# Patent

## Title
Protein fragment complementation assay for thermophiles

## Abstract

A protein fragment complementation assay for thermophiles is provided wherein a thermophilic bacteria having a temperature-sensitive adenylate kinase is transformed with one or more vectors having sequences encoding a first test peptide operatively fused to a first portion of a thermostable adenylate and a second test peptide operatively fused to a second portion of the thermostable adenylate kinase. Association of the first and second test peptides allows association of the first and second portions of the thermostable adenylate kinase and growth of the thermophilic bacteria at a temperature greater than 70° C.

## Background
PRIOR RELATED APPLICATIONS
The present invention claims priority to U.S. 61/245,124, entitled “Protein Fragment Complementation Assay for Thermophiles,” filed on Sep. 23, 2009.


FEDERALLY SPONSORED RESEARCH STATEMENT
This invention was made with government support under Grant Number (NNX08A020G) awarded by National Aeronautic and Space Administration. The government has rights in the invention.


FIELD OF THE INVENTION
The invention relates to a protein fragment complementation assay for thermophilic organisms.
BACKGROUND OF THE INVENTION
Microbes that grow optimally above 60° C. (thermophiles) and 80° C. (hyperthermophiles) populate ecosystems where many biomolecules have reduced stability. To support cellular growth, proteins from such organisms have evolved distinct amino acid compositions and higher thermostability compared to orthologs found in mesophilic organisms that grow at lower temperatures. This latter feature has been exploited for a variety of biotechnological applications.
For example, thermostable DNA polymerases have revolutionized molecular biology, xylanases have made paper processing greener, and oligosaccharide-modifying enzymes have been harnessed for corn syrup production. There is also a great interest in harnessing thermotolerant microbes and their proteins for other industrial processes, such as biomass conversion to bioethanol or biohydrogen. However, no in vivo screens have been developed to help achieve these high-temperature metabolic engineering and synthetic biology goals.
A comparison of the findings from mesophile and hyperthermophile proteomic studies suggests that the lack of high-temperature protein-protein interactions screens may limit the discovery of useful protein complexes. A high-throughput screen for pairwise protein-protein interactions among almost one thousand Pyrococcus horikoshii proteins found only 56 hetero-interactions using a two-hybrid assay implemented at a temperature (37° C.) far below that of its optimal growth of 98° C.
This finding can be contrasted with similar screens for protein complexes in bacteria and yeast under near physiological conditions, which invariably find protein-protein interactions at a frequency that is more than an order of magnitude higher. The creation of an assay that can be used to assess protein complex formation at thermophile growth temperatures would have multiple advantages over available assays in studying natural and engineered proteins. High temperature assays are predicted to be superior at discovering interactions among proteins that require extreme temperatures to adopt their native conformation and among proteins whose interactions weaken as temperature is decreased from the levels where hyperthermophiles grow.
Using split adenylate kinases in protein fragment complementation has been discussed by Nguyen, et al in “Thermostability promotes the cooperative function of split adenylate kinases,” published in Protein Engineering, Design & Selection, Vol. 21, pp. 303-310, 2008.
However, the system discussed in this paper was not in a high-temperature setting, and it is unclear whether the split fragments of adenylate kinases could still function or associate at high temperatures. Nor was the assay tested in a thermophilic organism. Therefore, there is a still need for a protein complementation assay capable of performing at high temperatures.
SUMMARY OF THE INVENTION
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context clearly dictates otherwise.
The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.
As used herein the term “thermophiles” is defined as microorganisms capable of surviving in an environment of temperatures higher than 50° C. “Hyperthermophiles” are an even more specialized form of thermophile, wherein such microorganisms are capable of surviving in a hyperthermal environment of temperatures greater than 70° C., and more preferably, 78° C. or more.
The term “temperature sensitive” means the activity of a protein decreases to at least 25%, and preferably 10%, of normal as the temperature increases to or beyond a thermophilic or hyperthermophilic temperature.
The term “thermostable” means that at least 50% activity, and preferably at least 60, 70, 75, or 80% of a protein's activity remains as the temperature increases to thermophilic temperatures.
When the adenylate kinase protein is described in terms of amino acid number, that number is to be ascertained by reference to SEQ ID NO. 1—in other words, the sequence in question is aligned with SEQ ID NO 1, e.g., using BLAST and the default parameters, and as shown in FIG. 7.
To establish an assay for studying protein complex formation within a living thermophile, Thermotoga neapolitana adenylate kinase (AKTn) was split into fragments that can be used as in a protein-fragment complementation assay (PCA) in Thermus thermophilus. 
FIG. 1 illustrates the rationale of using split adenylate kinase fragments in a complementation assay. Specifically, a living cell that lacks a functional AK at the assay temperature (e.g., the cell has a temperature sensitive AK mutant) can be transformed with one or more expression vectors containing split AK proteins, each AK fragment fused to test proteins, that may or may not be binding pairs. Because adenylate kinase function is required for survival, where the test binding proteins do not associate, neither with the AK fragments associate, and the cell will not live.
