Enhanced production of recombinant proteins in myeloma cells

Claim 2

A mammalian myeloma cell, the genome of which comprises a double-stranded DNA molecule containing: (i) a coding sequence encoding a non-immunoglobulin protein and bounded by a translation start codon at its 5' terminus and a translation stop codon on its 3' terminus; (ii) a non-immunoglobulin promoter sequence adjacent to the 5' terminus of said coding sequence, said promoter sequence containing a transcription initiation site and capable of initiating transcription of said coding sequence in a mammalian myeloma cell; and (iii) the 8-base pair nucleotide sequence 5'-ATTTGCAT-3' on the nontranscribed strand located 5' to said transcription initiation site and in a position whereby the rate of transcription of said coding sequence in said mammalian myeloma cell is increased.

Claim 3

The myeloma cell line of claim 1 wherein said double-stranded DNA molecule also contains a mammalian kappa light-chain immunoglobulin gene transcription enhancer element located so as to increase the rate of transcription of said coding sequence in said mammalian myeloma cell.

Claim 4

The myeloma cell of claim 2 wherein said enhancer is a murine enhancer.

Claim 5

The myeloma cell of claim 1 wherein said protein is a mammalian protein.

Claim 6

The myeloma cell of claim 2 wherein said protein is a mammalian protein.

Claim 7

The myeloma cell of claim 3 wherein said protein is a mammalian protein.

Claim 8

The myeloma cell of claim 6 wherein said 8-base pair sequence is located from about 50 to about 500 base pairs from said transcription initiation site.

Claim 9

The myeloma cell of claim 7 wherein said 8-base pair sequence is located from about 50 to about 200 base pairs from said transcription initiation site.

Claim 10

The myeloma cell of claim 6 wherein said transcription enhancer element is located 5' to said 8-base pair sequence.

Claim 11

The myeloma cell of claim 7 wherein said transcription enhancer element is located 5' to said 8-base pair sequence.

Claim 12

The myeloma cell of claim 8 wherein said transcription enhancer element is located 5' to said 8-base pair sequence.

Claim 13

A murine myeloma cell according to claim 3 wherein said protein is a mammalian protein, and said 8-base pair sequence is located from about 50 to about 200 base pairs from said transcription initiation site.

Claim 14

The myeloma cell of claim 12 wherein said promoter sequence is substantially homologous to the wild-type thymidine kinase promoter of the herpes simplex virus.

Claim 15

A murine myeloma cell according to claim 3.

Claim 16

A murine myeloma cell according to claim 6.

Claim 17

A murine myeloma cell according to claim 7.

Claim 18

A murine myeloma cell according to claim 8.

Claim 19

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 1 stably transformed by said doubly-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 20

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 2 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 21

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 3 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 22

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 17 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 23

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 13 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 24

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 14 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 25

A method of producing non-immunoglobulin proteins comprising: (a) providing a clone of myeloma cells according to claim 15 stably transformed by said double-stranded DNA molecule; (b) growing said clone under conditions whereby said non-immunoglobulin protein is expressed; and (c) recovering said non-immunoglobulin protein thereby produced.

Claim 26

A double-stranded DNA molecule containing a coding sequence expressable in a myeloma cell comprising: (i) a coding sequence encoding a non-immunoglobulin protein and bounded by a translation start codon at its 5' terminus and a translation stop codon on its 3' terminus; (ii) a non-immunoglobulin promoter sequence adjacent to the 5' terminus of said coding sequence, said promoter sequence containing a transcription initiation site and capable of initiating transcription of said coding sequence in a mammalian myeloma cell; (iii) the 8-base pair nucleotide sequence 5' -ATTTGCAT-3' on the nontranscribed strand located 5' to the transcription initiation site of said promoter sequence and in a position whereby the rate of transcription of said coding sequence in a mammalian myeloma cell is increased; and (iv) a transcription enhancer element of a mammalian kappa light-chain immunoglobulin gene located so as to increase the rate of transcription of said coding sequence in a mammalian myeloma cell.

Claim 27

A double-stranded DNA molecule containing a coding sequence expressable in a myeloma cell comprising: (i) a coding sequence encoding a non-immunoglobulin protein and bounded by a translation start codon at its 5' terminus and a translation stop codon on its 3' terminus; (ii) a non-immunoglobulin promoter sequence adjacent to the 5' terminus of said coding sequence, said promoter sequence containing a transcription initiation site and capable of initiating transcription of said coding sequence in a mammalian myeloma cell; (iii) the 8-base pair nucleotide sequence 5' -ATTTGCAT-3' on the nontranscribed strand located 5' to the transcription initiation site of said promoter sequence and in a position whereby the rate of transcription of said coding sequence in a mammalian myeloma cell is increased; and (iv) a transcription enhancer element of a mammalian kappa light-chain immunoglobulin gene located so as to increase the rate of transcription of said coding sequence in a mammalian myeloma cell.

Claim 28

The DNA molecule of claim 25 wherein said immunoglobulin gene enhancer is a murine enhancer.

Claim 29

The DNA sequence of claim 25 wherein said promoter sequence is substantially homologous to the wild-type thymidine kinase promoter of the herpes simplex virus.

Claim 30

The DNA sequence of claim 26 wherein said promoter sequence is substantially homologous to the wild-type thymidine kinase promoter of the herpes simplex virus.