# US 5,707,828 A · Expression of human serum albumin in Pichia pastoris

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- Number: US 5,707,828 A
- Document ID: US5707828A
- Status: Granted
- Kind: A
- Application number: 08/433,037
- Filed: 1995-05-03
- Granted: 1998-01-13
- Earliest claimed: 1991-04-25
- Assignees: Research Corporation Technologies, Inc.
- Inventors: Kotikanyadan Sreekrishna; Juerg F. Tschopp; Gregory P. Thill; Russell A. Brierley; Kathryn A. Barr
- Web page: https://patentgrep.com/patent/US5707828A

## CPC

- C12N 15/815: {for yeasts other than Saccharomyces}
- C07K 14/765: Serum albumin, e.g. HSA
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention is directed to expression cassettes comprising a 5' regulatory region from at least one of the Pichia pastoris AOXI gene, p40 gene, DASI gene or HIS4 gene, operably linked to an HSA structural gene including the HSA signal sequence. The HSA structural gene has a translational start codon within 0 to 11 deoxyribonucleotides from the 5' end of the HSA structural gene and is operably linked to a 3' termination sequence. Further, the adenine and thymine content of the intervening deoxyribonucleotides is in the range from about 55 to about 64%. The expression cassette may be an autonomously replicating vector or an integrative vector. Other embodiments of the present invention include Pichia pastoris strains transformed with the HSA expression cassettes and processes for secretion of HSA using the expression cassettes.

## Claims

### Claim 1

An expression cassette for the Human Serum Albumin (HSA) in Pichia pastoris comprising:
(a) the Pichia pastoris AOX1 5' regulatory region wherein said regulatory region is operably linked to
(b) a HSA structural gene encoding a HSA signal sequence and a mature HSA protein, wherein the HSA structural gene has an ATG start codon within no more than 11 intervening deoxyribonucleotides at the 5' end of said HSA structural gene and wherein the adenine and thymine content of the intervening deoxyribonucleotides is in the range of from about 55 to about 64 percent and wherein said HSA structural gene is operably linked to
(c) a functional 3' termination sequence.

### Claim 2 (depends on 1)

The expression cassette of claim 1 wherein the expression cassette is incorporated into a vector selected from the group consisting of circular plasmids and linear plasmids.

### Claim 3 (depends on 2)

The expression cassette of claim 2 wherein the vector is a integrative site-specific vector.

### Claim 4 (depends on 1)

The expression cassette of claim 1 wherein the expression cassette is incorporated into a vector comprising the following serial arrangement:
(a) a first insertable DNA fragment;
(b) at least one marker gene; and
(c) a second insertable DNA fragment;
wherein at least one expression cassette is incorporated either before or after the marker gene of component (b), and the first and second insertable DNA fragments employed are homologous with separate portions of the Pichia pastoris genome and the insertable fragments are in the same relative orientation as exist in the Pichia pastoris genome.

### Claim 5 (depends on 4)

The expression cassette of claim 4 wherein the first insertable DNA fragment and the second insertable DNA fragment are obtained from the DNA sequences of a gene isolated from Pichia pastoris selected form the group consisting of an AOX1 gene, a p40 gene, a DAS1 gene and an HIS4 gene.

### Claim 6 (depends on 4)

The expression cassette of claim 4 wherein the marker gene is selected from the group consisting of a Pichia pastoris HIS4 gene, a Pichia pastoris ARG4 gene, a Saccharomyces cerevisiae SUC2 gene, a G418.sup.R gene of bacterial transposon Tn601 and a G418.sup.R gene of bacterial transposon Tn903.

### Claim 7 (depends on 4)

The expression cassette of claim 4 wherein said vector comprises:
(a) a first insertable DNA fragment which is the Pichia pastoris AOX1 5' regulatory region operably linked to
(b) an HSA structural gene having an ATG start codon within no more than 8 deoxyribonucleotides of the end of the AOX1 regulatory region, operably linked to
(c) the 3' termination sequence of the Pichia pastoris AOX1 gene; operably linked to
(d) the Pichia pastoris HIS4 gene as a marker gene and operably linked to
(e) a second insertable DNA fragment which is about 0.65 kilobases of the AOX1 termination sequence.