On the other hand, if there is interaction between proteins to be tested, the two adenylate kinase fragments will be brought close enough together to also bind together and provide normal adenylate kinase function. Thus, the cell will survive, and hundreds of thousands of test proteins can easily be screened in this way.
AKTn has many characteristics that make it suitable for designing a split enzyme that reports on protein-protein interactions at extreme temperatures. AKTn is monomeric and extremely thermostable, exhibiting maximal phosphotransferase activity at 80° C. (ATP+AMP←→2ADP) and having a denaturing temperature of 99.7° C. Furthermore, AKTn can be split to generate fragments that spontaneously associate and cooperatively function within a mesophilic bacterium growing at 40° C.
In an aspect of the present invention, there is provided a protein fragment complementation assay for thermophilic bacteria. The assay comprises: culturing a thermophilic bacteria having a temperature-sensitive adenylate kinase, a first vector having an expressable first test gene operatively fused to a first portion of a thermostable adenylate kinase gene encoding a first portion of a thermostable adenylate kinase, and a second vector having an expressable second test gene operatively fused to a second portion of the thermostable adenylate kinase gene encoding a second portion of the thermostable adenylate kinase. The first and second test genes encode first and second test peptides, and the association of the first and second test peptides allows association of the first and second portions of the thermostable adenylate kinase and growth of the thermophilic bacteria at a temperature greater than 70° C.
The first and second vector can be different or the same—in other words the vector can be polycistronic, coding for both test protein-AK fragment fusions, or the vector can contain two separate operons, each coding for one half of the system.
The adenylate kinase used in the present invention can come from any thermophilic bacteria, provided it has an adenylate kinase with at least about 50% protein identity to AAN86272 (SEQ ID NO: 1), and about 70% positive matches, preferably about 60% identity and about 75-80% positives matches and most preferably, at least 92% identity. Such adenylate kinases include but are not limited to the adenylate kinases from Thermotoga neapolitana, Thermotoga maritime, Thermotoga naphthophila, Thermotoga petrophila, or Marinitoga piezophila. 
The adenylate kinase used in the present invention can be split to comprise: a. residues 1-79 or 80-220 from Thermotoga neapolitana (AAN86272) (SEQ ID NO: 1); b. residues 1-79 or 80-220 from Thermotoga maritime (NP—229279) (SEQ ID NO: 2); c. residues 1-79 or 80-220 from Thermotoga naphthophila (ZP—05471476) (SEQ ID NO: 3); d. residues 1-79 or 80-220 from Thermotoga petrophila (YP—001244903) (SEQ ID NO: 4); e. residues 2-80 or 81-220 from Marinitoga piezophila (ZP—05098624) (SEQ ID NO: 5) or combinations and homologs thereof.
FIG. 7 shows a multi-alignment of the adenylate kinases from 5 thermophilic bacteria. As can be seen from the alignment, the adenylate kinase sequences from Thermotoga maritima (SEQ ID NO: 2), Thermotoga naphthophila (SEQ ID NO: 3), Thermotoga petrophila (SEQ ID NO: 4), and Marinitoga piezophila (SEQ ID NO: 5); have a 92% or higher identity to the AK sequence from Thermotoga neapolitana (SEQ ID NO: 1), indicating very high conservation of the adenylate kinase gene among thermophilic bacteria, and can thus be used in the protein complementary assay of the present invention.
FIG. 8 (SEQ ID NOS: 6-31) shows adenylate kinases from other bacteria that have at least 50% identity and at least about 70% positive matches that may also be used in the protein fragment complementation assay of the present invention.
In another aspect of the present invention, there is provided a kit for performing a protein complementation assay. The kit comprises a first expression vector encoding a first portion of a thermostable adenylate kinase and a second vector encoding a second portion of the thermostable adenylate kinase, wherein the first and second portions of the thermostable adenylate kinase are functional at temperatures higher than 60° C. when the first and second portions associate. Preferably, the vector contains suitable cloning sites for adding in the test protein sequences, and the test sequences can be added at either end of the AK fragments and be directly conjugated thereto, but are preferably separated from the adenylate kinase portion(s) by a short (5-15 amino acids) flexible linker sequence.
In a preferred embodiment, the first vector and the second vector are same vector, and the first and second portions of the thermostable adenylate kinase gene are contained in two operons, and more preferred the two portions are polycistronic on the vector with a cleavage site therebetween.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the rationale of a protein fragment complementation assay.
FIGS. 2A-2C illustrate the creation of PQN1 mutant strain from Thermus thermophilus HB8, the growth rate measurement of the wild type HB8 and mutant PQN1 strains, and the result of growth complementation by different fragments of adenylate kinase.
FIGS. 3A-3B illustrate the design and creation of vectors encoding different adenylate kinase fragments.
FIGS. 4A-4B illustrate the detection of chemotaxis protein-protein interaction.
FIGS. 5A-5C illustrate the constructs of different combinations of adenylate kinase fragments and CheA/CheX genes and the results of complementation assay.
FIGS. 6A-6B illustrate the constructs of different combinations of adenylate kinase fragments and CheA/CheY genes and the results of complementation assay.
FIG. 7 is an alignment comparison of adenylate kinases from Thermotoga neapolitana, Thermotoga maritime, Thermotoga naphthophila, Thermotoga petrophila, and Marinitoga piezophila (SEQ ID NOS: 1-5 respectively).
FIG. 8 is a table listing adenylate kinases from other bacteria that have at least about 50% identity and at least about 70% positives. (SEQ ID NOS: 6-31)
FIG. 9 shows the strategy used to create vectors that express AK fragment fusions from a single polycistronic transcript.



DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The following examples are illustrative only, and are not intended to unduly limit the scope of the invention.
Example 1
Materials
Agar was from DIFCO,TM Gelrite was from RESEARCH PRODUCTS INTERNATIONAL,TM and all other bacterial growth media components were from BD BIOSCIENCESTM and SIGMA-ALDRICH.TM
QuikchangeTM mutagenesis reactions were performed using PFUTURBOTM DNA polymerase from STRATAGENE,TM genes were amplified for cloning using VENTRTM DNA Polymerase from NEW ENGLAND BIOLABS,TM and restriction endonucleases were obtained from ROCHE BIOCHEMICAL,TM NEW ENGLAND BIOLABS,TM and PROMEGA.TM
Synthetic oligonucleotides were from OPERON BIOTECHNOLOGY,TM pETTM vectors were from EMD BIOSCIENCES,TM Escherichia coli XL1-BLUETM cells used for plasmid amplification and cloning were from STRATAGENE,TM and kits for DNA purification were from ZYMO RESEARCHTM and QIAGEN.TM
Example 2
Methods
Thermus thermophilus HB8 (ATCC #27634) was used as the parent strain for adk gene replacement. Liquid growth was performed in Evian-Thermus Medium (EvTM), which contains 8 g tryptone, 4 g yeast extract, and 3 g NaCl per liter of Evian mineral water. EvTM-agar plates containing 3% agar were used for growth at temperatures below 75° C., whereas EvTM-Gelrite plates containing 1.5% Gelrite were used for growth at higher temperatures to minimize desiccation.
Electrocompetent HB8 cells were prepared using a protocol similar to that previously described (de Grado, et al. 1999). Cells (500 mL) were grown to mid-logarithmic phase in EvTM medium (A600≈0.7) at 65° C., concentrated by centrifugation at 5,000 rpm for 30 min, washed with 10% glycerol (250 mL) two times at room temperature, resuspended in 10% glycerol (2 mL), and frozen at −80° C. in aliquots (100 μL).
Transformations were performed by mixing 3 μg of vector (100-800 μg/μL) and 100 μL of electrocompetent cells, incubating the mixture on ice for one hour, and electroporating the cells with a pulse of 12.5 kV/cm in 0.1 cm cuvettes using a BIORADTM MICROPULSER.TM Cells were immediately transferred to EvTM medium (5 mL) that had been prewarmed to 60° C. and incubated for 4 hrs at 60° C. in a 50 mL flask shaking at 150 rpm.
Cells transformed with pJJS-derived expression vectors (100 μL) were plated onto EvTM-agar plates containing 15 μg/mL bleocin (CALBIOCHEMTM) and incubated at 65° C. for 72 hours. Cells transformed with the adk gene replacement vectors were plated onto EvTM-agar plates containing 250 μg/mL kanamycin and incubated at 60° C. for 72 hours, and colonies identified as inviable at 80° C. (e.g. the native AK gene was replaced with a temperature sensitive mutant AK), were cured of their plasmid by restreaking them twice onto EvTM-agar plates lacking antibiotic at 60° C.
Desiccation was minimized during incubation by placing agar plates into ZIPLOCTM bags, removing extra air from the bags, and sealing all but one inch of the bag. To avoid condensation on plates during incubation, freshly poured plates were incubated with the agar facing up at 37° C. for 1-2 hours prior to use.
FIG. 2 illustrates how the vector used for adk gene replacement was built. A 1,600 bp segment of T. thermophilus HB8 genomic DNA including approximately 1,000 bp adjacent to the adk start codon, the adk gene, and 45 bp after the adk stop codon was PCR amplified from genomic DNA and cloned into pUC18 using hindIII and xbaI. In addition, approximately 1,000 bp of genomic DNA including the last 121 bp of the adk gene and sequence adjacent to the adk stop codon was PCR amplified from genomic DNA using and cloned into the pUC18-derived vector containing the first amplicon using xbaI and ecoRI to create pTT1.
The gene encoding a highly thermostable kanamycin nucleotidyltransferase (htk) (Hoseki, et al. 1999) with only a RBS was PCR amplified from pMK-18 (de Grado, et al. 1999) and cloned into pTT1 using xbaI to obtain pTT2. This plasmid was modified by two QUIKCHANGETM mutagenesis reactions to create pTT2-BK, a plasmid that has a unique bmtI site 8 bp prior to the adk start codon and a kpnI site 147 bp before the adk stop codon.
The adk gene from Geobacillus stearothermophilus (adkGs) was PCR amplified from genomic DNA and cloned into pTT2-BK using bmtI and kpnI to obtain pTT-GsteAK. Selection of HB8 transformed with pTT-GsteAK on EvTM-agar plates containing 250 μg/mL kanamycin yielded multiple colonies. After curing these strains of their plasmid by sequentially streaking them onto two EvTM-agar plates lacking antibiotic, PCR amplification with primers complementary to the HB8 adk gene revealed that the native adk had not been removed from the chromosome (data not shown), suggesting that the small amount of genomic DNA separating the adk and htk genes in pTT-GsteAK had facilitated off-pathway recombination. To remove this DNA, the htk resistance cassette was PCR amplified and cloned into the kpnI and xbaI sites in pTT-GsteAK to create pTTΔ200-GsteAK, which was the vector successfully used for gene replacement, as shown in FIG. 2A.
Cultures of HB8 and PQN1 cells grown overnight at 60° C. in EvTM were used to inoculate 50 mL flasks of prewarmed EvTM. Cells were grown over a range of temperatures (60 to 85° C.), and the optical density at 600 nm was measured to acquire growth curves until the stationary phase. Each data trace was fit in Kaleidagraph to a modified Gompertz equation, ln(N/N0)=A*(exp(−exp((μm*(2.71828183)/A)*(λ−t)+1))), where N is the population size as determined by A600 measurements, N0 is the initial A600 upon inoculation, A is the asymptotic value of the curve, λ is the lag period, and μm is the maximum specific growth rate. For each strain and temperature, we report the mean and standard error for the maximum specific growth rate calculated from three independent measurements, as shown in FIG. 2B.
The E. coli-T. thermophilus shuttle vector pWUR112 (Brouns, et al. 2005) containing a thermostable bleomycin selectable marker (shble) was modified through QuikChange mutagenesis to remove the unique ecoRI and xbaI restriction sties and create pWUR112-ΔEX, as shown in FIG. 3A. An ampicillin resistance cassette (bla) PCR amplified with primers that incorporated flanking kpnI-ecoRI-notI-xbaI and speI-notI-pstI-kpnI sites was cloned into the kpnI site of pWUR112-ΔEX to create pJJS. The strong constitutive slpA promoter (Faraldo, et al. 1992) was PCR amplified from genomic DNA and subcloned into pJJS using xbaI and pstI, replacing the bla gene, to create pJJS-Pro, as shown in FIG. 3A. In addition, the slpA transcriptional terminator TslpA (Faraldo, et al. 1992) was PCR amplified from T. thermophilus HB8 genomic DNA using primers that incorporate flanking ecoRI-notI-xbaI and speI-notI-pstI sites, digested with ecoRI and pstI, and cloned into pJJS digested with ecoRI and pstI to yield pJJS-Term. The pJJS, pJJS-Pro, and pJJS-Term vectors are all compatible with a modular subcloning assembly strategy, which was used for construction of the majority of protein expression vectors.
The Thermotoga neapolitana adk gene was PCR amplified from pET21d-TnAK (Vieille, et al. 2003) using primers that incorporate flanking ecoRI-notI-xbaI and speI-notI-pstI restriction sites, digested with xbaI and pstI, and cloned into pJJS-Pro that had been digested with speI and pstI to create pJJS-TnAK. In addition, fragments of the T. neapolitana adk gene that encode residues 1-79 (TnN) and 80-220 (TnC) were PCR amplified from pET21d-TnN and pET24d-TnC (Nguyen, et al. 2008), respectively, using primers that incorporate flanking ecoRI-notI-xbaI and speI-notI-pstI restriction sites and cloned into pJJS-Pro using a similar protocol to create pJJS-TnN and pJJS-TnC, respectively, as shown in FIG. 3B.
A vector for coexpressing TnN and TnC, pJJS-N+C, was created by digesting pJJS-TnC with xbaI and pstI, and subcloning the excised gene into pJJS-TnN that had been digested with speI and pstI. QuikChange mutagenesis was used to modify pJJS-TnC to create vectors that express TnC with C133A (pJJS-TnC133) and C156A (pJJS-TnC156) mutations. These plasmids were then used to create the coexpression vectors pJJS-N+C133 and pJJS-N+C156, using a cloning scheme similar to that described for pJJS-N+C.
The strategy used to create vectors that express AK fragment fusions from a single polycistronic transcript is shown in FIG. 9. The genes encoding the P1 and P2 domains of CheA (CheAP1P2; residues 1-264), full-length CheX, and full-length CheY were PCR amplified from Thermotoga maritima MSB8 genomic DNA (ATTC #43589D-5), and pJJS-Pro vectors were generated that encode each of these genes fused in frame to the N-terminus of TnN and TnCC156A mutation through a linker that is predicted to be flexible (GASGGGSSGGHM)) (SEQ ID NO: 32).
These gene fusions were PCR amplified using primers that incorporate ecoRI-notI-xbaI sites upstream of the ribosomal binding site (RBS) and speI-notI-pstI restriction sites adjacent to the stop codon. The RBS in the amplified gene fusions is identical in sequence to that found adjacent to the T. thermophilus HB8 slpA promoter (Faraldo, et al. 1992), and it includes twenty nucleotides upstream of the annotated RBS.
PCR amplified gene fusions encoding the TnN fragments were cloned into pJJS, whereas the gene fusions encoding TnCC156A were cloned into pJJS-Term. Upon sequence verification, the rbs-cheA/X/Y-tnCC156A-TslpA gene fusions were excised from their vectors using XbaI and PstI and subcloned into the vectors harboring the rbs-cheA/X/Y-tnN gene fusions that had been digested with SpeI and PstI. Table 1 lists all vector intermediates, and Table 2 lists the vectors used for complementation analysis.







