WO2024101755A1

WO2024101755A1 - Method for preparing taq dna polymerase in pichia pastoris

Info

Publication number: WO2024101755A1
Application number: PCT/KR2023/017140
Authority: WO
Inventors: Suk-Tae Kwon; Sung Suk Cho; Myung Il Kim; Sang-Hyuk Lee; Ki Jong Song; In Hwa Song; Ju-Hee GWON; Gyu Bi Lee
Original assignee: Seegene, Inc.
Priority date: 2022-11-10
Filing date: 2023-10-31
Publication date: 2024-05-16

Abstract

The present disclosure relates to a novel method for preparing Taq DNA polymerase, and more specifically, to a method for preparing Taq DNA polymerase in Pichia pastoris, Taq DNA polymerase produced thereby, and a method for amplifying one or more target nucleic acids in a sample using the same. Conventional methods for extracellular expression of Taq DNA polymerase using an expression vector containing a secretion signal peptide can hardly be accomplished due to the large size of Taq DNA polymerase, whereas the method of the present disclosure allows for intracellular expression of Taq DNA polymerase in high yield.

Description

METHOD FOR PREPARING TAQ DNA POLYMERASE IN PICHIA PASTORIS

The present disclosure relates to a novel method for preparing Taq DNA polymerase, and more specifically, to a method for preparing Taq DNA polymerase in Pichia pastoris, Taq DNA polymerase produced thereby, and a method for amplifying one or more target nucleic acids in a sample using the same.

Nucleic acid amplification is mainly used in molecular biology and biotechnology to detect and analyze a small quantity of target nucleic acids in a sample. For nucleic acid amplification, PCR (polymerase chain reaction) has been widely used, which involves repeating a series of steps of denaturing double-stranded DNAs into single-stranded DNAs, hybridizing primers with the single-stranded DNAs, and then extending the primers along the single-stranded DNAs by DNA polymerase.

DNA polymerase (deoxyribonucleic acid polymerase, E.C. number 2.7.7.7) as a key component in PCR plays a role in synthesizing DNA in the 5' to 3' direction depending on template DNA. Since PCR is performed at a high temperature of 50℃ or higher, the use of thermostable DNA polymerase is required. As a representative thermostable DNA polymerase, Taq DNA polymerase having a molecular weight of about 94 kDa, isolated from a thermophilic bacterium, Thermus aquaticus YT-1, has been widely used (Ishino, Y., et al., 1994).

Taq DNA polymerase is typically produced by culture and purification using a recombinant E. coli expression system. However, due to its powerful DNA-binding properties, Taq DNA polymerase is likely to bind to the genomic DNA of recombinant E. coli during its purification process. Therefore, commercially available Taq DNA polymerases are known to be contaminated with E. coli DNA.

The use of Taq DNA polymerases contaminated with E. coli DNA in an amplification reaction (e.g., PCR) for detecting a target nucleic acid from E. coli may lead to amplification of the contaminated E. coli DNA and thus false positives.

Therefore, there remains a need in the art to prepare Taq DNA polymerase not contaminated with E. coli DNA.

In addition to E. coli, yeasts such as Saccharomyces cerevisiae have been widely used to express heterologous proteins. Recently, Pichia pastoris has been attracting attention.

P. pastoris can be engineered as easily as E. coli or S. cerevisiae, and unlike E. coli, it has an expression system unique to eukaryotic cells, including protein processing, protein folding, and protein post-translational modification. Both P. pastoris and S. cerevisiae have some eukaryotic and prokaryotic characteristics, but the proteins expressed in P. pastoris have conformations that are closer to humans and animals than those expressed in S. cerevisiae. Thus, P. pastoris has an advantage over S. cerevisiae in producing proteins that will actually act on animal cells. Also, P. pastoris is advantageous in that it produces a higher amount of soluble protein per the same cell mass than S. cerevisiae.

Korean Patent No. 10-1546358 discloses a method for preparing Taq DNA polymerase, comprising the steps of transforming P. pastoris with pPIC9 vector containing Taq DNA polymerase and an α-mating factor (αA) as a secretion signal peptide; culturing the transformed P. pastoris in a methanol-containing medium; heating the culture; and purifying the Taq DNA polymerase from the culture.

However, the method requires special measures and inspections in factory installation and considerable attention to fire and health issue, due to the use of highly combustible methanol, which discourages commercial availability of the method. In addition, although the present inventors made considerable efforts to extracellularly secret Taq DNA polymerase with reference to the above method, it was found that Taq DNA polymerase was not secreted outside the cells but accumulated within the cells.

Therefore, there is a need to develop a novel method for preparing Taq DNA polymerase in Pichia pastoris.

Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entirety are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

The present inventors have endeavored to develop a method for preparing Taq DNA polymerase in Pichia pastoris. As a result, the present inventors have developed a method for preparing Taq DNA polymerase not contaminated with nucleic acids derived from E. coli, comprising incorporating a polynucleotide encoding the Taq DNA polymerase into an expression vector lacking a secretion signal peptide, transforming P. pastoris with the expression vector, culturing the transformed P. pastoris, lysing the cultured P. pastoris, and purifying the Taq DNA polymerase from the lysate.

Thus, it is an object of the present disclosure to provide a method for preparing Taq DNA polymerase.

It is another object of the present disclosure to provide Taq DNA polymerase prepared by the method as described above.

It is still another object of the present disclosure to provide a method for amplifying one or more target nucleic acids in a sample using the Taq DNA polymerase as described above.

It is still another object of this disclosure to provide a transformed Pichia pastoris for preparing the Taq DNA polymerase as described above.

In an aspect of the present disclosure, there is provided a method for preparing Taq DNA polymerase, comprising: (a) providing an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide; (b) transforming Pichia pastoris with the expression vector to obtain a transformed P. pastoris; (c) culturing the transformed P. pastoris in a culture medium under conditions permitting intracellular expression of the Taq DNA polymerase; and (d) lysing the cultured P. pastoris and purifying the intracellularly expressed Taq DNA polymerase from the lysate.

In certain embodiments, the polynucleotide encoding the Taq DNA polymerase is codon-optimized for P. pastoris.

In certain embodiments, the polynucleotide encoding the Taq DNA polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto.

In certain embodiments, the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the polynucleotide encoding the Taq DNA polymerase.

In certain embodiments, the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)_n linker between the polynucleotide encoding the Taq DNA polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.

In certain embodiments, the expression vector further comprises an antibiotic resistance gene as a selectable marker.

In certain embodiments, the antibiotic resistance gene is a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.

In certain embodiments, the expression vector comprises a plurality of the nucleic acid constructs.

In certain embodiments, the plurality of the nucleic acid constructs is 6 to 10 in total.

In certain embodiments, the method further comprises linearizing the expression vector prior to the step (b).

In certain embodiments, the step (b) induces homologous recombination between the expression vector and the genome of P. pastoris.

In certain embodiments, the genome of P. pastoris after homologous recombination has 1-10 copies of the nucleic acid constructs integrated thereinto.

In certain embodiments, the purified, intracellularly expressed Taq DNA polymerase is produced in an amount of 6 to 15 mg per 100 ml of the culture medium.

In certain embodiments, the purified, intracellularly expressed Taq DNA polymerase does not comprise a secretion signal peptide.

In certain embodiments, the purified, intracellularly expressed Taq DNA polymerase is free of nucleic acids derived from E. coli.

In certain embodiments, the purified, intracellularly expressed Taq DNA polymerase does not lead to any false positives from a non-target E. coli in an amplification reaction.

In another aspect of the present disclosure, there is provided Taq DNA polymerase, prepared by the method as described above.

In certain embodiments, the Taq DNA polymerase is free of nucleic acids derived from E. coli.

In certain embodiments, the Taq DNA polymerase does not lead to any false positives from a non-target E. coli in an amplification reaction.

In another aspect of the present disclosure, there is provided a method of amplifying one or more target nucleic acids in a sample using the Taq DNA polymerase as describe above.

In certain embodiments, at least one of the one or more target nucleic acids is from E. coli.

In another aspect of the present disclosure, there is provide a transformed Pichia pastoris, prepared by transforming P. pastoris with an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

The features and advantages of the present disclosure are summarized as follows:

(a) Taq DNA polymerase produced by E. coli expression systems may be contaminated by E. coli genomic DNA and thus lead to false positives due to non-target nucleic acids from the contaminated E. coli in amplification of target nucleic acids from E. coli, whereas Taq DNA polymerase prepared by the method of the present disclosure is free of any nucleic acids from E. coli, thereby preventing false positives.

(b) Conventional methods for preparing Taq DNA polymerase in a transformed Pichia pastoris require use of highly flammable and toxic methanol to induce promoter transcription, whereas the method of the present disclosure can avoid the use of methanol, enabling safer production of Taq DNA polymerase.

(c) Conventional methods for extracellular expression of Taq DNA polymerase using an expression vector containing a secretion signal peptide can hardly be accomplished due to the large size of Taq DNA polymerase, whereas the method of the present disclosure allows for intracellular expression of Taq DNA polymerase in high yield.

(d) The use of an expression vector lacking a secretion signal peptide according to the method of the present disclosure can increase the production yield of Taq DNA polymerase, compared with the use of an expression vector containing a secretion signal peptide.