### Claim 8 (depends on 7)

The expression cassette of claim 7 wherein the HSA structural gene has an ATG start codon with the deoxyribonucleotides AGGAATTC 5' of said ATG start codon.

### Claim 9 (depends on 7)

The expression cassette of claim 7 wherein the HSA structural gene has no deoxynucleotides 5' of the ATG start codon.

### Claim 10 (depends on 1)

A Pichia pastoris cell transformed with an expression cassette of claim 1.

### Claim 11 (depends on 10)

The Pichia pastoris cell of claim 10 wherein the Pichia pastoris cell which is to be transformed is selected from the group consisting of Pichia pastoris GS115 (NRRL Y-15851), Pichia pastoris GS190 (NRRL Y-18014), Pichia pastoris PPF1 (NRRL Y-18017), Pichia pastoris (NRRL Y-11430) and Pichia pastoris (NRRL Y-14431).

### Claim 12 (depends on 10)

The Pichia pastoris cell of claim 10 wherein the Pichia pastoris cell which is to be transformed is Pichia pastoris GS115 (NRRL Y-15851).

### Claim 13 (depends on 10)

The Pichia pastoris cell of claim 10 wherein the expression cassette used for transformation utilizes the 3' termination sequence isolated from a Pichia pastoris gene selected from the group consisting of an AOX1 gene, a DAS1 gene, a p40 gene and a HIS4 gene.

### Claim 14 (depends on 10)

The Pichia pastoris cell of claim 10 wherein the expression cassette is incorporated into a vector selected from the group consisting of circular plasmids and linear plasmids.

### Claim 15 (depends on 14)

The Pichia pastoris cell of claim 14 wherein the vector utilized for transformation is an integrative site-specific vector.

### Claim 16 (depends on 10)

The Pichia pastoris cell of claim 10 wherein the Pichia pastoris cell is transformed with an expression cassette which is incorporated into a vector comprising the following serial arrangement:
(a) a first insertable DNA fragment;
(b) at least one marker gene; and
(c) a second insertable DNA fragment;
wherein at least one expression cassette is incorporated either before or after the marker gene of component (b), and the first and second insertable DNA fragments employed are homologous with separate portions of the Pichia pastoris genome wherein the insertable fragments are in the same relative orientation as exist in the Pichia pastoris genome.

### Claim 17 (depends on 16)

The Pichia pastoris cell of claim 16 wherein the Pichia pastoris cell is transformed with a vector having the first insertable DNA fragment and the second insertable DNA fragment obtained from the DNA sequences of a gene isolated from Pichia pastoris selected from the group consisting of an AOX1 gene, a p40 gene, a DAS1 gene and a HIS4 gene.

### Claim 18 (depends on 16)

The Pichia pastoris cell of claim 16 wherein the Pichia pastoris cell is transformed with the vector which has a marker gene selected from the group consisting of a Pichia pastoris HIS4 gene, a Pichia pastoris ARG4 gene, a Saccharomyces cerevisiae SUC2 gene, a G418.sup.R gene of bacterial transposon Tn601 and a G418.sup.R gene of bacterial transposon Tn903.

### Claim 19 (depends on 7)

A Pichia pastoris cell transformed with the vector of claim 7.

### Claim 20 (depends on 19)

The expression cassette of claim 19 wherein the HSA structural gene has an ATG start codon with the deoxyribonucleotides AGGAATTC 5' of said start codon.

### Claim 21 (depends on 19)

The expression cassette of claim 19 wherein the HSA structural gene has no deoxynucleotides 5' of the ATG start codon.

### Claim 22

A process for the secretion of Human Serum Albumin (HSA) comprising: transforming a Pichia pastoris cell with at least one vector having at least one expression cassette comprising:
(a) the Pichia pastoris AOX1 5' regulatory region wherein said regulatory region is operably linked to
(b) an HSA structural gene encoding a HSA signal and a mature HSA protein, wherein the HSA structural gene has an ATG start codon within no more than 11 intervening deoxyribonucleotides at the 5' end of said HSA structural gene and wherein the adenine and thymine content of the intervening deoxyribonucleotides is the range of from about 55 to about 64 percent and wherein said HSA structural gene is operably linked to
(c) a functional 3' termination sequence and thereafter culturing the resulting transformed Pichia pastoris cell under suitable conditions to obtain the production of HSA.

