Process for the production of naturally folded and secreted...

Details Process for the production of naturally folded and secreted... Process for the production of naturally folded and secreted...

2000-04-20

2001-10-30

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

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

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

C435S071100, C435S071200, C435S849000, C435S877000, C530S402000, C530S404000, C530S412000, C530S414000, C536S023500, C536S023600, C536S023700

Reexamination Certificate

active

06309861

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field
The invention concerns a process for the production of water-soluble, naturally folded and secreted polypeptides after expression in prokaryotic cells.
2. Description
Protein synthesis in prokaryotic organisms, which is also called translation, takes place on the ribosomes in the cytoplasm. When recombinant DNA is expressed in prokaryotic host organisms, it is often desirable to secrete the recombinant gene product or protein that is obtained in this process from the cytoplasm through the inner bacterial membrane into the periplasmic space between the inner and outer membrane. Secreted proteins can then be released from the periplasm into the nutrient medium for example by osmotic shock. A disadvantage of this process is that the secreted polypeptides often do not form the native, biologically active conformation (Hockney, TIBTECH 12 (1994) 456-463; Baynex, Curr. Opin. Biotechnol. 10 (1999) 411-421).
Compounds such as urea or urea derivatives, formamide, acetamide or L-arginine are used in methods for the in vitro renaturation of insoluble protein aggregates (inclusion bodies) which are formed during the cytoplasmic expression of recombinant DNA in prokaryotic cells. L-arginine as an additive can considerably improve the yield of natively folded proteins in the renaturation in vitro (Rudolph et al., U.S. Pat. No. 5,593,865; Buchner & Rudolph, Bio/Technology 9 (1991)157-162; Brinkmann et al., Proc. Natl. Acad. Sci USA 89 (1992) 3075-3079; Lin & Traugh, Prot. Express. Purif. 4 (1993) 256-264). Thiol reagents such as glutathione are known to improve the yield of natively folded proteins when recombinant DNA is expressed in prokaryotic cells (Glockshuber et al., EP-A 0 510 658).
Recently molecular chaperones and folding catalysts such as peptidyl-prolyl-cis/trans-isomerases or protein disulfide isomerases (Glockshuber et al., EP-A 0 510 658) have been used to increase the yield of native recombinant protein when folded in vivo (Thomas et al., Appl. Biochem. Biotechnol. 66 (1997) 197-238). In some cases this has led to considerable improvements in the expression e.g. of ribulose bisphosphate carboxylase (RUBISCO; Goloubinoff et al., Nature 337 (1989) 44-47), human procollagenase (Lee & Olins, J. Biol. Chem. 267 (1992) 2849-2852) or neuronal nitrogen oxide synthase from rats (Roman et al., Proc. Natl. Acad. Sci. USA 92 (1995) 8428-8432). In these examples GroEL/ES or the DnaK system from
E. coli
was co-overexpressed in the cytosol. The positive effect is usually an increased yield of the desired protein in a soluble form.
The co-expression of chaperones has also been examined when recombinant proteins are secreted into the periplasm of
E. coli.
However, in this case only a cytosolic overexpression of chaperones was evaluated in order to optimize secretion into the periplasm (Perez-Perez et al., Biochem. Biophys. Res. Commun. 210 (1995) 524-529; Sato et al., Biochem. Biophys. Res. Commun. 202 (1994) 258-264; Berges et al., Appl. Environ. Microbiol. 62 (1996) 55-60). Molecular chaperones are used in the prior art to stabilize proteins and thus to protect them from aggregation and inactivation (Buchner et al., EP-A 0 556 726 A1). Previous attempts at cosecretion in
E. coli
have only concerned folding catalysts, such as protein disulfide isomerase (PDI; Glockshuber et al., EP-A 0 510 658) or peptidyl-prolyl-cis/trans-isomerases or Dsb proteins from
E. coli
(Knappik et al., Bio/Technology 11 (1993) 77-83; Qiu et al., Appl. Environm. Microbiol. 64 (1998) 4891-4896 and Schmidt et al., Prot. Engin. 11 (1998) 601-607). Recently, co-overexpression of the periplasmic Skp protein led to more efficient folding of phase display and higher yield of antibody fragments secreted to the periplasm (Bothman and Plutckthun, Nat. Biotechnol. 16 (1998) 376-380; Hayhurst and Harris, Prot. Expr. Purif. 15 (1999) 336-343).
SUMMARY OF THE INVENTION
The subject invention provides a process for producing a water-soluble, naturally-folded eukaryotic polypeptide containing at least two cysteines linked by disulfide bridges. This process comprises culturing in a nutrient medium prokaryotic cells which contain an expression vector that (i) encodes the polypeptide and (ii) contains a prokaryotic signal sequence at its N-terminus. The culturing is under conditions such that the polypeptide is secreted into the periplasm of the prokaryotic cells or into the medium. The culturing is in the presence of an amount of arginine or a compound of the formula:
R
2
—CO-NRR
1
  (I)
wherein
R and R
1
are each independently hydrogen or a saturated or unsaturated, branched or unbranched C
1
-C
4
alkyl chain, and
R
2
is hydrogen, NHR
1
, or a saturated or unsaturated, branched or unbranched C
1
-C
3
alkyl chain.
The amount of arginine or the compound of formula I is sufficient to minimize the formation of inclusion bodies.Preferably, the arginine or the compound of formula I is present at a concentration greater than 0.1 mole per liter, for example, from 0.1 to 1.5 moles per liter.
The signal sequence is cleaved from the polypeptide and the polypeptide is isolated. Preferably, the signal sequence is derived from gram-negative bacteria.
Preferably, the arginine is added in its hydrochloride or other titrated form. A reducing thiol reagent, such as glutathione, can also be added to the nutrient medium. The prokaryotic cell may also contain an additional expression vector which encodes a molecular chaperone, such as DnaJ from
E. coli
or Hsp25. The additional expression vector can contain recombinant DNA encoding for the molecular chaperone in opeative linkage with DNA encoding a signal peptide for penetrating the inner bacterial membrane. The additional expression vector can further contain DNA encoding a secreted molecular chaperone.
The DNA encoding the secreted protein is under the control of an inducible expression signal. While not limiting the choice of polypeptide, the polypeptide can be an antibody, antibody fragment, interferon, protein hormone, or a protease.


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Derwent Abstract for EP510 658.

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