(e) The use of an expression vector containing 6 to 10 nucleic acid constructs according to the method of the present disclosure can further increase the production yield of Taq DNA polymerase.

Figure 1 depicts a map of a commercially available expression vector, "pGAPZαA", which is used to prepare an expression vector according to an embodiment of the present disclosure.

Figure 2 shows 0.8% agarose gel electrophoresis to confirm that Pichia pastoris obtained in Example 1 has been transformed with an expression vector containing a tetrameric Taq DNA polymerase. In Fig. 2, lanes 1-1, 1-2, and 1-3 show the results for the expression vector "pGAPZαA Taq 4M", which includes a tetramer of an α-mating factor as a secretion signal peptide and the Taq DNA polymerase; lanes 2-1, 2-2, and 2-3 show the results for the expression vector "pGAPZMAF Taq 4M", which includes a tetramer of a modified α-mating factor (MAF) as a secretion signal peptide and the Taq DNA polymerase; lanes 5-1, 5-2, and 5-3 show the results for the expression vector "pGAPZpkS Taq 4M", which includes a tetramer of a proteinase K (pKS) as a secretion signal peptide and the Taq DNA polymerase; and lanes 6-1 and 6-2 show the results for the expression vector "pGAPZaqS Taq 4M", which includes a tetramer of an aqualysin I (aqS) as a secretion signal peptide and the Taq DNA polymerase. For size comparison, 7^th, 8^th, and 9^th lanes show the results for the expression vectors "pGAPZpkS Taq 1M", which includes a monomer of a pKS and the Taq DNA polymerase, "pGAPZpkS Taq 2M", which includes a dimer of a pKS and the Taq DNA polymerase, and "pGAPZpkS Taq 3M", which includes a trimer of a pKS and the Taq DNA polymerase, respectively.

Figure 3 shows 10% SDS-PAGE for culture supernatants and cell lysates to confirm extracellular secretion or intracellular expression of Taq DNA polymerase in P. pastoris obtained in Example 1. In Figure 3, 1^st lane represents a ladder as a molecular weight marker; 2^nd lane represents the loading result for the culture supernatant (abbreviated as "S") of an untransformed P. pastoris; 3^rd and 4^th lanes represent the loading results for the culture supernatant and the cell lysate (abbreviated as "L"; heated at 80℃ for 30 min) of P. pastoris, transformed with the expression vector "pGAPZαA Taq 4M", which includes a tetramer of an α-mating factor as a secretion signal peptide and the Taq DNA polymerase, respectively; 5^th and 6^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZMAF Taq 4M", which includes tetramer of a modified α-mating factor (MAF) as a secretion signal peptide and the Taq DNA polymerase, respectively; 7^th and 8^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZpkS Taq 4M", which includes a tetramer of a proteinase K signal sequence (pKS) as a secretion signal peptide and the Taq DNA polymerase; and 9^th and 10^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZaqS Taq 4M", which includes a tetramer of an aqualysin I signal sequences (aqS) as a secretion signal peptide and the Taq DNA polymerase.

Figure 4 shows 0.7% agarose gel electrophoresis for the expression vectors prepared according to Example 2, which were each digested with BglII and BamHI. In Figure 4, 1^st lane represents a 1 kb ladder as a molecular weight marker; 2^nd to 4^th lanes represent the loading results for the expression vector "pGAPZ Taq GSG C-His 8M"; and 5^th lane represents the loading result for the expression vector "pGAPZ Taq GSG C-His 4M" for comparison. In each lane, the upper band corresponds to an octameric or tetrameric Taq DNA polymerase, and the lower band corresponds to remaining backbone of the expression vector except for the octameric or tetrameric Taq DNA polymerase.

Figures 5A and 5B show 10% SDS-PAGE for the cell lysates of the transformed P. pastoris obtained in Example 2.

Figure 6 shows 1% agarose gel electrophoresis for PCR products using the cell lysates of the transformed P. pastoris obtained in Example 2.

Figure 7 shows the amplification results for target E. coli genomic DNA using Taq DNA polymerase according to the present disclosure. In the figure, the leftmost lane represents a 1 kb ladder as a molecular weight marker; 1^st lane shows the amplification result for E. coli DH5α genomic DNA using Taq DNA polymerase according to the present disclosure; and 2^nd lane shows the amplification result using only the Taq DNA polymerase in the absence of E. coli DH5α genomic DNA.

I. Method for preparing Taq DNA polymerase

In an aspect of the present disclosure, there is provided a method for preparing Taq DNA polymerase, comprising:

(a) providing an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide;

(b) transforming Pichia pastoris with the expression vector to obtain a transformed P. pastoris;

(c) culturing the transformed P. pastoris in a culture medium under conditions permitting intracellular expression of the Taq DNA polymerase; and

(d) lysing the cultured P. pastoris and purifying the intracellularly expressed Taq DNA polymerase from the lysate.

Hereafter, the steps of the method according to the present disclosure is described in detail.

Step (a): Preparing an expression vector

In step (a) of the method of the present disclosure, provided is an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

The expression vector according to the present disclosure is described below.

Taq DNA polymerase

As used herein, the term "Taq DNA polymerase" refers to a thermostable DNA polymerase, which is derived from a thermostable eubacterium, Thermus aquaticus YT-1.

The Taq DNA polymerase herein may be a wild-type Taq DNA polymerase or variants thereof. The term "variant" as used herein refers to a Taq DNA polymerase having substitution, insertion, or deletion of amino acid(s) relative to the wild-type Taq DNA polymerase, for the purpose of improving the function of wild-type Taq DNA polymerase. The Taq DNA polymerase variant may have a sequence identity of at least 90%, e.g., at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% to the nucleotide sequence of the wild-type Taq DNA polymerase. The wild-type Taq DNA polymerase or variants thereof are widely known in the art. For example, see GenBank Accession no. See J04639.1.

Polynucleotide encoding Taq DNA polymerase

As used herein, the term "codon optimization" or "codon-optimized" refers to a process of changing the codons of a heterologous protein based on codon usage bias of a host organism being transformed to increase expression of the heterologous protein.

The polynucleotide encoding the Taq DNA polymerase herein is codon-optimized by changing their codons based on codon usage bias of P. pastoris. The sequence of the codon-optimized polynucleotide is different from that of the polynucleotide before codon-optimization, but their amino acid sequences are the same.

The codon usage bias of P. pastoris can be found, e.g., in a codon usage table, known in the art.

Amino acid	Codon	Usage	Amino acid	Codon	Usage
F	UUU	0.54	Y	UAU	0.47
F	UUC	0.46	Y	UAC	0.53
L	UUA	0.16	STOP	UAA	0.51
	UUG	0.33	STOP	UAG	0.29
	CUU	0.16	H	CAU	0.57
	CUC	0.08	H	CAC	0.43
	CUA	0.11	Q	CAA	0.61
	CUG	0.16	Q	CAG	0.39
I	AUU	0.50	N	AAU	0.48
	AUC	0.31	N	AAC	0.52
	AUA	0.18	K	AAA	0.47
M	AUA	1.00	K	AAG	0.53
V	GUU	0.42	D	GAU	0.58
	GUC	0.23	D	GAC	0.42
	GUA	0.15	E	GAA	0.56
	GUG	0.19	E	GAG	0.44
S	UCU	0.29	C	UGU	0.64
	UCC	0.20	C	UGC	0.36
	UCA	0.18	STOP	UGA	0.20
	UCG	0.09	W	UGG	1.00
P	CCU	0.35	R	CGU	0.17
	CCC	0.15		CGC	0.05
	CCA	0.42		CGA	0.10
	CCG	0.09		CGG	0.05
T	ACU	0.40	S	AGU	0.15
	ACC	0.26	S	AGC	0.09
	ACA	0.24	R	AGA	0.48
	ACG	0.11	R	AGG	0.16
A	GCU	0.45	G	GGU	0.44
	GCC	0.26		GGC	0.14
	GCA	0.23		GGA	0.33
	GCG	0.06		GGG	0.10