TABLE 1







Plasmids derived from pJJS that were used to construct protein expression vectors.


The protein coding region cloned into pJJS is indicated as well as the regulatory


elements cloned adjacent to that region










Name
5′ DNA
Protein coding region
3′ DNA





pJJS
—
—
—


pJJS-Pro
PslpA-RBS
—
—


pJJS-Term
—
—
TslpA


pJJS-TnN
PslpA-RBS
TnN
—


pJJS-TnC
PslpA-RBS
TnC
—


pJJS-TnC133
PslpA-RBS
TnCC133A
—


pJJS-TnC156
PslpA-RBS
TnCC156A
—


pJJS-AN
PslpA-RBS
CheAP1P2-GASGGGSSGGHM-TnN
—


pJJS-XN
PslpA-RBS
CheX-GASGGGSSGGHM-TnN
—


pJJS-YN
PslpA-RBS
CheY-GASGGGSSGGHM-TnN
—


pJJS-AC156
PslpA-RBS
CheAP1P2-GASGGGSSGGHM-TnCC156A
—


pJJS-XC156
PslpA-RBS
CheX-GASGGGSSGGHM-TnCC156A
—


pJJS-YC156
PslpA-RBS
CheY-GASGGGSSGGHM-TnCC156A
—


pJJS-rbsTnC156-TslpA
RBS
TnCC156A
TslpA


pJJS-rbsAC156-TslpA
RBS
CheAP1P2-GASGGGSSGGHM-TnCC156A
TslpA


pJJS-rbsXC156-TslpA
RBS
CheX-GASGGGSSGGHM-TnCC156A
TslpA


pJJS-rbsYC156-TslpA
RBS
CheY-GASGGGSSGGHM-TnCC156A
TslpA


pJJS-rbsTnN
RBS
TnN
—


pJJS-rbsAN
RBS
CheAP1P2-GASGGGSSGGHM-TnN
—


pJJS-rbsXN
RBS
CheX-GASGGGSSGGHM-TnN
—


pJJS-rbsYN
RBS
CheY-GASGGGSSGGHM-TnN
—
















TABLE 2







Vectors used for complementation studies in T. thermophilus PQN1









Name
Proteins expressed
Expression





pJJS-TnAK
AKTn
monocistronic


pJJS-N + C
TnN & TnC
monocistronic


pJJS-N + C133
TnN & TnCC133A
monocistronic


pJJS-N + C156
TnN & TnCC156A
monocistronic


pJJS-N + C156-poly
TnN & TnCC156A
polycistronic


pJJS-YN + AC156
CheY-TnN & CheAP1P2-TnCC156A
polycistronic


pJJS-YN + XC156
CheY-TnN & CheX-TnCC156A
polycistronic


pJJS-YN + YC156
CheY-TnN & CheY-TnCC156A
polycistronic


pJJS-YN + C156
CheY-TnN & TnCC156A
polycistronic


pJJS-N + AC156
TnN & CheAP1P2-TnCC156A
polycistronic


pJJS-XN + XC156
CheX-TnN & CheX-TnCC156A
polycistronic


pJJS-N + XC156
TnN & CheX-TnCC156A
polycistronic


pJJS-XN + C156
CheX-TnN & TnCC156A
polycistronic


pJJS-XN + AC156
CheX-TnN & CheAP1P2-TnCC156A
polycistronic









Complementation analysis involving monocistronic constructs was assessed by streaking colonies obtained from transformations on solid medium in glass petri dishes, and incubating these plates at temperatures (≧78° C.) where PQN1 cannot grow like parental HB8 because it has a temperature sensitive adenylate kinase. Complementation studies involving the polycistronic constructs were performed by spotting a defined titer of cells grown at 65° C. onto EvTM-Gelrite plates, and evaluating growth after 24 hours at 78° C. In the spotting experiments, 5 mL EvTM liquid cultures containing 5 μg/mL bleocin were inoculated with colonies obtained from transformations and cultured overnight at 65° C. while shaking at 150 rpm.
The optical density of each culture was measured after 24 hours, and each culture was diluted to an A600 of 0.5, pelleted by centrifugation, and resuspended in 25% glycerol so that it was concentrated 4 fold. Serial dilutions (1×, 10×, 100×, and 1000×) of the resuspended cells (10 μL each) were spotted onto EvTM-Gelrite plates. After incubation at 78° C. for 24 hours, growth at each spot was analyzed using a Fluor Chem 5500 imager (Alpha-Innotech), and the program ImageJ was used to quantify the relative growth of spots.
In order to test our system we chose to use various chemotaxis proteins that are known to self assemble in vivo. T. maritima MSB8 CheAP1P2, CheX, and CheY were overexpressed in E. coli Rosetta cells (NOVAGENTM) using plasmids kindly provided by the Crane lab (Park, et al. 2004, Park, et al. 2004) that express these proteins with N-terminal (His)6 tags. Cells transformed with these plasmids were grown at 37° C. in LB supplemented with 25 μg/mL kanamycin, protein expression was induced at an A600 of  ̃0.5 bp adding 1 mM isopropylthio-β-D-galactoside (IPTG), cells were harvested by centrifugation after 3 hours of growth, and cells were lysed by resuspending them in PSI buffer (50 mM phosphate pH 7.5, 150 mM NaCl, 10 mM imidazole) containing 1 mM MgCl2, 300 μg/mL lysozyme, and 2 U/mL DNase I.
Cells were frozen at −80° C., thawed, and centrifuged at 15k×g for 1 hr. Cleared lysate was filtered through a sterile 0.2 micron syringe filter, applied to a 2 mL nickel talon affinity column (Qiagen) equilibrated with PSI buffer, washed with 10 column volumes of PSI buffer, and bound protein was eluted using PSI containing 250 mM imidazole. The elution containing CheAP1P2 (theoretical pI=4.6) was diluted 100 fold into 50 mM phosphate pH 7.5 buffer, applied to a 5 mL HiTrap-Q HP anion exchange column (GE Healthcare), and eluted using a linear gradient from 0 to 500 mM NaCl in 50 mM phosphate pH 7.5. All purified proteins were dialyzed overnight into PS buffer (50 mM phosphate pH 7.5 and 150 mM NaCl), and stored at −80° C.
An ÄKTA FPLC was used for all protein purification. Protein concentrations were determined by measuring their absorbance with a Cary 50 spectrophotometer, using the calculated extinction coefficients ε280(CheAP1P2)=5,120 M−1cm−1 and ε280(CheY)=2,560 M−1cm−1, as calculated from the primary sequence by the PEPSTATS algorithm. Dynamic light scattering was performed with a Malvern Instruments Zetasizer Nano ZS using samples that contained 115 μM of each protein in PS buffer (50 mM Phosphate pH 7.5 and 150 mM NaCl). These samples were analyzed in triplicate at 78° C. using a 1 cm quartz cuvette at a backscattering angle of 173° after a 5 minute incubation at 78° C. Data processing was performed using the high resolution analysis model provided with Zetasizer software v6.01.
To generate a Thermus thermophilus HB8 mutant for evaluating AK fragment complementation above 75° C., homologous recombination was used to replace its adk gene with adkGs, an ortholog that encodes a protein that is not functional above 70° C. in vitro (Bae and Phillips 2004). Gene replacement was performed by transforming HB8 cells with the pTTΔ200-GsteAK plasmid and selecting for growth at 60° C. on EvTM-agar plates containing kanamycin as previously described (Cameron, et al. 2004). Since HB8 cells cannot replicate this plasmid, they are only able to grow if the htk gene is integrated into the chromosome as illustrated in FIG. 2A.
Kanamycin-resistant colonies were screened for growth at 80° C. in EvTM-liquid culture, and strains identified as nonviable at this temperature were cured of their plasmid. PCR analysis of genomic DNA from one of the strains obtained (designated PQN1) confirmed integration of adkGs into the targeted chromosome, and DNA sequencing of these amplicons revealed that PQN1 expresses an AKGs with an E70V mutation.
To better characterize the temperature-sensitive phenotype of PQN1, we compared its specific maximal growth rate in liquid medium to the parental strain used for recombination. FIG. 2B shows that T. thermophilus HB8 grows over a broad temperature range (60 to 85° C.), as was previously known. In contrast, PQN1 only displayed detectable growth up to 75° C., ten degrees lower than the maximal growth temperature of the parental HB8 strain. We also investigated the effect of temperature on the growth of PQN1 on solid medium over a range of temperatures. As in liquid medium, PQN1 did not grow above 75° C. under these conditions. However, this temperature-sensitive phenotype could be rescued by transformation with a vector (pJJS-TnAK) that constitutively expresses the hyperthermophilic AKTn using the strong constitutive slpA promoter (Faraldo, et al. 1992).
Example 3
Results
