group consisting of circular or linear plasmids.

Claim 27

The process of claim 22 wherein the plasmid is linear.

Claim 28

The process of claim 23 wherein the linear plasmid is a site directed integration vector.

Claim 29

The process of claim 24 wherein said site directed integration vector contains the following serial arrangement: (a) a first insertable DNA fragment, (b) a marker gene, and expression cassette containing a polypeptide coding region coding for a hepatitis B surface antigen operably linked to a regulatory region and a transcription termination fragment, and (c) a second insertable DNA fragment; wherein the marker gene and cassette of component (b) are positioned between the 3' end of the first insertable DNA fragment and the 5' end of the second insertable DNA fragment.

Claim 30

The process of claim 25 wherein the first insertable DNA fragment and the second insertable DNA fragment are derived from the DNA sequence of a gene isolated from Pichia pastoris.

Claim 31

The process of claim 25 wherein said marker gene is isolated from Pichia pastoris.

Claim 32

The process of claim 25 wherein the transformed Pichia pastoris capable of growth on methanol as a carbon and energy source is Pichia pastoris GS115.

Claim 33

The process of claim 25 wherein the transformed Pichia pastoris capable of growth on methanol as a carbon and energy source is Pichia pastoris GS190.

Claim 34

The process of claim 25 wherein the transformed Pichia pastoris is Pichia pastoris PPF1.

Claim 35

The process of claim 25 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 hepatitis B surface antigen gene operably linked to (c) the transciption termination fragment 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 AOX1 transcription termination fragment.