In certain embodiments, the polynucleotide encoding the Taq DNA polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto. In one example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence of SEQ ID NO: 1. In another example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence having at least 95% sequence identity to SEQ ID NO: 1. In another example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence having at least 96% sequence identity to SEQ ID NO: 1. In another example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence having at least 97% sequence identity to SEQ ID NO: 1. In another example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence having at least 98% sequence identity to SEQ ID NO: 1. In another example, the polynucleotide encoding the Taq DNA polymerase comprises or consists of a sequence having at least 99% sequence identity to SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1 is as follows:

atgaggggta tgttaccact gtttgaacct aagggtaggg ttcttctagt agatggacat 60

cacttggctt atagaacctt tcatgctctg aaggggttaa caactagtag aggtgagcca 120

gttcaggccg tatatggatt tgctaaatct ttacttaaag cattgaagga ggacggcgat 180

gcggtaatag tggtctttga cgctaaggct ccttccttca gacatgaagc atacggtgga 240

tataaagcag gaagagcccc tactcccgag gacttccctc gtcaattggc cctgatcaaa 300

gaacttgtcg atttattggg gcttgctaga ctagaagtgc caggctatga ggccgatgat 360

gtcctcgcat cattggcaaa aaaggctgag aaagaaggct atgaggtaag gattctaacc 420

gctgacaaag atttatacca actgttgtcc gatcgaatac acgtccttca cccagaggga 480

tacctaataa cccctgcttg gctgtgggaa aagtatggct tgaggcccga tcaatgggcg 540

gattatcgag ctctgactgg agacgagtcg gataatttgc ctggagttaa gggtattggg 600

gaaaagacag caagaaaact gcttgaagag tggggttctt tagaagctct gctaaagaac 660

ttagatagat tgaaaccagc aatacgtgaa aaaattctgg cacacatgga cgatctgaaa 720

ctttcttggg atttagcaaa agttagaacg gacctcccgt tagaagtgga ttttgccaag 780

agacgggaac cagatcgaga gcgtctgaga gcatttttgg aaagattgga atttggtagc 840

cttttacacg aatttggttt gctcgaatcc ccaaaggcat tggaggaggc cccgtggcca 900

ccaccagagg gcgcattcgt tggtttcgtt ctttcacgca aagaacccat gtgggcagac 960

cttctcgcct tggctgccgc gagaggtgga agggtacaca gagctcctga accatacaaa 1020

gcattaaggg atttgaaaga agctagggga ttgctcgcta aggatctgtc agttctggcg 1080

ctaagagaag gtctaggtct accaccaggc gatgatccta tgcttttagc ctacttactt 1140

gatcctagca acacgactcc agagggagtt gccagacgat acggaggtga atggactgag 1200

gaggctggtg aacgagccgc attgtcagag cgactttttg ctaatttatg gggtcgcttg 1260

gaaggagagg aaagattact ctggctatac cgtgaggtcg aaagaccatt atccgcagtt 1320

ctcgcccaca tggaggccac cggagtccgt ttggacgtag cttatcttcg ggcactttct 1380

ctggaagttg cagaggaaat tgctagattg gaggccgagg tgttccgttt agctggtcac 1440

ccgtttaatt taaattctag agatcaattg gagagggtgt tgttcgatga gcttggactt 1500

ccagcaatcg gtaagacaga aaagacggga aagagatcaa ccagtgcagc tgtgctagaa 1560

gcgttgagag aggctcatcc catcgttgaa aagattttgc agtatcgtga gctaaccaaa 1620

ctgaaaagca cttacatcga cccattgcct gatttgattc atccgagaac cgggagactt 1680

cacactagat ttaaccaaac tgctactgct acagggagat tgtccagttc tgaccctaat 1740

cttcagaaca tccccgttcg tactccactg ggtcaaagga tcagacgggc tttcatcgcc 1800

gaagagggat ggcttttggt ggccttagat tactctcaaa ttgaactccg cgttttggca 1860

catttaagtg gggatgaaaa cctaattaga gtttttcaag aaggcaggga cattcacact 1920

gaaacagctt cttggatgtt cggtgtccct agagaagctg ttgaccctct aatgcgtaga 1980

gctgccaaaa ccattaattt tggagtcctg tacggcatgt ctgctcaccg attatcgcaa 2040

gaattggcta ttccctatga ggaagcccag gcctttatcg aacgttattt ccagagtttc 2100

cctaaggtca gagcgtggat tgagaagaca ctagaagaag gacgtaggag aggttacgtc 2160

gaaacactat tcggtcggcg aaggtacgtt ccagacttgg aagcaagagt gaagtcggtt 2220

agagaagctg ccgaacgcat ggccttcaac atgcccgttc aaggtacagc agctgacctg 2280

atgaaattgg ctatggttaa gttgtttcct agacttgagg agatgggagc aaggatgctc 2340

ttgcaggtgc atgacgaatt ggtattggaa gctcctaaag aaagagccga ggccgttgct 2400

agactggcta aagaggtcat ggagggggta tatcctctgg ctgtgccatt ggaagtcgaa 2460

gttggtatag gagaagactg gttgtcagct aaagaa 2496

(SEQ ID NO: 1)

Nucleic acid construct

As used herein, the term "nucleic acid construct" refers to an artificially designed segment of DNA containing all the elements required for self-expression. The nucleic acid construct can typically include a promoter, a transcriptional terminator, a ribosome binding site, and a translation terminator, operably linked to transgenic genes. The nucleic acid construct may be in the form of a self-replicable expression vector.

In an embodiment, the nucleic acid construct herein comprises (i) a polynucleotide encoding the Taq DNA polymerase, and (ii) a promoter operably linked thereto.

As used herein, the term "promoter" refers to a nucleic acid sequence encoding an amino acid containing a binding site for RNA polymerase and having activity of initiating transcription of downstream genes into mRNA.

The promoter as used herein includes various promoters known to be capable of driving the transcription of proteins of interest in P. pastoris. The promoter may be an inducible promoter or a constitutive promoter, depending on the expression pattern of the protein of interest. Examples include, without limitation, AOX1 (alcohol oxidase 1) promoter (Lin-Cereghino J, et al (2000) FEMS Microbiol Rev24:45-66), ADH3 (alcohol dehydrogenase) promoter (Karaoglan M, et al (2016) Protein Expr Purif 121:112-117), DAS (Dihydroxyacetone hormone) promoter (Tschopp JF, et al (1987) Nucleic Acids Res 15:3859-3876), FLD1 (Formaldehyde dehydrogenase) (Shen S, et al (1998) Gene 216:93-102), PEX8 (Peroxisomal matrix protein) promoter (Lin-Cereghino J, et al (2000) FEMS Microbiol Rev 24:45-66), ICL1 (Isocitrate lyase) promoter (Menendez J, et al (2003) Yeast 20:1097-1108), LRA3 (L-rhamnonate dehydratase) promoter (Liu B, et al (2016) Sci Rep 6:27352), LRA4 (L-KDR aldolase) promoter (Liu B, et al (2016) Sci Rep 6:27352), THI11 (Thiamine biogenic protein) promoter (Stadlmayr G, et al (2010) J Biotechnol 150:519-529), GTH1 (High affinity glucose transporter) promoter (Koller A, et al (2000) Yeast 16:651-656), CUP1 (Copper-binding metallothionein protein) (Landes N, et al (2016) Biotechnol Bioeng 113(12):2633-2643), PPLCC1 (Laccase) promoter (Landes N, et al (2016) Biotechnol Bioeng 113(12):2633-2643), GAP (Glyceraldehyde-3-phosphate dehydrogenase) promoter (Waterham HR, et al (1997) Gene 186:37-44), YPT1 (GTPase involved in secretion) promoter (Sears IB, et al (1998) Yeast 14:783-790), TEF1 (Translation elongation factor-1 alpha) promoter (Stadlmayr G, et al (2010) J Biotechnol 150:519-529), GCW14 (Glycosylphosphatidylinositol) promoter (Liang S, et al (2013) Biotechnol Lett 35 (11):1865-1871), and PGK1 (Phosphoglycerate kinase). In certain embodiments, the promoter included in the nucleic acid construct is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.

Depending on the promoter used, the nucleic acid construct or expression vector as used herein can be termed as AOX1 promoter-, ADH3 promoter-, DAS promoter-, FLD1 promoter-, PEX8 promoter-, ICL1 promoter-, LRA3 promoter-, LRA4 promoter-, THI11 promoter-, GTH1 promoter-, CUP1 promoter-, PPLCC1 promoter-, GAP promoter-, YPT1 promoter-, TEF1 promoter- GCW14 promoter-, or PGK1 promoter-based nucleic acid construct or expression vector. In certain embodiments, the nucleic acid construct or expression vector as used herein is a GAP promoter-based nucleic acid construct or expression vector.

As used herein, the term "operably linked" refers to arrangement of fragments such that transcription is initiated by a promoter, and proceeds to the termination code through amino acid coding sequences.

In addition to the polynucleotide encoding the Taq DNA polymerase and a promoter operably linked thereto, the nucleic acid construct as used herein may include various elements known to be involved in helping the expression/secretion of Taq DNA polymerase.

In an embodiment, the nucleic acid construct herein further comprises a transcription terminator. The transcription terminator is essential for the processing and polyadenylation of messenger RNA. The transcription terminator includes those known to be used in P. pastoris, example of which includes AOX1 terminator.

In certain embodiments, the nucleic acid construct herein further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the polynucleotide encoding the Taq DNA polymerase. The histidine tag is used to facilitate protein purification using affinity chromatography. The addition of histidine to the terminus of the protein of interest leads to a significantly increased metal ion affinity of the protein, enabling easy purification. Contacting a protein having a histidine tag with a column onto which metal ions such as nickel are immobilized under conditions of pH 8.0 or higher allows the histidine tag to chelate the metal ions and bind to the column, thereby recovering the target protein with high purity.

The nucleic acid sequence encoding the histidine tag can be linked to the C-terminus or N-terminus of the polynucleotide encoding the Taq DNA polymerase. In certain embodiments, the nucleic acid sequence encoding the histidine tag is linked to the C-terminus of the polynucleotide encoding the Taq DNA polymerase.