To be useful as a protein complementation assay, fragments of a split protein must exhibit impaired function in the absence of protein tags (test proteins fused thereto). Otherwise, the split AK fragments would self associate, even without the fusion proteins, and this would add to background noise and decrease sensitivity
There are two ways to address this problem—one being to encode with the test proteins a selectable marker, and the second to modify the AK fragments so that they are less active without the test proteins fused to them.
To evaluate whether the AK fragments self associate at 78° C. without a test protein fused thereto (as observed with homologous fragments of B. subtilis AK at 40° C.), we evaluated the growth of PQN1 transformed with pJJS-N+C, a vector that constitutively coexpresses monocistronic transcripts encoding each fragment. FIG. 2C shows that PQN1 growth was complemented by TnN and TnC above 75° C. without the assistance of protein tags that drive their association.
We next examined whether point mutations within the tetracysteine motif that coordinates zinc abrogate the association and cooperative function of TnC and TnN sufficiently to allow them to be used as a more effective protein complementation assay in PQN1. These cysteines were targeted for mutagenesis since previous studies have shown zinc binding by the tetracysteine motif in the LID domain enhances AK thermostability.
We found that coexpression of adenylate kinase TnN with TnC fragments harboring either a Cys133Ala (TnCC156A) or Cys156Ala (TnCC156A) mutation could not complement PQN1 growth at 78° C. This suggests that disruption of zinc binding in the TnCC133A and TnCC156A polypeptides causes a decrease in the association of TnC with TnN and/or a decrease in the thermostability of AKTn-fragment complexes, as observed previously upon loss of zinc from full-length AKTn.
To test whether adenylate kinase TnN and TnCC156A can be used with PQN1 to report on protein-protein interactions, we examined whether PQN1 growth could be complemented at 78° C. by vectors that express these polypeptides fused to different combinations of the Thermotoga maritima MSB8 chemotaxis proteins (CheAP1P2, CheX, and CheY). These proteins were chosen because biochemical and biophysical studies have provided evidence for interactions between CheAP1P2 and CheY (Park, et al. 2004), CheX and CheY (Park, et al. 2004), and CheX and CheX (Park, et al. 2004). Among these pairwise interactions, only the CheAP1P2 and CheY interaction has been studied at a temperature where thermophiles grow, although the maximum temperature where this interaction has previously been analyzed (70° C.) is lower than the minimum temperature (78° C.) where our assay was performed (Park, et al. 2004).
FIG. 4A compares the growth of untransformed PQN1 with cells harboring vectors that coexpress TnN and TnCC156A fused to different combinations of chemotaxis proteins via a twelve amino acid linker. For these experiments, both adenylate kinase TnN and TnCC156A fragments were expressed from a single polycistronic transcript with the thermostable bleomycin selectable marker.
We found that PQN1 coexpressing CheY-TnN and CheAP1P2_TnCC156A grew after 24 hours of incubation at 78° C. at all titers analyzed, similar to that observed with PQN1 harboring a vector that expresses full-length AKTn. The average magnitude of the growth was calculated for the highest titers analyzed in each experiment by integrating the intensity of replicate spots using ImageJ.
This analysis revealed that PQN1 coexpressing CheY-TnN and CheAP1P2-TnCC156A grows 72% as dense as cells expressing full-length AKTn. In contrast, the growth detected with PQN1 expressing AK fragments fused to only CheY (CheY-TnN and TnCC156A) or CheAP1P2 (TnN and CheAP1P2-TnCC156A) is ≧4-fold lower than that of cells expressing AK fragments fused to CheY and CheAP1P2 at the highest titer and undetectable at the lowest titer analyzed. Similarly, little growth was observed after 24 hours with PQN1 transformed with vectors that express TnN and TnCC156A fused to nothing, CheY and CheX, and CheY and CheY.
Taken together, these findings provide direct evidence that the complementation observed with PQN1 coexpressing CheY-TnN and CheAP1P2-TnCC156A arises because the association of these chemotaxis proteins drives the cooperative function of the AKTn fragments at the physiological growth temperatures of T. maritima as predicted. CheY and CheX are also predicted to interact in T. maritima. However, the lack of growth with CheY and CheX protein fusions is consistent with studies which find that CheY must be phosphorylated to associate strongly with CheX.
Since CheX is a dimer in its crystal structure, we also investigated whether two CheX molecules could enhance AKTn-fragment complementation of PQN1 at 78° C. like CheAP1P2 and CheY. FIG. 4B shows that PQN1 cells coexpressing CheX-TnN and CheX-TnCC156A display detectable growth after a 24 hour incubation at 78° C. At the highest titer analyzed, this growth is ≧5.5-fold higher than that of PQN1 cells harboring vectors that only express one of the two AKTn fragments as a fusion with CheX, i.e., CheX-TnN and TnCC156A or TnN and CheX-TnCC156A.
In addition, PQN1 cells coexpressing CheX-TnN and CheX-TnCC156A grow to a density that is  ̃8-fold higher than that of cells expressing CheX-TnN and CheAP1P2-TnCC156A chemotaxis protein fusions that are not expected to interact and promote AK-fragment function. However, PQN1 were not complemented by CheX-TnN and CheX-TnCC156A to the same extent as with CheY-TnN and CheAP1P2-TnCC156A Growth was only observed with cells expressing CheX protein fusions at the highest titer of cells analyzed even after incubation for an additional day, whereas growth could be detected with CheAP1P2 and CheY protein fusions at all titers analyzed after 24 hours.
To examine if the split AKTn harboring the C156A mutation can report on protein-protein interactions in a living thermophile, we next created vectors for expressing each fragment fused to the Thermotoga maritia chemotaxis proteins CheA, CheX, and CheY using a protocol similar what is mentioned above and assessed their ability to complement PQN1 growth defects. Structural studies have shown that CheX homodimerizes and CheA and CheY form a stable complex, suggesting that AK complementation of PQN1 should be enhanced through fusion to these proteins. FIGS. 5A-5C show the results from complementation studies examining pairwise interactions between CheA and CheX. PQN1 growth defects at 78° C. were complemented when the N- and C-terminal adenylate kinase fragments were both fused to CheX as predicted.
To confirm that this enhanced complementation arose because of CheX dimerization, we also examined whether the split AKTn could complement the growth of PQN1 when only one of the fragments was fused to CheX (e.g., coexpressing AK-N and CheX::AK-CC156A). As shown in FIGS. 5A-5C, PQN1 harboring plasmids that express all combinations of the AK-N and AK-CC156A fragments fused to CheA, CheX, or no protein were streaked on EvTM-Gelrite plates and incubated at 65° C. (FIG. 5B) and 78° C. (FIG. 5C) for 48 hrs. These experiments revealed that CheX only assists in AK fragment complementation when both polypeptide fragments are fused to CheX. Consistent with previous studies that suggest CheA and CheX do not interact, AK fragments fused to CheA and CheX (or CheA and CheA) could not complement PQN1.
