Polypeptide factor from a thermophilic eubacterial species and use thereof in the production of functional, heterologous proteins in an expression host

Abstract

A polypeptide factor derived from the thermophilic eubacterial species Thermus thermophilus has universal protein expression-assisting activity. The polypeptide factor has been named the CzrB protein active in full length or truncated form has the potential to act as a universal protein expression-assisting molecule which can increase the yields of all heterologous proteins produced in E. coli by a mechanism that is independent of the protein being expressed.

Description

[0001] The present application is a continuation application of co-pending application Ser. No. 10 / 389,771, file on Mar. 18, 2003, priority of which is claimed under 35 U.S.C. §120 and the contents of which are hereby incorporated by reference.FIELD OF THE INVENTION [0002] This invention relates to the production of heterologous proteins in a host organism and to protein expression-assisting molecules which result in said heterologous proteins having functional activity, due to a correct folding thereof. BACKGROUND AND PRIOR ART [0003] The production of heterologous proteins in bacterial hosts such as the bacterium Escherichia coli (hereinafter referred to collectively as E. coli and exemplified by E. coli except where otherwise expressly stated) is a powerful tool in the generation of many important biotechnological and medical products. This technique involves inserting the DNA encoding the product in question into an E. coli cell and using the cell to convert the genetic informat...

AI Technical Summary

Benefits of technology

[0021] The CzrB protein from T. thermophilus either in its full length or truncated forms described herein has the potential to act as a universal protein expression-assisting molecule which can increase the yields of all heterologous proteins produced in E. coli as hereinafter described.

[0022] It is expected that the truncated version of CzrB, containing a putative 92 amino acids as opposed to the 291 of the mature CzrB protein will lead to significantly higher improvements in protein yields upon over-expression from a better regulated promoter. The truncated protein is less than one-third the size of the mature protein and thus is likely to accumulate to much higher levels and at lower metabolic expense to the expressing cell. Furthermore, the truncated protein is also unlikely to be inserted into the cell membrane in the host bacterial cell and, thus, less likely to interfere with normal cell functioning if expressed at greatly elevated levels in the cell under the control of a strong promoter.

[0030] According to a further embodiment of the invention there is provided a method for increasing production of heterologous protein in a bacterial host cell, which method comprises cultivating said host cell under conditions permitting expression of a DNA sequence as hereinbefore defined.

[0034] According to a further embodiment of the invention there is provided a method of reducing stress in an expressing bacterial cell, which method comprises co-expressing a heterologous protein and a polypeptide factor as hereinbefore defined.

Problems solved by technology

Furthermore, the nutritional (and sterility) requirements of E. coli are uncomplicated, relative to higher organisms.

A major disadvantage of E. coli as an expression host, however, is the fact that the yields attainable with this organism are relatively low, while it frequently also exhibits difficulties in synthesising proteins derived from eucaryotic sources.

These difficulties can take the shape of an inability to carry out particular post-translational modifications of the translated polypeptide or, more fundamentally, an inability of the E. coli cellular machinery to fold the peptide in the first place.

In instances such as the latter, the available solutions have been to translate the polypeptide in E. coli, followed by refolding in vitro—a time-consuming and highly inefficient process—or to switch expression host to a higher organism which can carry out the expression efficiently, but with the concomitant loss of advantages of E. coli, as outlined above.

While E. coli carries out the process of gene expression and protein production very efficiently with its own, natural proteins, it is considerably less productive when expressing proteins from other species.

This is most likely due to an inability to correctly fold the translated polypeptide, or to successfully transport it to the appropriate subcellular compartment for assembly or folding.

Such a deficiency may result from the E.

In such a scenario, the expressed protein typically forms large, insoluble aggregates consisting of multiple copies of the protein, which is non-functional and may be destroyed by the normal cellular machinery.

Furthermore, expression of heterologous genes in E. coli appears to frequently subject the cells to severe stress, leading to damage to the outer membrane of the host E. coli cell and leaking of the contents of the cell into the culture medium.

With some foreign genes, E. coli has been found to be incapable of producing any functional protein; in cases in which E. coli folds the translated protein inefficiently or is overly stressed as a result of its expression, yields of the heterologous protein are dramatically reduced.

(1999) Prot. Eng. 12:605-611) but remains severely limited by the fact that solutions to expression problems that result from mutagenic modification are likely to be highly specific for the particular protein being expressed—whereas solutions that could be applied to all heterologous proteins expressed in E. coli would eliminate the need for labour-intensive, highly time consuming mutagenic studies to be repeated for each protein being produced.

To date, no single molecule has been identified, however, that improves the production of all heterologous proteins studied and, thus, again the difficulty arises of having to individually optimise expression for each heterologous protein.

Images