The histidine tag may consist of at least 6 histidine residues. In one embodiment, the histidine tag consists of 6 histidine residues (hexahistidine). In another embodiment, the histidine tag consists of 7 histidine residues (heptahistidine). In another embodiment, the histidine tag consists of 8 histidine residues (octahistidine). In another embodiment, the histidine tag consists of 9 histidine residues (nonahistidine). In another embodiment, the histidine tag consists of 10 histidine residues (decahistidine). The number of histidine constituting the histidine tag is readily adjustable by those skilled in the art.

In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)_n linker between the polynucleotide encoding the Taq DNA polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.

The (GSG)_n linker allows for interaction between the polynucleotide encoding the Taq DNA polymerase and the nucleic acid sequence encoding the histidine tag or increases the spatial separation between the two domains. Direct fusion of the polynucleotide encoding the Taq DNA polymerase with the nucleic acid sequence encoding the histidine tag may cause undesirable results, such as misfolding of the fusion protein, low protein yield, or dysfunction.

It was found by the inventors that direct fusion of a histidine tag to Taq DNA polymerase leads to poor purification, because Taq DNA polymerase does not bind well to the column during purification (data not presented).

Although various linkers composed of glycine and serine, such as GS linkers, GSG linkers, GSSG linkers, GGGG linkers, and GGGGS linkers, are typically known in the art, it was found by the inventors that GSG linkers are the most effective in the expression and purification efficiency of Taq DNA polymerase.

The length of the (GSG)_n linker can be optimized by adjusting the copy number n. In certain embodiments, the copy number n in the (GSG)_n linker is 1. In certain embodiments, the copy number n in the (GSG)_n linker is 2.

In one embodiment, the nucleic acid construct used herein further comprises an intervening sequence between the polynucleotide encoding the Taq DNA polymerase and the nucleic acid sequence encoding the histidine tag. In certain embodiments, the intervening sequence includes a single or multiple cloning site, such as a restriction site. The intervening sequence is used for inserting the polynucleotide encoding the Taq DNA polymerase in front of the nucleic acid sequence encoding the histidine tag.

According to the present disclosure, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter and the polynucleotide encoding the Taq DNA polymerase.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the Taq DNA polymerase, and a transcriptional terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the Taq DNA polymerase, the nucleic acid sequence coding the histidine tag, and a transcriptional terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, the polynucleotide encoding the Taq DNA polymerase, and a transcriptional terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the Taq DNA polymerase, a nucleic acid sequence encoding a (GSG)_n linker, the nucleic acid sequence encoding the histidine tag, and a transcriptional terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, a nucleic acid sequence encoding a (GSG)_n linker, the polynucleotide encoding the Taq DNA polymerase, and a transcription terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the polynucleotide encoding the Taq DNA polymerase, a nucleic acid sequence encoding a (GSG)_n linker, an intervening sequence, the nucleic acid sequence coding the histidine tag, and a transcription terminator.

In certain embodiments, the nucleic acid construct used herein includes, from N-terminus to C-terminus, a promoter, the nucleic acid sequence encoding the histidine tag, a nucleic acid sequence coding a (GSG)_n linker, the polynucleotide encoding the Taq DNA polymerase, and a transcription terminator.

In certain embodiments, a plurality of such nucleic acid constructs are included in an expression vector. In certain embodiments, 6 to 10 of such nucleic acid constructs are included in an expression vector.

Expression vector

The expression vector used herein includes the nucleic acid construct(s) described above.

As used herein, the term "expression vector" refers to a vehicle designed to express a protein, such as Taq DNA polymerase, in a cell, such as a cell of P. pastoris.

The expression vector herein comprises a nucleic acid construct, which is fundamental to expression, and may also comprise various other components.

In certain embodiments, the expression vector further comprises an origin of replication.

In certain embodiments, the expression vector further comprises a selectable marker. The selectable marker may include a nucleic acid sequence encoding an antibiotic resistance gene. Examples of such antibiotic resistance genes include, but are not limited to, a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.

Bleomycin and phleomycin are glycopeptide antibiotics of bleomycin family, isolated from a mutant strain of Streptomyces verticillus. Phleomycin binds to DNA and is intercalated into it, destroying the integrity of the double helix. Phleomycin is known to be active against most bacteria, filamentous fungi, yeast, plant, and animal cells.

Zeocin is a member of bleomycin/phleomycin family of antibiotics and is known to show strong toxicity against bacteria, fungi (including yeast), and plants and mammalian cell lines (Calmels et al., 1991; Drocourt et al., 1990; Gatignol et al., 1987; Mulsant et al., 1988; Perez et al., 1989).

In one embodiment, the antibiotic resistance gene as described above is BleoR. In one embodiment, the BleoR is a gene encoding an antibiotic binding protein that imparts resistance to bleomycin, phleomycin, and zeocin, and specifically a Shble gene derived from Streptoalloteichus hindustanus.

The expression vector of the present disclosure is characterized in that it lacks a polynucleotide encoding a secretion signal peptide. Typically, a secretion signal peptide plays a role in promoting the secretion of proteins expressed inside a host cell to the outside of the cell, i.e., into the medium, during the culture of the transformed cell. Conventionally, several secretion signal peptides have been reported in the art, including Saccharomyces cerevisiae α-mating factor (Poirier, N. et al. J. Agric. Food Chem. 2012, 60, 9807-9814), Saccharomyces cerevisiae modified α-mating factor (MAF) (Xiong, A.S. et al. J. Biochem. Mol. Biol. 2004, 37, 282-291), α-amylase (Paifer, E. et al. Yeast 1994, 10, 1415-1419), Exg1p (Liang, S. et al. Biotechnol. Lett. 2013, 35, 97-105), inulinase (Massahi, A. et al. J. Theor. Biol. 2015, 364, 179-188), lysozyme (Oka, C. et al. Biosci. Biotechnol. Biochem. 1999, 63, 1977-1983), serum albumin (Xiong, R. et al. Biotechnol. Appl. Biochem. 2008, 51, 129-134), invertase (Kuberl, A. et al. J. Biotechnol. 2011, 154, 312-320), proteinase K (Gunkel, F. A and Gassen H. G. Eur. J. Biochem. 1989, 179, 185-194), or aqualysin I (Oledzka, G, et al. Protein Expr. Purifi. 2003, 29, 223-229) to express heterologous proteins extracellularly in P. pastoris.

However, the present inventors have found that Taq DNA polymerase, upon expression using an expression vector containing a secretion signal peptide in P. pastoris, is not secreted extracellularly, but remains within the cell in a state bound to the secretion signal peptide. Instead, it has been found that the use of an expression vector lacking a secretion signal peptide can increase the expression of Taq DNA polymerase into cells of P. pastoris. This is contrary to previous reports that it is effective to use expression vectors with secretion signal peptides for expression of target proteins in P. pastoris.

Without wishing to be bound by theory, this is probably because Taq DNA polymerase cannot be secreted out of the cell due to its large size, even with the help of secretion signal peptides, and the presence of secretion signal peptides has an adverse effect on the expression of Taq DNA polymerase.

Expression vectors lacking a secretion signal peptide may be prepared by various engineering techniques known in the art. As an example, expression vectors with a secretion signal peptide, such as pGAPZαA vector (J. Wu et al. (2016); J. S. Lee et al. (2018); Sams et al. (2017)) can be digested with appropriate restriction enzymes, such as BstI and SalI, to remove only the secretion signal peptide contained therein, such as α-mating factor. As another example, expression vectors lacking a commercially available secretion signal peptide, such as pPICZ (Thermo Fisher Scientific) and pGAPZ (Thermo Fisher Scientific), can be directly used.

The expression vector according to the present disclosure may be one selected from various vectors known in the art to be applicable to P. pastoris. Examples include, without limitation, pPIC9K vector having His4, Kan, and Amp as selectable markers (Fu, Zhao, Xiong, Tian, and Peng (2011), Tu et al. (2013), X. Chen et al. (2012), Apte-Deshpnade, Mandal, Soorapaneni, Prasad, Kumar, and Padmanabhan (2009)); pPICZα vector having Shble as a selectable marker (Goodrick et al. (2001), Chan et al. (2018); Prabhu, Veeranki, and Dsilva (2016); J. Li et al. (2017); Baeshen et al. (2016); Bardiya and Chang (2017)); pHIL-S1 vector having His4 and Amp as selectable markers (Ben Azoun, Belhaj, Gongrich, Gasser, and Kallel (2016); Satomura, Kuroda, and Ueda (2015); Chahardooli, Niazi, Aram, and Sohrabi (2016)); pGAPZαA vector having Shble as a selectable marker (J. Wu et al. (2016); J. S. Lee et al. (2018); Sams et al. (2017)); pJL-Sx vector having FLD1 and Amp as selectable markers (Sunga and Cregg (2004)), pBLHIS-SX vector having His4 and Amp as selectable markers (Li et al. (2010)), or variants thereof.

As described above, the expression vector used herein may comprise a plurality of the nucleic acid constructs, i.e., one or more copies of the nucleic acid constructs. In an embodiment, the plurality of the nucleic acid constructs is 6 to 10 in total, and thus the expression vector used herein comprises 6 to 10 nucleic acid constructs, i.e., 6-10 copies of the nucleic acid constructs. In certain embodiments, the plurality of the nucleic acid constructs is 8 in total, and thus the expression vector used herein comprises 8 nucleic acid constructs, i.e., 8 copies of the nucleic acid constructs.