Additionally, complementation of PQN1 by coexpressing T. Maritima CheA and CheY proteins was also tested. FIG. 6A shows the results from complementation studies examining interaction between T. maritima CheA and CheY, proteins that have been shown to bind at temperatures as high as 70° C. While coexpression of AK fragments fused to CheA and CheY supported the growth of PQN1, as predicted, this complementation cannot be interpreted as arising from CheA-CheY interaction since growth was observed when only one of the AK fragments was fused to CheY. We hypothesized that CheY alone promotes PQN1 growth because its extreme thermostability (Tm>100° C.) stabilizes the AK fragments and the strong slpA promoter (PslpA) without transcriptional terminators leads to high transcription and fragment accumulation.
To test this, vectors were created that produce both AK fragments from a single transcript with multiple ribosomal binding sites. FIG. 6B shows that with this expression strategy AK fragments fused to a single CheY were not able to support PQN1 growth at 78° C. In contrast, AK fragments fused to CheA and CheY complement PQN1, indicating that AK fragment complementation can be used to detect CheA and CheY binding at 78° C. by simply tuning down protein expression.
Example 4
AK Variants
In addition to the adenylate kinase split at position 79 tested above, we have made a great many additional mutants and tested their ability to self complement at mesophilic temperatures. Unlike the adenylate kinase protein from mesophilic bacteria, the adenylate kinase from the thermophilic bacteria is much more tolerant of fragmentation, and will still self assemble to produce function adenylate kinase activity. In fact, we identified a higher fraction of functional variants in the T. neapolitana library (41%) than with the B. subtilis library (27%). This data is extremely voluminous, and is therefore not shown, but only summarized herein.
Many functional variants were found that were split throughout the AMP binding site, the core, and the lid domains of T. neapolitana adenylate kinase, but DNA sequencing of unselected variants from the libraries identified fragmentation sites dispersed throughout the entire adk coding sequence.
We also discovered that the T. neapolitana adenylate kinase is more tolerant of truncation. Indeed, strongly functional variants were discovered that lack up to 31 residues, which correspond to α-helix 9, β-sheet 8, and a portion of α-helix 8 observed in the B. subtilis AK crystal structure. In contrast, B. subtilis adenylate kinase truncations lacking 22 and 31 residues are not able to support E. coli CV2 growth at 40° C.
Therefore, we believe that a great many variations in both thermophilic AK sequence will function in the claimed assay, and also that the fragmentation can occur at most points throughout the adenylate kinase, and that many truncations all also functional.
To summarize—We have provided evidence that AK fragment complementation in T. thermophilus is proportional to the thermostability of the protein being fragmented, as previously observed in E. coli. Whereas AKTn fragments alone could complement PQN1 at 78° C., fragments with mutations in the cysteines that coordinate a thermostabilizing Zn+2 (Glaser, et al. 1992, Vieille, et al. 2003) could not support PQN1 growth at this temperature. However, AKTn fragments harboring the destabilizing C156A mutation were able to complement PQN1 when they were fused to chemotaxis proteins that homodimerize (CheX) and heterodimerize (CheAP1P2 and CheY). Therefore, the complementation assay was successful in a living thermophile at physiological temperatures.
This indicates that covalent linkage to associating proteins can drive the reassembly of the AKTn fragments harboring the C156A mutation at 78° C., greatly extending the temperature range where a split AK can be used as a protein complementation assay from mesophile to thermophile to hyperthermophile temperatures.
The finding that the TnN and TnC fragments cooperatively function at 78° C. upon heterologous expression also reinforces the hypothesis that the ability of a protein to fold and function at extreme temperatures is intrinsically encoded by its amino acid sequence, and this suggests that these AKTn polypeptides may be useful for creating a protein complementation assay that functions at even higher temperatures than those tested herein, although at the highest temperatures it is expected that the CYS mutations to decrease zinc binding may not be needed.
Our studies examining the effects of T. maritima chemotaxis proteins on AKTn-fragment complementation increases the maximum temperature (78° C.) where an interaction has been observed between the kinase CheAP1P2 and its target CheY. PQN1 growth was complemented by a vector that expresses AKTn fragments fused to CheAP1P2 and CheY but not by vectors that express AKTn fragments fused to only one of these chemotaxis proteins.
This is consistent with our findings from light scattering measurements performed at 78° C., which revealed that a mixture of purified CheAP1P2 and CheY exhibits an average hydrodynamic diameter that is greater than either of the individual proteins. Measurements of CheX-assisted fragment complementation also provides evidence that the CheX phosphatase self-associates at the temperature where it functions in T. maritima. PQN1 growth was complemented at 78° C. by a vector that coexpresses CheX-TnN and CheX-TnCC156A to a much greater extent than vectors that express only one of the two AKTn fragments as a fusion with CheX.
The high-temperature protein fragment complementation assay described herein is expected to have several advantages in discovering interactions among natural and engineered proteins compared with previously described approaches, such as mesophilic two-hybrid and PCA screens. The AKTn-based complementation assay is implemented within a T. thermophilus HB8 strain growing at a temperature where many thermophiles and hyperthermophiles grow, making it better suited for discovering interactions among proteins that have evolved to fold and function at extreme temperatures. In addition, this selection is expected to be more sensitive than existing assays at detecting interactions among proteins whose free energy of binding increases as temperature decreases below the physiological growth range of thermotolerant microbes.
Our AKTn-based protein complementation assay should also aid protein design efforts working to create artificial protein complexes that are stable at the temperatures where thermophiles and hyperthermophiles grow. This novel protein complementation assay can be used as a high-throughput screen to evaluate the association of proteins created through rational design and laboratory evolution.
In addition, this protein complementation assay can be used to investigate the robustness of protein complexes to different mutational processes and to examine why some hyperthermophilic protein complexes weaken in affinity as temperature is decreased below the physiological growth range of hyperthermophiles, whereas other do not. The latter problem can be addressed by mining libraries of engineered (or natural) proteins for interactions that occur at 78° C. and re-screening discovered variants for decreased association at 40° C., using a structurally-related protein complementation assay that was developed for use in E. coli at 40° C. By comparing the sequences of the protein complexes that support protein complementation to different extents at high and low temperatures, hypotheses about the biophysical origin of these observations can be tested.
The following references are incorporated by reference herein in their entirety:

