Preparation of a recombinant protein in a prokaryotic host cell

Details Preparation of a recombinant protein in a prokaryotic host cell Preparation of a recombinant protein in a prokaryotic host cell

2001-07-27

2004-11-23

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

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

Using a micro-organism to make a protein or polypeptide

C435S252300, C435S252330, C435S252800, C435S069400, C435S069700

Reexamination Certificate

active

06821755

ABSTRACT:

The instant application claims the benefit of provisional U.S. Appl. No. 60/229,010, filed Aug. 30, 2000, which is herein incorporated by reference and DE 100 37 111.6, filed July 27, 2000 (in the German language), which is herein incorporated by reference.
The present invention relates to advantageous processes for preparing heterologous proteins in prokaryotic host cells by improved codon use and/or expression of tRNAs which code for rarely occurring codons in the said host cell. Numerous bacteria, particularly Gram-positive bacteria, for example, have already been used as host cells for the preparation of heterologous recombinant proteins, e.g. secreted proteins, in an endotoxin-free environment (references 8, 9, 19). The bacterium
Staphylococcus carnosus
(
S. carnosus
), for example, which is used in the food industry for the fermentation of meat and fish, is a suitable bacterium for this purpose. It is free from virulence factors and proteolytic activities in the supernatant and is capable of secreting large amounts of protein (10). Moreover, only small amounts of host cell-coded proteins are secreted, which makes it easier to purify the protein produced. Bacterial enzymes (7, 4, 17), for example, have already been recombinantly produced and expressed in
S. carnosus
. Recombinant
S. carnosus
strains which express antigens such as the Streptococcus protein G on their surface,are particularly promising as live vaccines (5, 12).
One crucial disadvantage of numerous bacterial systems is their use of rare codons, which is very different from the codon preference in human genes. The presence of rare codons in
E. coli
led to delayed and reduced expression of recombinant genes (2, 6). The problem of the present invention is therefore to overcome this disadvantage of the prior art and provide a prokaryotic expression system with improved properties.


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