To our knowledge, there are no known expression vectors containing 6 or more, particularly 8 or more nucleic acid constructs. On the other hand, the present inventors have found that expression vectors containing a multimer of nucleic acid constructs of considerable size are effective for high expression of target proteins, particularly Taq DNA polymerase.

The expression vector containing the plurality of the nucleic acid constructs may be prepared by any method known in the art.

As an example, an expression vector containing a single nucleic acid construct is digested with an appropriate restriction enzyme to remove the nucleic acid construct. Then, a plurality of the nucleic acid constructs are linked together to produce a multimer of a desired size. Subsequently, the multimer is incorporated into the expression vector to produce an expression vector containing a plurality of the nucleic acid constructs (a multimer of the nucleic acid constructs).

As a specific example, an expression vector containing a single nucleic acid construct comprising a GAP promoter, a polynucleotide encoding Taq DNA polymerase, a nucleic acid sequence coding a histidine tag, and a transcriptional terminator is digested with restriction enzymes BglII and BamHI to obtain the nucleic acid construct. Thereafter, the obtained nucleic acid construct is linked to the same nucleic acid construct. Since the sticky end generated by BglII digestion can be linked to the sticky end generated by BamHI digestion, the end of BamHI-digested nucleic acid construct can be linked to the end of BglII-digested nucleic acid construct. However, since linkage products between the sticky end generated by BglII digestion and the sticky end generated by BamHI digestion are not digested even by BglII or BamHI digestion, it is possible to construct a multimer, such as a dimer.

An expression vector containing a desired number of nucleic acid constructs can be chosen by treating host cells containing expression vectors with increasing concentrations of an antibiotic or by checking the sizes of the nucleic acid constructs from the host cells, for example, by electrophoresis.

As described above, the use of an expression vector containing a plurality of nucleic acid constructs can increase the copy number of an expressed target protein to improve its expression efficiency. In the present disclosure, inclusion of a plurality of nucleic acid constructs can be referred to as increased copy number or multimerization of a gene encoding for a target protein.

Step (b): Transforming Pichia pastoris

In step (b), Pichia pastoris is transformed with the expression vector to obtain a transformed P. pastoris.

Pichia pastoris

The host cell used to produce Taq DNA polymerase herein is a strain of P. pastoris.

The P. pastoris strain may be one of a variety of strains known to be capable of producing foreign proteins. Examples include CBS7435 (Kuberl A et al., (2011) J Biotechnol 154:312-320); DSMZ 70382 (CBS 704) (Mattanovich D et al., (2009) Microb Cell Fact 8:29); X-33 (Life Technologies); GS115 (Cregg JM et al., (1985) Mol Cell Biol 5:3376-3385); GS190; JC220; JC254 (Cregg JM et al., Methods Mol Biol 103:17-26); KM71 (Cregg JM et al., (1987) In: Biological Research on Industrial Yeasts (Stewart GG, Russell I, Klein RD and Hiebsch RR, Eds.), Vol. 2, pp. 1-18. CRC Press, Boca Raton, FL.); KM71H (Life technologies); SMD1163; SMD1165; SMD1168 (Gleeson MA et al., (1998) Methods Mol Biol 103:81-94); kex1 (Boehm T et al., (1999) Yeast 15: 563-572; Ni Z et al., (2007) Yeast 25:1-8); kex2 (Werten MWT et al., (2005) Appl Environ Microbiol 71:2310-2317); ysp1(Yao XQ et al., (2009) J Biotechnol 139:131-136; Werten MWT et al., (2005) Appl Environ Microbiol 71:2310-2317); GlycoSwitch-Gal2 (Jacobs PP et al., Nat Protoc 4:58-70); GlycoSwitch-Man5 (Jacobs PP et al., Microb Cell Fact 9:93); YSH597 (Hamilton SR et al., (2006) Science 313:1441-1443); YGLY4140 (Choi B-K et al., (2003) Proc Natl Acad Sci USA 100:5022-5027); and ku70 (Naatsaari L et al., (2012) PLoS One 7:e39720).

In certain embodiments, the strain of P. pastoris for the transformation is P. pastoris X-33 (Thermo Fisher Scientific).

Transformation

According to the method of the present disclosure, P. pastoris is transformed with an expression vector having a nucleic acid construct which comprises a polynucleotide encoding the Taq DNA polymerase, and a promoter operably linked thereto.

The transformation of P. pastoris can be performed by any of various methods known in the art.

As used herein, the term "transformation" means that a foreign DNA is imported into host cells and integrated into host chromosomal DNA (homologous recombination). The transformation of P. pastoris according to the present disclosure can be performed, for example, by electroporation, protoplasmic fusion, calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂) precipitation, agrobacterium-mediated transformation, PEG (polyethylene glycol), dextran sulfate, lipofectamine, or particle bombardment.

The step (b) according to the present disclosure, i.e., transformation, induces homologous recombination between the expression vector and the genome of P. pastoris.

The homologous recombination described above arises from the similarity of sequences between the expression vector and the genome of P. pastoris.

In one embodiment, the homologous recombination is based on sequence similarity between a promoter found in the expression vector and a promoter within the genome of P. pastoris. Examples of the promoters can be found in this specification, including GAP (glycerde-3-hydrogenase) promoter.

In one embodiment, the method of the present disclosure further comprises linearizing the expression vector prior to the step (b), i.e., transformation.

Linearization of the expression vector enables the expression vector to be incorporated into the genomic DNA of P. pastoris via homologous recombination to generate stable transformants.

In one embodiment, linearization of the expression vector can be achieved by using a restriction site present in the expression vector.

In one embodiment, linearization of the expression vector can be achieved by using a restriction site upstream of the nucleic acid construct.

In certain embodiments, linearization of the expression vector can be achieved by using a restriction site upstream of the promoter in the nucleic acid construct.

In certain embodiments, linearization of the expression vector can be achieved by using a restriction site 3 to 150 bp, 5 to 100 bp, 5 to 50 bp, 5 to 40 bp, 5 to 30 bp, or 5 to 20 bp upstream from the promoter in the nucleic acid construct.

Examples of restriction enzymes for the restriction site include, but are not limited to, BglII or NsiI.

The step (b) as described above allows transformants of P. pastoris to be obtainable.

In one embodiment, the genome of P. pastoris after homologous recombination has 1-10 copies of the nucleic acid constructs integrated thereinto.

Specifically, the genome of the transformed P. pastoris in which the homologous recombination was induced, contains (promoter-polynucleotide encoding Taq DNA polymerase)_m derived from the expression vector, where m is an integer of 1 to 10.

Step (c): Culture of P. pastoris

In this step, the transformed P. pastoris is cultured in a culture medium under conditions permitting intracellular expression of the Taq DNA polymerase.

The culture medium may be a conventional medium which contains a carbon source and a nitrogen source required for culturing yeast. In one embodiment, the culture medium does not contain methanol.

Examples of the culture medium include, but are not limited to, YPD (Yeast extract Peptone Dextrose) medium, YPDS (Yeast extract Peptone Dextrose Sorbitol) medium, MGY (Minimal Glycerol) medium, MGYH (Minimal Glycerol Histidine) medium, MD (Minimal Dextrose) medium, MDH (Minimal dextrose Histidine) medium, MM (Minimal Methanol) medium, MMH (Minimal Methanol Histidine) medium, BMGH (Buffered Minimal Glycerol) medium, BMMH (Buffered Minimal Methanol) medium, BMGY (Buffered Glycerol complex) medium, and BMMY (Buffered Methanol complex) medium.

The culture medium may further comprise an antibiotic to select transformants transformed with the polynucleotide encoding Taq DNA polymerase according to the present disclosure. The antibiotic is one corresponding to a selectable marker contained in the expression vector.

In certain embodiments, the culture medium according to the present disclosure is YPDS medium containing an antibiotic, such as zeocin.

The composition of the YPDS medium typically includes 1% yeast extract, 2% peptone, 2% dextrose (glucose), and 1 M sorbitol, but it will be understood by those skilled in the art that the composition can be adjusted as needed.

Such antibiotic, such as zeocin, may typically be contained in a culture medium in an amount of 100 μg/ml, but the amount is readily adjustable by those skilled in the art.

The conditions for culturing P. pastoris may typically be at 20 to 40°C, preferably at 30°C, for 1 to 10 days, particularly for 1 to 7 days, and more particularly for 2 to 3 days.

Step (d): Purification of intracellularly expressed Taq DNA polymerase

In step (d), the cultured P. pastoris is lysed and intracellularly expressed Taq DNA polymerase is then purified from the lysate.

The lysis of the cultured P. pastoris may be performed using any of methods known in the art.

In one embodiment, the lysis of the cultured P. pastoris is performed mechanically after separating cells from the culture of P. pastoris.

The mechanical cell lysis may include lysis by sonication, lysis by French press and lysis by a bead mill.