    Bae, E., and Phillips, G. N., Jr., Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases, J. Biol. Chem. 279: 28202-28208 (2004).
    Brouns, S. J., et al., Engineering a selectable marker for hyperthermophiles, J. Biol. Chem. 280: 11422-11431 (2005).
    Cameron, D. M., et al., Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly, J. Bacteriol. 186: 5819-5825 (2004)
    Faraldo, M. M., et al., Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli, J. Bacteriol. 174:7458-7462 (1992).
    Glaser, P., et al., Zinc, a novel structural element found in the family of bacterial adenylate kinases, Biochemistry 31: 3038-3043 (1992)
    Hoseki, J., et al., Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus, J. Biochem. (Tokyo) 126, 951-956. (1999)
    Nguyen, P. Q., et al., Thermostability promotes the cooperative function of split adenylate kinases, Protein Eng. Des. Sel. 21:303-310 (2008)
    Park, S. Y., et al., In different organisms, the mode of interaction between two signaling proteins is not necessarily conserved, Proc. Natl. Acad. Sci. USA 101: 11646-11651 (2004).
    Park, S. Y., et al., Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX, Mol. Cell. 16: 563-574.
    Vieille, C., et al., Thermotoga neapolitana adenylate kinase is highly active at 30 degrees C., Biochem. J. 372:577-585 (2003)

