WO1997018321A1

WO1997018321A1 - Photosynthetic production of stable isotopically labeled recombinant proteins

Info

Publication number: WO1997018321A1
Application number: PCT/US1996/018229
Authority: WO
Inventors: F. C. Thomas Allnutt
Original assignee: Martek Corp
Current assignee: Martek Corp
Priority date: 1995-11-15
Filing date: 1996-11-14
Publication date: 1997-05-22
Anticipated expiration: 1998-05-15
Also published as: AU7731696A

Abstract

An inexpensive method for producing stable isotope, uniformly labeled recombinant proteins employs photosynthetic cells or organisms such as algae grown on simple inorganic substrates which are labeled with stable isotopes.

Description

PHOTOSYNTHETIC PRODUCTION OF STABLE ISOTOPICALLY LABELED RECOMBINANT PROTEINS

BACKGROUND OF THE INVENTION

Stable isotope incoφoration into biologically important compounds has become a common technique Recent work has utilized uniformly, stable isotopically-labeled complex growth media that has been derived from algae (either bluegreen or green) to produce uniformly labeled recombinant proteins for use in structural determinations This has also been done using minimal media supplemented with glucose, also of algal origin The reason for use of algally-derived materials as the feedstock for this approach is that algae are capable of incorporation of very simple, and therefore cheaply made, compounds Simple carbon and nitrogen sources, such as ¹⁵NH₃, ¹⁵NO₂, ¹⁵NO₃, H CCy, ¹⁵ N, , ¹³ CQ , oi² 1^ O can be incorporated via photosynthesis into complex biomolecules such as proteins, lipids, amino acids, and nucleic acids An alternative approach is now available in methyltrophic organisms capable of growth on simple carbon sources such as methanol and methane However, these grow very poorly on these simple substrates Pichia, the most commonly used methyltrophic yeast, can be grown on glycerol or glucose and then have an expression vector induced by methanol addition This means that the biomass needs to be grown up on labeled complex carbon sources prior to induction with the simple carbon source

Thus there is a continuing need in the art for methods of producing proteins which are labeled with stable isotopes for use in structural studies SUMMARY OF THE INVENTION

It is an object ofthe invention to provide a method for producing recombinant proteins which are uniformly labeled with a stable isotope

This and other objects of the invention are provided by one or more embodiments described below In one embodiment a method of producing recombinant protein which is uniformly labeled with a stable isotope is provided The method comprises the steps of: photosynthetically culturing a recombinant cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate selected from the group consisting of a simple ¹⁵N-containing compound, ¹³CO₂, and ²H₂O, to produce said foreign protein which is labeled with a stable isotope, harvesting said recombinant cell or organism from said culture medium, and isolating said foreign protein from said harvested recombinant cell or organism or from said culture medium

Another method for producing recombinant protein which is uniformly labeled with a stable isotope, is also provided The method comprises the steps of autotrophically culturing a recombinant photosynthetic cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate, to produce said foreign protein which is labeled with the stable isotope; harvesting said photosynthetic cell or organism, and isolating said foreign protein from said harvested photosynthetic cell or organism or from said culture medium

These and other embodiments of the invention provide the art with an inexpensive means of preparing uniformly stable isotope labeled proteins of interest DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is a discovery of the present invention that stable isotopically labelled proteins can be made very inexpensively by production in photosynthetic cells or organisms, e.g., algal cells. The photosynthetic cells or organisms can be grown on simple inorganic substrates which are labeled with a stable isotope. For example, one or more of ¹⁵N₂, ¹ CO₂, or ²H, O can be used in the culture medium of the algae to achieve proteins which are uniformly labeled in their nitrogen, carbon or hydrogen atoms. Other simple substrates employing ^{l 5}N, C, or ²H, may be used, as well as other stable isotopes, such as ¹⁸0, ³³S, ³⁴S, ⁵⁸Fe, "Fe, ⁵⁴Fe, ^7ϋZn, ⁶⁷Zn, ⁷⁴Se, ⁷⁶Se, ⁷⁷Se, ⁷⁸Se, ⁸²Se, ²⁹Si, ³⁰Si. Preferably the isotope is oxygen, iron, or silicon Most preferably the photosynthetic cells or organisms are cultured autotrophically.