Cell lysis by sonication refers to a process of using sound energy greater than 16 kHz to disrupt the cell wall and cell membrane. However, sonication has the disadvantage of denaturing heat-sensitive enzymes. Meanwhile, cell lysis by French press refers to a process of filling cells into a hollow stainless-steel cylinder, which is often used on a laboratory scale, then extruding the cells under high pressure to an atmospheric pressure state through a needle valve at the bottom of the cylinder. Also, cell lysis by a bead mill refers to a process of stirring beads made of glass or iron to dissolve cells with high shearing force and impact force.

In certain embodiments, the lysis of the cultured P. pastoris is achieved by a process comprising the steps of: (i) centrifuging a culture of P. pastoris to obtain cell pellets, and (ii) adding glass beads to the obtained cell pellets to lyse cells.

For example, P. pastoris may be lysed by adding about 1 to 3 times as much glass beads (e.g., commercially available from Merck) and a suitable buffer (e.g., Breaking buffer) to the cell pellets and repeatedly vortexing.

In another embodiment, the lysis of the cultured P. pastoris is performed non-mechanically. Examples include treating the culture with an enzyme such as lysozyme to lyse cells or slowly freezing cells and then thawing them to lyse the cell membrane.

The purification of the Taq DNA polymerase from the lysed P. pastoris (lysate) may be performed by any of chromatographic methods known in the art.

Examples of chromatographic methods that can be used include, but are not limited to, immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC), and reverse phase HPLC.

In one embodiment, purification according to the present disclosure is performed using affinity chromatography for histidine protein. Examples of such chromatography include, but are not limited to, Ni-NTA affinity chromatography (e.g., using Cytiva HisTrap^TM Fast Flow column).

In one embodiment, purification according to the present disclosure is performed using anion exchange chromatography (e.g., using Cytiva HiPrep^TM Q FF column), followed by affinity chromatography for histidine protein.

Using the method of the present disclosure, Taq DNA polymerase expressed within the cells of P. Pastoris can be obtained.

According to the method of the present disclosure, the purified, intracellularly expressed Taq DNA polymerase is obtained at a purification yield of 6 to 15 mg per 100 ml of the culture medium.

To date, Taq DNA polymerase has never been expressed in P. pastoris at such a high yield. Also, considering that the yield of Taq DNA polymerase in E. coli is 1.33 mg per 100 ml of the culture medium (according to the experimental results of the inventor, data not presented), it can be seen that the yield of the method of the present disclosure is somewhat high.

According to the method of the present disclosure, the purified, intracellularly expressed Taq DNA polymerase does not contain any secretion signal peptide.

Using an expression vector containing a secretion signal peptide leads to accumulation of Taq DNA polymerase bound to the secretion signal peptide within a cell without secreting Taq DNA polymerase outside the cell, whereas the method of the present disclosure accumulates Taq DNA polymerase without a secretion signal peptide within a cell. As described above, Taq DNA polymerase bound to a secretion signal peptide has no commercial value because its structure is unstable, while the Taq DNA polymerase obtained by the method of the present disclosure is of high commercial value due to having no secretion signal peptide bound.

According to the method of the present disclosure, the purified, intracellularly expressed Taq DNA polymerase is free of nucleic acids derived from E. coli. In other words, the purified, intracellularly expressed Taq DNA polymerase is not contaminated with nucleic acids derived from E. coli.

Thus, the purified, intracellularly expressed Taq DNA polymerase does not lead to any false positives due to a non-target E. coli in an amplification reaction. That is, an amplification reaction using the purified, intracellularly expressed Taq DNA polymerase does not result in false positives due to E. coli-derived non-target nucleic acids. For example, the purified, intracellularly expressed Taq DNA polymerase does not generate a signal at all or generates a signal having a Ct value of 40 or higher, in the absence of a target nucleic acid in a nucleic acid amplification reaction, such as a PCR reaction. A signal having a Ct value of 40 or higher is considered to indicate the absence of the target nucleic acid.

According to our experiments, subjecting each of a sample containing the uidA gene from E. coli and a sample without the gene (negative control) to an amplification reaction using Taq DNA polymerase according to the present disclosure both results in amplification products, but a sample not containing the gene does not generate any amplification product (see Figure 7).

In an aspect, the present disclosure provides Taq DNA polymerase, prepared by the method as described above.

In one embodiment, the Taq DNA polymerase is free of nucleic acids derived from E. coli.

In one embodiment, the Taq DNA polymerase does not lead to any false positives from a non-target E. coli in an amplification reaction.

Since the detailed description of the Taq DNA polymerase described above is described elsewhere in this specification, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

In an aspect, the present disclosure provides a method of amplifying one or more target nucleic acids in a sample using the Taq DNA polymerase as described above.

The detection of the target nucleic acids may be performed by a nucleic acid amplification reaction known in the art, such as PCR or real-time PCR. The nucleic acid amplification reaction may include the steps of amplifying a target nucleic acid in a sample using a primer and detecting the amplified target nucleic acid. The amplified target nucleic acid may be detected by measuring a fluorescence signal generated from a probe to which a label, such as a fluorescent label, is linked.

The method described above is not limited to any specific detection method known in the art, as long as the Taq DNA polymerase prepared according to the method of the present disclosure is used to amplify target nucleic acids.

In one embodiment, at least one of the one or more target nucleic acids is from E. coli.

Since commercially available Taq DNA polymerases are produced by an E. coli expression system, they are likely to be contaminated with DNA derived from E. coli. Therefore, it is difficult to determine whether a positive result in a nucleic acid amplification reaction using such contaminated Taq DNA polymerase is due to the presence of target E. coli or the presence of contaminated E. coli.

On the other hand, since the Taq DNA polymerase obtained by the method of the present disclosure does not contain nucleic acids from contaminated E. coli, positive results in the nucleic acid amplification reaction can be assured that they originate from amplification of target nucleic acids in target E. coli.

In an aspect, the present disclosure provides a transformed Pichia pastoris, prepared by transforming P. pastoris with an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.

Since the details described above are described elsewhere in this specification, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative, and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

Example 1: Confirmation of intracellular accumulation of Taq DNA polymerase using expression vectors containing secretion signal peptide

<1-1> Construction of expression vectors containing various secretion signal peptides

In order to secrete Taq DNA polymerase outside the Pichia pastoris cells, expression vectors containing each of secretion signal peptides were prepared as follows.

First, the polynucleotide sequence of the wild-type Taq DNA polymerase was codon-optimized according to the codon usage of P. pastoris (see Table 1). The codon-optimized sequence was shown in SEQ ID NO: 1.

Subsequently, the codon-optimized polynucleotide (SEQ ID NO: 1) encoding the Taq DNA polymerase was inserted into pGAPZαA vector (Thermo Fisher Scientific, see Fig. 1) having the α-mating factor (αA) signal peptide sequence of Saccharomyces cerevisiae (282 bp; 89 amino acids; see SEQ ID NO: 2) using EcoRI and NotI restriction sites. The resulting expression vector was used to transform E. coli DH10B by electroporation. The transformants were then grown on a low-salt LB agar medium supplemented with zeocin (25 μg/ml) until colonies were formed. The colonies were used to isolate the plasmid, which was sequenced to confirm the correct construction of the expression vector "pGAPZαA Taq 1M" containing a monomeric polynucleotide encoding a single Taq DNA polymerase.

Then, in order to further construct expression vectors containing a polynucleotide encoding multimeric Taq DNA polymerase, the expression vector, pGAPZαA Taq 1M (about 5.62 kb) containing monomeric Taq DNA polymerase was digested with BglII and then treated with an alkaline phosphatase to prevent self-ligation. Also, the pGAPZαA Taq 1M was digested with BglII and BamHI to recover a fragment containing pGAP promoter-Taq DNA polymerase (about 3.7 kb) by agarose gel electrophoresis. Thereafter, the BglII-digested product and the BglII- and BamHI-digested fragment were ligated with each other, and the ligation product was used to transform E. coli DH10B. Then, the plasmid was isolated from the transformant and digested with BglII and BamHI to select the expression vector "pGAPZαA Taq 2M" (about 9.3 kb) containing dimeric pGAP promoter-Taq DNA polymerase (about 7.4 kb) by agarose gel electrophoresis.

In order to construct tetrameric pGAP promoter-Taq DNA polymerase, the pGAPZαA Taq 2M was digested with BglII and treated with alkaline phosphatase. Also, the pGAPZαA Taq 2M was digested with BglII and BamHI to recover a fragment of dimeric pGAP promoter-Taq DNA polymerase (about 7.4 kb). The BglII-digested product and the BglII- and BamHI-digested fragment were ligated with each other, and the ligation product was used to transform E. coli DH10B. Then, the plasmid was isolated from the transformant and digested with BglII and BamHI to select the expression vector "pGAPZαA Taq 4M" (about 16.7 kb) containing tetrameric pGAP promoter-Taq DNA polymerase (about 14.8 kb) by agarose gel electrophoresis. The pGAPZαA Taq 4M is an expression vector containing a tetramer of a pGAP promoter, an α-mating factor signal sequence as a secretion signal peptide, and the polynucleotide encoding Taq DNA polymerase as a target protein.

