Study of Recombinant protein expression

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There are provided methods for the expression of a recombinant protein of interest, said methods comprising, in additional to various additional steps:

a) culturing a host cell which expresses: i) one or more genes encoding the recombinant protein(s) of interest; ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GRoES, Dnak, DnaJ, GRpe, ClpB and their homologs; under conditions suitable for protein expression; and separating said recombinant protein of interest from the host cell culture. Also provided are methods for increasing the degree of refolding of a recombinant protein of interest by ading a composition containing a chaperone protein to a preparation of the recombinant protein of interest in vitro.

So far, no systematic approach has been made, to analyse whether the combination of all three chaperones systems expressed together with target genes in E. coli cells enhances solubility of recombinant proteins.

Furthermore, none of the above-described studies allows the widespread optimisation of expression systems that is required to improve yields of soluble proteins on a general level.

For example, each of the prior investigations focused on only one or a very small number of target proteins. These investigations also focused on the use of only one or two combined chaperone systems. In addition, none of these investigations addressed the issue of the importance of the ratio of the chaperones to one another and to the recombinant target protein. The previous studies therefore did not provide any understanding of the relationship between different chaperone proteins with respect to the folding/refolding of recombinant target proteins.

Accordingly, there remains a great need in the art for a general method to improve the yield of soluble recombinant protein in a given expression system. Such a method would allow the optimisation of expression systems to give maximal yields of soluble target proteins, and be of obvious industrial and commercial benefit.

The present invention is based upon the systematic engineering of cells for the controlled co-overexpression of different combinations of chaperone genes and taxget genes. In addition, it was investigated whether in vivo disaggregation and refolding of recombinant proteins from aggregates/inclusion bodies could be stimulated by enhanced levels of chaperones when the production of the target protein is stopped. As a result, the invention provides novel methods of optimising a given expression system in order to achieve higher yields of the desired soluble recombinant protein.

According a first aspect of the present invention, there is provided a method for the expression of a recombinant protein of interest, said method comprising:

a) culturing a host cell which expresses:

i) one or more genes encoding one or more recombinant proteins) of interest;

ii) at least two genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, CIpB and their homologs (for example, Hsp104, Ydjl and Ssal in yeast); under conditions suitable for protein expression; and b) separating said recombinant protein of interest from the host cell culture.

Through the recombinant engineering of host cells in this manner, the invention provides novel methods for producing a recombinant protein of interest, which have been found to lead to significant improvements in the levels of protein produced in the system. The mechanism is thought to be through increasing the folding rates of particular proteins using the co-expression of particular chaperones in controlled amounts. Using this system, very high yields of the desired soluble recombinant proteins of interest can be obtained.

Any recombinant protein of interest may be produced using the system of the invention.

Preferred examples of proteins of interest will be apparent to the skilled reader. Particularly preferred recombinant proteins are those for which it is desirable to produce a large amount, and those of commercial interest.

Furthermore, the invention is readily applicable to a wide range of known expression systems by alterations in the cell culture techniques employed. For example, anaerobic fermenter-based cell culture would be appropriate for the culture of obligate anaerobes, whereas standard aerobic cell culture techniques would be appropriate for obligate aerobes.

The nutrient composition of the culture medium may also be varied in accordance with the chosen expression system. The most suitable method of cell culture for a given expression system will be readily apparent to the skilled man.

Preferably, the genes selected in step a) ii) include DnaK, DnaJ and GrpE or homologs thereof, and may additionally include CIpB or a homolog thereof.

In another preferred aspect of the invention, the genes selected in step a) ii) include GroES

and GroEL or homologs thereof.

More preferably, the genes selected in step a) ii) include the DnaI~, DnaJ, GrpE, CIpB, GroES and GroEL genes or homologs thereof.

The above combinations of chaperone proteins have been found to be particularly suitable for use in the methods according to the invention.

According to a further embodiment of the first aspect of the present invention, there is provided a method for the expression of a recombinant protein of interest, said method 5 comprising:

a) culturing under conditions suitable for protein expression a host cell which expresses:

i) one or more genes encoding one or more recombinant proteins) of interest;

ii) one or more genes encoding proteins selected from the group consisting of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, CIpB and their homologs;

iii) one or more genes encoding proteins selected from the group consisting of the small heatshock proteins of the IbpA family and/or the IbpB

family and/or their homologs; and b) separating said recombinant protein of interest from the host cell culture.

The inclusion of a small heatshock protein of the IbpA family and/or the IbpB

family with one or more of the chaperone proteins GroEL, GroES, DnaK, DnaJ, GrpE, ClpB in a host cell with a gene encoding a protein of interest has been shown to bestow significant beneficial effects on the level of expression of the recombinant protein.

For the purposes of this patent specification, two genes or proteins are said to be 'homologs' if one of the molecules has a high enough degree of sequence identity or similarity to the sequence of the other molecule to infer that the molecules have an equivalent function. 'Identity' indicates that at any particular position in the aligned sequences, the amino acid or nucleic acid residue is identical between the sequences.