### Claim 23 (depends on 22)

The process of claim 22 wherein the Pichia pastoris cell utilized to produce HSA is transformed with said at least one expression cassette which utilizes the AOX1 5' regulatory region from Pichia pastoris.

### Claim 24 (depends on 22)

The process of claim 22 wherein the Pichia pastoris cell utilized to produce HSA is transformed with an expression cassette containing a 3' termination sequence isolated from a Pichia pastoris gene selected from the group consisting of an AOX1 gene, a DAS1 gene, a p40 gene and a HIS4 gene.

### Claim 25 (depends on 22)

The process of claim 22 wherein the Pichia pastoris cell which is to be transformed is selected from the group consisting of Pichia pastoris GS115 (NRRL Y-15851), Pichia pastoris GS190 (NRRL Y-18014), Pichia pastoris PPF1(NRRL Y-18017), Pichia pastoris (NRRL Y-11430) and (NRRL Y-11431).

### Claim 26 (depends on 25)

The process of claim 25 wherein Pichia pastoris cell which is to be transformed is Pichia pastoris GS115 (NRRL Y-15851).

### Claim 27 (depends on 22)

The process of claim 22 wherein the vector utilized to transform said Pichia pastoris cell is selected from the group consisting of circular plasmids and linear plasmids.

### Claim 28 (depends on 27)

The process of claim 27 wherein the vector utilized to transform said Pichia pastoris cell is an integrative site-specific vector.

### Claim 29 (depends on 27)

The process of claim 27 wherein the integrative site-specific vector utilized to transform said Pichia pastoris cell contains the following serial arrangement:
a) a first insertable DNA fragment;
b) at least one marker gene; and
c) a second insertable DNA fragment;
wherein at least one expression cassette is incorporated either before or after the marker gene of component (b), and the first and second insertable DNA fragments employed are homologous with separate portions of the Pichia pastoris genome and the insertable fragments are in the same relative orientation as exist in the Pichia pastoris genome.

### Claim 30 (depends on 29)

The process of claim 29 wherein the first insertable DNA fragment and the second insertable DNA fragment are obtained from the DNA sequence of a gene isolated from Pichia pastoris and selected from the group consisting of an AOX1 gene, a p40 gene, a DAS1 gene and a HIS4 gene.

### Claim 31 (depends on 29)

The process of claim 29 wherein the marker gene is selected from the group consisting of a Pichia pastoris HIS4 gene, a Pichia pastoris ARG4 gene, a Saccharomyces cerevisiae SUC2 gene, a G418.sup.R gene of bacterial transposon Tn601 and a G418.sup.R gene of bacterial transposon Tn903.

### Claim 32 (depends on 29)

The process of claim 29 wherein said vector comprises:
a) a first insertable DNA fragment which is about one kilobase of the AOX1 5' regulatory region isolated from Pichia pastoris operably linked to
b) an HSA structural gene encoding an HSA signal sequence and a mature HSA protein having a 5' end and a 3' end wherein the HSA structural gene has an ATG start codon within no more than 8 deoxyribonucleotides of the 5' end of said HSA structural gene; operably linked to
c) the 3' termination sequence of the AOX1 gene isolated from Pichia pastoris; operably linked to
d) a marker gene which is the HIS4 gene isolated from Pichia pastoris; operably linked to
e) a second insertable DNA fragment which is about 0.65 kilobases for the AOX1 3' termination sequence.

### Claim 33 (depends on 32)

The expression cassette of claim 32 wherein the HSA structural gene has an ATG start codon with the deoxyribonucleotides AGGAATTC 5' of said ATG start codon.

### Claim 34 (depends on 32)

The expression cassette of claim 32 wherein the HSA structural has no deoxynucleotides 5' of the ATG start codon.