Additionally, in order to construct expression vectors containing different secretion signal peptides, the expression vector, pGAPZαA Taq 4M as prepared above was digested with BstI and EcoRI to remove the α-mating factor signal sequence. The digested product was ligated with a modified α-mating factor signal sequence (MAF; 321 bp; 104 amino acids; see SEQ ID NO: 3), a proteinase K signal sequence (pKS; 60 bp; 18 amino acids; see SEQ ID NO: 4) and an aqualysin I signal sequence (aqS; 60 bp; 18 amino acids; see SEO ID NO: 5) to construct expression vectors containing various secretion signal peptides, "pGAPZMAF Taq 4M," "pGAPZpkS Taq 4M," and "pGAPZaqs Taq 4M," respectively.

The electrophoresis results for the expression vectors constructed above were shown in Fig. 2.

In Fig. 2, lanes 1-1, 1-2, and 1-3 show the results for the expression vector "pGAPZαA Taq 4M", which includes a tetramer of an α-mating factor as a secretion signal peptide and the Taq DNA polymerase; lanes 2-1, 2-2, and 2-3 show the results for the expression vector "pGAPZMAF Taq 4M", which includes a tetramer of a modified α-mating factor (MAF) as a secretion signal peptide and the Taq DNA polymerase; lanes 5-1, 5-2, and 5-3 show the results for the expression vector "pGAPZpkS Taq 4M", which includes a tetramer of a proteinase K (pKS) as a secretion signal peptide and the Taq DNA polymerase; and lanes 6-1 and 6-2 show the results for the expression vector "pGAPZaqS Taq 4M", which includes a tetramer of an aqualysin I (aqS) as a secretion signal peptide and the Taq DNA polymerase. For size comparison, 7^th, 8^th, and 9^th lanes show the results for the expression vectors "pGAPZpkS Taq 1M", which includes a monomer of a pKS and the Taq DNA polymerase, "pGAPZpkS Taq 2M", which includes a dimer of a pKS and the Taq DNA polymerase, and "pGAPZpkS Taq 3M", which includes a trimer of a pKS and the Taq DNA polymerase, respectively.

As shown in Fig. 2, it was found that the transformants obtained in Examples <1-1> and <1-2> contains expression vectors containing tetramers of various secretion signal peptides and Taq DNA polymerase.

<1-2> Transformation of P. pastoris

Each of the four expression vectors constructed in Example <1-1> was linearized by digesting it with BglII, and then was used to transform P. pastoris X-33 (Thermo Fisher Scientific).

Specifically, the P. pastoris strain was cultured in a 5 ml YPD medium in a 50 ml conical tube at 30℃ overnight, and 0.1-0.5 ml of the culture was inoculated into 500 ml of a fresh medium to allow P. pastoris to grow overnight up to OD₆₀₀ 1.3-1.5. Then, the cells were centrifuged at 3000 × g at 4℃ for 10 minutes to obtain cell pellets. The cell pellets were added with 20 ml of a transfection buffer [1 M LiAc 2 ml, 1 M DTT 0.2 ml, 1 M sorbitol 12 ml, 1 M Tris-HCl (pH 7.6) 0.2 ml, sterile water 5.6 ml], transferred to a 50 ml conical tube, placed at room temperature for 30 minutes, and centrifuged at 3000 × g at 4℃ for 7 minutes to recover cell pellets. Then, the cell pellets was added with 5-10 ml of 1 M sorbitol to suspend it and the suspension was centrifuged. This process was repeated 2 times to completely remove any salt from the medium. The cell pellets were then resuspended in 1 ml of cold 1 M sorbitol.

80 μl of the P. pastoris cell suspension was mixed with a linearized product (1-10 μg), which was obtained by digesting the expression vector with BglII, removing salts, and dissolving it in 5-10 μl of sterile distilled water. The mixture was transferred to a cold electroporation cuvette and an electrical pulse of 1500 V-2400 V was applied to the cuvette. Then, the cuvette was added with 1 ml of cold 1 M sorbitol and transferred to a sterile 50 ml tube. The tubes were incubated at 30℃ for 1 hour without agitation. Then, the tube was added with 1 ml of YPD medium and incubated at 30℃ for 1 hour while stirring at 200 rpm. Approximately 200 μl of the culture was plated onto YPDS medium containing 100 μg/ml of zeocin and incubated at 30℃ for 2-3 days to obtain resulting colonies.

<1-3> Confirmation of intracellular accumulation of Taq DNA polymerase in transformed P. pastoris

It was investigated whether the transformed P. pastoris obtained in Example <1-2> expresses Taq DNA polymerase extracellularly or intracellularly.

Specifically, each of the transformants obtained in Example <1-2> was cultured in 3 ml YPD medium containing zeocin for 48 hours while stirring at 250 rpm at 30℃. Then, approximately 0.8-1 ml of the culture was centrifuged. The supernatant was used to confirm extracellular secretion of Taq DNA polymerase, and the precipitated pellet was used to confirm intracellular accumulation of Taq DNA polymerase.

32 μl of the supernatant was loaded onto SDS-PAGE to confirm extracellular secretion of Taq DNA polymerase.

Meanwhile, in order to confirm intracellular expression of Taq DNA polymerase, cell lysates of each of these transformants were obtained. Specifically, to the cell pellets, about 2 times glass beads (Merck G9268) per cell weight, 100 μl of breaking buffer (50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA, and 5% glycerol) and about 2 μl PMSF (phenylmethanesulfonylfluoride, 100 mM) were added. The mixture was vortexed at maximum speed for 30 seconds, and then cooled on ice for 1 minute, which was repeated 5 times. Then, 100 μl of breaking buffer and about 2 μl PMSF (100 mM) were added again, vortexed at maximum speed for 30 seconds, and then cooled on ice for 1 minute, which was repeated 3 times. After heating at 80℃ for 30 minutes, the mixture was vortexed and centrifuged to recover the supernatant. The supernatant was loaded onto SDS-PAGE to confirm intracellular expression of Taq DNA polymerase.

SDS-PAGE results for the supernatants and the cell lysates are shown in Fig. 3.

In Figure 3, 1^st lane represents a ladder as a molecular weight marker; 2^nd lane represents the loading result for the culture supernatant (abbreviated as "S") of an untransformed P. pastoris; 3^rd and 4^th lanes represent the loading results for the culture supernatant and the cell lysate (abbreviated as "L"; heated at 80℃ for 30 min) of P. pastoris, transformed with the expression vector "pGAPZαA Taq 4M", which includes a tetramer of an α-mating factor as a secretion signal peptide and the Taq DNA polymerase, respectively; 5^th and 6^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZMAF Taq 4M", which includes tetramer of a modified α-mating factor (MAF) as a secretion signal peptide and the Taq DNA polymerase, respectively; 7^th and 8^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZpkS Taq 4M", which includes a tetramer of a proteinase K signal sequence (pKS) as a secretion signal peptide and the Taq DNA polymerase; and 9^th and 10^th lanes represent the loading results for the culture supernatant and the cell lysate of P. pastoris, transformed with the expression vector "pGAPZaqS Taq 4M", which includes a tetramer of an aqualysin I signal sequences (aqS) as a secretion signal peptide and the Taq DNA polymerase.

As shown in Fig. 3, it was found that P. pastoris transformed with expression vectors containing various secretion signal peptides and the Taq DNA polymerase is unable to secrete Taq DNA polymerase outside cells. Also, it was found that the Taq DNA polymerase expressed in the transformed P. pastoris is bound to the secretion signal peptide. As shown in Fig. 3, any band corresponding to the Taq DNA polymerase was not visible in the supernatant of P. pastoris transformed with pGAPMAF Taq 4M, while a band corresponding to the molecular weight of about 100,000 Da (MAF M.W 10.99 kDa + Taq polymerase M.W 94.74 kDa = 105.73 kDa), which is larger than the molecular weight 94.74 kDa of Taq DNA polymerase, was not visible in heat-treated cell lysates. Also, any band corresponding to Taq DNA polymerase was not visible in the supernatant of P. pastoris transformed with pGAPZpkS Taq 4M, while a band corresponding to a molecular weight of about 96,000 Da (pKS M.W 1.94 kDa + Taq polymerase M.W 94.74 kDa = 96.68 kDa) was visible in heat-treated cell lysates.

Unlike previous reports, the above results show that the Taq DNA polymerase is not secreted extracellularly by the P. pastoris expression system which contains a secretion signal peptide.

Example 2: Intracellular expression of Taq DNA polymerase using expression vector not containing secretion signal peptide

From the results of Example 1, it was confirmed that after being expressed within the P. pastoris cells, the Taq DNA polymerase is intracellularly accumulated without being secreted out of the cell even with the help of a secretion signal peptide. Since an intact, structurally stable Taq DNA polymerase would be more desirable than a structurally unstable Taq DNA polymerase fused with a secretion signal peptide, expression vectors lacking any secretion signal peptide were constructed and transformed into P. pastoris to measure the intracellular expression of Taq DNA polymerase.

<2-1> Construction of expression vectors

For intracellular expression of Taq DNA polymerase in P. pastoris, expression vectors lacking a secretion signal peptide were constructed as below.