The gene encoding the desired foreign protein for labelling can be cloned into a vector according to techniques which are known in the art. Foreign proteins according to the invention include any which are not found in nature in the particular organism being used. The vector can be self-replicating in the photosynthetic cell or organism or it can incorporate into the genome The gene-loaded vector can then be introduced into the target photosynthetic cell or organism via standard transformation procedures. These include natural uptake, conjugation, microprojectile bombardment, eiectroporation, and physical disruption by particulates such as glass beads or silicon whiskers. Transformed cells can be plated on selective medium to select specific clones containing the desired genes. Clones which produce high amounts of the desired protein can also be selected or screened. The desired foreign protein can be isolated from the harvested biomass or from the culture medium, if the protein is secreted from the host cell into the culture medium. Purification can be accomplished using affinity chromatography, or other suitable protein separation methods, as are known in the art.

Host strains for the desired recombinant protein can be any photosynthetic cell or organism known in the art. These include cyanobacteria and eukaryotic algae The cyanobacteria (blue-green algae) are prokaryotic organisms that have similar characteristics to other gram-negative bacteria. The eukaryotic algae include the green as well as the brown algae, both of which can be used as the host cells for the recombinant protein In addition, diatoms and other chrysophytes can be used, as well as dinoflagellates, red algae, cryptomonads, and euglenoids Other photosynthetic bacteria can be used as well, including non-oxygenic purple sulfur and non-sulfur bacteria and nitrogen-fixing bacteria Single cell culture of higher plants may also be used

The recombinant foreign protein can be targeted for deposition in specific cells that are always anaerobic (the heterocysts), the cytoplasm, the periplasmic space, or the external medium Specific vectors for incorporation of the gene into either the chloroplast or nuclear genomes can be used, or autonomously replicating vectors can be used The gene for the desired foreign protein can be expressed either constitutively or by induction Thus vectors having sites of insertion downstream from suitable promoters for such control can be used, as is appropriate Typically, genes for toxic proteins are introduced to a cell in a repressed state, induction is required for expression, but typically only after sufficient biomass is achieved in the culture Alternatively, genes encoding toxic proteins can be targeted for expression in specialized cells or organelles which will effectively sequester the protein Desired proteins can be expressed in the cytoplasm, be directed to specific sites in the cell by leader sequences, or be directed for export from the cell Any such techniques as are known in the art can be used After isolation ofthe stable isotopically labeled protein from the recombinant photosynthetic cell or organism, the protein may be used to determine the structure of the protein, or may be used as a tracer in metabolic studies Typically such proteins are subjected to nuclear magnetic resonance spectroscopy NMR) to obtain structural information, according to techniques well known in the art

Claims

1 A method of producing recombinant protein which is uniformly labeled with a stable isotope, compnsing the steps of photosynthetically culturing a recombinant cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate selected from the group consisting of a simple ¹⁵ -contaιnιng compound, ^I3CO₂, and ²H₂O, to produce said foreign protein which is labeled with a stable isotope, harvesting said recombinant cell or organism from said culture medium, and isolating said foreign protein from said harvested recombinant cell or organism or from said culture medium

2 The method of claim I wherein the recombinant cell or organism is an alga 3 The method of claim 2 wherein the alga is prokaryotic

4 The method of claim 2 wherein the alga is eukaryotic

5 The method of claim 3 wherein the alga is blue-green

6 The method of claim 4 wherein the alga is green

7 The method of claim 4 wherein the alga is brown 8 The method of claim 4 wherein the alga is selected from the group consisting of diatoms, cryptomonads, dinoflagellates, euglenoids, and chrysophytes 9 The method of claim 1 further comprising the step of subjecting said stable isotopically labelled foreign protein to nuclear magnetic resonance spectroscopy to determine its structure 10 A method of producing recombinant protein which is uniformly labeled with a stable isotope, comprising the steps of autotrophically cultuπng a recombinant photosynthetic cell or organism which encodes a foreign protein in a culture medium which comprises a stable isotopically labelled substrate, to produce said foreign protein which is labeled with the stable isotope; harvesting said photosynthetic cell or organism, and isolating said foreign protein from said harvested photosynthetic cell or organism or from said culture medium 1 1 The method of claim 10 wherein said stable isotope is ^{l 5}N

12 The method of claim 10 wherein said stable isotope is "C

13 The method of claim 10 wherein said stable isotope is ^:H₂

14 The method of claim 10 wherein the photosynthetic cell or organism is an alga 15 The method of claim 14 wherein the ala is prokaryotic

16 The method of claim 14 wherein the alga is eukaryotic

17 The method of claim 15 wherein the alga is blue-green

18 The method of claim 16 wherein the alga is green

19 The method of claim 16 wherein the alga is brown 20 The method of claim 16 wherein the alga is selected from the group consisting of. diatoms, cryptomonads, dinoflagellates, euglenoids, and chrysophytes 21 The method of claim 10 further comprising the step of subjecting said stable isotopically labelled foreign protein to nuclear magnetic resonance spectroscopy to determine its structure