## Claims

1. A protein fragment complementation assay for thermophilic bacteria, comprising:
a) culturing a thermophilic bacteria at a temperature greater than 70° C.,
i) wherein said thermophilic bacteria comprises a native adenylate kinase that is replaced with a temperature-sensitive adenylate kinase that does not function at temperatures of above 70° C.,
ii) a first vector having an expressable first test gene fused in frame to a first portion of a nucleic acid encoding a first portion of a thermostable adenylate kinase having at least 50% activity at temperature greater than 70° C.,
iii) a second vector having an expressable second test gene fused in frame to a nucleic acid encoding a second portion of a thermostable adenylate kinase having at least 50% activity at temperature greater than 70° C.,
iv) wherein association of a first test peptide and a second test peptide allows association of the first and second portions of the thermostable adenylate kinases and growth of the thermophilic bacteria at a temperature greater than 70° C.,

b) and assaying for growth of said thermophilic bacteria, wherein growth indicates that said first test and said second test peptides associate in vivo.

2. The protein complementation assay of claim 1, wherein the first and second portions are split at residue 79 or 80, said residue numbered in accordance with SEQ ID NO. 1, and said first and second portions originate from the same adenylate kinase or from different adenylate kinases.

3. The protein complementation assay of claim 1, wherein the thermostable adenylate kinase has about 50% identity to SEQ ID NO. 1.

4. The protein complementation assay of claim 1, wherein the thermostable adenylate kinase has at least 60% identity to SEQ ID NO. 1.

5. The protein complementation assay of claim 1, wherein the thermostable adenylate kinase has at least 92% identity to SEQ ID NO. 1.

6. The protein complementation assay of claim 1, wherein the thermostable adenylate kinase has a sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3, 4, and 5.

7. The protein complementation assay of claim 1, wherein the first and second portions are selected from residues 1-79 and 80-220 of the thermostable adenylate kinase of SEQ ID NOs. 1, 2, 3, 4, and 5, said residues numbered in accordance with SEQ ID NO. 1.

8. The protein complementary assay of claim 1, wherein the first and second test genes are polycistronic.

9. The protein complementation assay of claim 1, wherein the thermostable adenylate kinase comprises a mutated cysteine residue and binds less zinc than a wild type thermostable adenylate kinase.

10. The protein complementation assay of claim 1, wherein the second portion comprises Cys133Ala or Cys156Ala, said Cys numbered in accordance with SEQ ID NO. 1.

11. The protein complementation assay of claim 8, wherein the second portion comprises Cys133Ala or Cys156Ala, said Cys numbered in accordance with SEQ ID NO. 1.

12. The protein complementary assay of claim 1, wherein said test genes are each separated from said adenylate kinase portions with a flexible linker sequence.

13. The protein complementary assay of claim 1, wherein the bacteria is hyperthermophilic, the temperature sensitive adenylate kinase does not function at temperatures of at least 78° C., and the association of the first and second portion of the thermostable adenylate kinase allows growth of the hyperthermophilic bacteria at temperatures of at least 78° C.

14. The protein complementary assay of claim 1, wherein the first and second vectors are the same vector and the first and second portions of the thermostable adenylate kinase are polycistronic.

15. The protein complementary assay of claim 1, wherein the first and second portions of the thermostable adenylate kinase are selected from the group consisting of:
a) residues 1-79 or 80-220 from SEQ ID NO. 1;
b) residues 1-79 or 80-220 from SEQ ID NO. 2;
c) residues 1-79 or 80-220 from SEQ ID NO. 3;
d) residues 1-79 or 80-220 from SEQ ID NO. 4;
e) residues 2-80 or 81-220 from SEQ ID NO. 5; and mixed species combinations thereof.