Enhancer for eukaryotic expression systems

Details Enhancer for eukaryotic expression systems Enhancer for eukaryotic expression systems

1994-11-09

2001-04-17

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S455000, C435S456000, C435S320100, C435S325000, C435S091400, C435S091410, C536S023100, C536S024100

Reexamination Certificate

active

06218140

ABSTRACT:

The invention is directed to an enhancer for eukaryotic expression systems, containing DNA from the upstream region of the major immediate early (IE) region of human cytomegalovirus (HCMV). According to certain embodiments, the enhancer is obtainable by sonication of the DNA from the IE region of HCMV with the formation of about 300 bp fragments, co-transfection of CV1 monkey cells and enhancerless SV40 genome, isolation of the recombinants which show lytic growth, and isolation of the inserted HCMV DNA, and enhancer-active mutants of this DNA. A process for improvement of eukaryotic expression systems by incorporating the enhancer upstream or downstream of the structural gene or of the regulation region is also provided. According to certain embodiments, the enhancer is incorporated not more than about 7,000 bp, or about 3,000 bp, upstream or downstream of the sites specified. Other advantageous embodiments follow.
The “enhancer trap” is described in F. Weber et al., Cell 36 (1984) 983-992; is respect of HCMV DNA, see G. Jahn et al., J. Virology, Feb. 1984, Vol. 49, 363-370 and literature quoted there, also D. R. Thomsen et al., Proc. Natl. Acad. Sci. USA, 81 (1984), 659-663, and P. J. Greenaway et al., Gene 18 (1982) 355-360.
In the HCMV DNA, the enhancer is located in the Hind III E fragment (Greenaway et al., loc. cit.), which includes the Pst I m fragment (about 2.1 kb).
Two recombinants were isolated by sonication of the DNA from the IE region of HCMV with the formation of about 300 bp fragments, co-transfection of CV1 monkey cells and enhancerless SV40 genome, isolation of the recombinants which show lytic growth, and isolation of the inserted HCMV DNA, and enhancer-active mutants of this DNA. Those recombinants contained 341 and 262 bp of HCMV DNA, located at positions −
118
to −
458
and −
263
to −
524
respectively on the published DNA sequence (Greenaway et al., loc. cit.). The overlap of 196 bp contains an essential part of the enhancer. Deletion mutants, for example obtained by Aha II and religation of the fragments in various combinations, are likewise enhancer-active.
The invention also relates to DNA which is a sequence homolog of reisolated HCMV specific enhancer DNA to the extent of at least 75, preferably at least 80, %, or is hybridized therewith.
The enhancer increases the expression of rabbit beta-globin in HeLa cells, after incorporation downstream of the appropriate gene, by at least two orders of magnitude, irrespective of the orientation. Thus the enhancer is superior to that of SV40 by the factor 3 to 5, dependent on the host system.
The HCMV enhancer has activity in a broad spectrum of host cells (cells of primates, mice, rats and frogs). It stimulates the expression of proteins in eukaryotic systems and thus facilitates the production of modified proteins, for example glycoproteins.
It is also possible to eliminate the promoter instrinsic to HCMV, for example by deletion of about 100 bp using Bal 31 beyond the Sac I restriction site. Where appropriate, the enhancer sequence can be modified by the attachment of adaptors of linkers.
When used with the intrinsic promoter, it is possible for a eukaryotic promoter to be substituted, for example by incorporation with inclusion of the first splice donor consensus sequence of the IE gene before the splice acceptor sequence of the gene which is to be expressed.
The invention is illustrated in detail in the Example which follows.


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