First, using the codon-optimized polynucleotide sequence of the Taq DNA polymerase (SEQ ID NO: 1; see Example 1) as a template, PCR was carried out using Taq BstBI forward primers (5'-ACGTATTCGAACGATGAGGGGTATGTTACCACTGTTTG-3'; SEQ ID NO: 6) to generate BstBI restriction site and Taq GSG SalI reverse primers (5'-TGATGGTCGACGCCGCTGCCTTCTTTAGCTGACAACCAGTCTTCTC-3'; SEQ ID NO: 7) to generate GSG-SalI restriction site. The PCR product consists of BstBI site-codon-optimized Taq DNA polymerase-GSG linker-SalI site.

Thereafter, the pGAPZαA vector (see Example 1) was digested with BstBI and SalI to remove a secretion signal peptide, the α-mating factor. The vector lacking the secretion signal peptide was ligated with the PCR product treated with the same restriction enzyme to construct "pGAPZ Taq GSG C-His 1M". The resulting expression vector includes a nucleic acid construct containing, from N-terminus to C-terminus, GAP promoter-polynucleotide encoding Taq DNA polymerase-GSG linker-histidine tag-AOX1 terminator. After transforming Escherichia coli DH10B with the expression vector pGAPZ Taq GSG C-His 1M, the transformant was grown on a low-sodium LB agar medium containing zeocin. The plasmid was isolated from the grown transformants and sequenced (Macrogen Co., Ltd.) to confirm the correct construction of the pGAPZ Taq GSG C-His 1M.

Meanwhile, in order to construct expression vectors containing a multimer of the nucleic acid constructs, the expression vector pGAPZ Taq GSG C-His 1M (about 5.27 kb) was digested with BglII and then treated with an alkaline phosphatase. Then, the pGAPZ Taq GSG C-His 1M was digested with BglII and BamHI to obtain a fragment (about 3.36 kb) containing the pGAP promoter-Taq DNA polymerase. The BglII-digested product and the BglII- and BamHI-digested fragment were ligated with each other, and the ligation product was transformed into Escherichia coli DH10B. The plasmid was then isolated and digested with BglII and BamHI to select "pGAPZ Taq GSG C-His 2M" (about 8.62 kb) containing a dimer of pGAP promoter-Taq DNA polymerase by agarose gel electrophoresis.

Again, in order to construct a tetramer of pGAP promoter-Taq DNA polymerase, a BglII-digested pGAPZ Taq GSG C-His 2M was ligated with a BglII- and BamHI-digested pGAPZ Taq GSG C-His 2M containing a dimer of pGAP promoter-Taq DNA polymerase. The ligation product was used to transform E. coli DH10B. The plasmid was isolated from the transformants and digested with BglII and BamHI to select "pGAPZ Taq GSG C-His 4M" (about 15.34 kb) containing a tetramer (about 13.44 kb) of pGAP promoter-Taq DNA polymerase by agarose gel electrophoresis.

Afterwards, above process was repeated for the pGAPZ Taq GSG C-His 4M to obtain "pGAPZ Taq GSG C-His 8M" (28.7 kb). The pGAPZ Taq GSG C-His 8M contains an octamer of the pGAP promoter and the polynucleotide encoding Taq DNA polymerase as a target protein.

To confirm the correct construction of the expression vectors, the expression vectors were each digested with BglII and BamHI, followed by electrophoresis.

The electrophoresis results are shown in Fig. 4.

In Figure 4, 1^st lane represents a 1 kb ladder as a molecular weight marker; 2^nd to 4^th lanes represent the loading results for the expression vector "pGAPZ Taq GSG C-His 8M"; and 5^th lane represents the loading result for the expression vector "pGAPZ Taq GSG C-His 4M" for comparison. In each lane, the upper band corresponds to an octameric or tetrameric Taq DNA polymerase, and the lower band corresponds to remaining backbone of the expression vector except for the octameric or tetrameric Taq DNA polymerase.

<2-2> Obtaining transformants of P. pastoris

Each of the four expression vectors constructed in Example <1-1> was linearized by digesting it with BglII and used to transform P. pastoris X-33.

The transformation of P. pastoris was performed as described in Example <1-2>. After the transformation, colonies of transformants were obtained.

<2-3> Confirmation of intracellular expression of Taq DNA polymerase in transformed P. pastoris

It was investigated that the transformed P. pastoris obtained in Example <2-2> expresses Taq DNA polymerase intracellularly.

Specifically, each of the transformants obtained in Example <1-2> was inoculated into 3 ml YPD medium containing 3 μl of zeocin (100 mg/ml) and cultured for 48 hours while stirring at 250 rpm at 30℃. The culture was then centrifuged to obtain cell pellets. The cell pellets were added with about twice glass beads (Merck G9268), 50 μl of breaking buffer (50 mM sodium phosphate pH 7.4, 1 mM PMSF, 1 mM EDTA, and 5% glycerol), and about 2 μl PMSF (phenylmethanesulfonylfluoride, 100 mM). The mixture was vortexed at maximum speed for 30 seconds, then cooled on ice for 1 minute, which was repeated 5 times. Then, another 50 μl of breaking buffer was added, vortexed, and heated at 80℃ for 30 minutes. After centrifugation, 32 μl of supernatant was loaded onto SDS-PAGE to confirm intracellular expression of Taq DNA polymerase.

SDS-PAGE results for cell lysates of each transformant are shown in Figs. 5A and 5B.

As seen in the figures, it was found that the transformed P. pastoris obtained in Example <2-2> expresses a significant amount of Taq DNA polymerase intracellularly.

<2-4> Analysis of polymerase activity in cell lysates

It was investigated that cell lysates from the transformed P. pastoris obtained in Example <2-2> have Taq DNA polymerase activity.

As described in Example <2-3>, the cell lysate of each transformant was subjected to PCR.

The PCR was performed by mixing 2 mM dNTPs 2 μl, λ DNA 0.8 μl, 2 kb λ forward primer (SEQ ID NO: 8), 2 kb λ reverse primer (SEQ ID NO: 9), 2 μl of 10X Taq buffer, 1 μl of the cell lysate and 13.2 μl of sterile water and subjecting it to 2 minutes at 94℃ and 25 cycles of 10 seconds at 94℃, 10 seconds at 56℃ and 50 seconds at 72℃.

The PCR product was electrophoresed to confirm whether the λ DNA was normally amplified.

The electrophoresis results are shown in Fig. 6.

As shown in Figure 6, it was found that the cell lysates of the transformed P. pastoris obtained according to this Example effectively amplify target nucleic acids in PCR. Thus, it was confirmed that the cell lysates of the transformed P. pastoris obtained according to this Example have normal Taq DNA polymerase activity.

<2-5> Mass production and purification of Taq DNA polymerase

The colony (transformed with pGAPZ Taq CMP GSG C-His 8M) obtained in the Example <2-2> was inoculated into 3 ml YPD medium containing 3 μl of zeocin (100 mg/ml) and cultured at 30℃ (250 rpm) for about 24 hours. After adding 100 μl zeocin to each of two 400 ml YPD medium and adding each of 2 ml of the culture into the media, the media were cultured at 30℃ (250 rpm) for 24 hours. 800 ml of the culture was aliquoted into 11 tubes in an amount of about 70 ml per tube and the aliquots were each centrifuged to recover cell pellets. Each cell pellet was suspended in sterile water, and the suspension was then centrifuged again to recover an average of 1.8 g of cell pellets per tube.

To 1.8 g of the cell pellets, two times glass beads, 1 ml of breaking buffer (50 mM sodium phosphate pH 7.4, 1 mM EDTA, 5% glycerol) and 20 μl PMSF (100 mM) were added. The mixture was vortexed at maximum speed for about 50 seconds, and then cooled in ice water for 1.5 minutes, which was repeated 5 times.

Next, another 1 ml of breaking buffer was added to the mixture to suspend it. Then, the suspension was centrifuged at 9000 rpm for 10 minutes, and the supernatant was collected in a separate tube and heated at 80℃ for 30 minutes. To the pellet was added 1 ml of breaking buffer and 20 μl PMSF (100 mM), and the suspension was vortexed at maximum speed for 40-50 seconds and cooled in ice water for 1.5 minute, which was repeated 3 times.

After adding 1 ml of breaking buffer to suspend it, the suspension was centrifuged at 9000 rpm for 10 minutes, the supernatant was collected in a separate tube and heated at 80℃ for 30 minutes. To the pellet was added 2 ml of washing buffer (50 mM Tris-HCl, pH 8.0) and 20 μl PMSF (100 mM), the suspension was immediately centrifuged to recover the supernatant. It was possible to obtain an average of 6 ml of heat-treated solution per tube, and to obtain about 66 ml of heat-treated solution from 11 tubes.

After centrifuging the supernatant again at 15000 rpm for 20 minutes, 65 ml of the supernatant was recovered. The supernatant was added with 1.95 ml of 5% PEI, and the suspension was left at 4℃ overnight and centrifuged at 9000 rpm for 20 minutes to recover the supernatant. Then, after adding 1.75 ml of 1 M Tris-HCl (pH 8.0) to adjust the final concentration to 50 mM, approximately 70 ml of the supernatant solution was loaded onto a HiPrep^TM Q column (20 ml) equilibrated with 20 mM Tris-HCl (pH 8.0) buffer. After eluting the bound protein with a 0.1-1 M KCl gradient in 20 mM Tris-HCl (pH 8.0), each fraction was electrophoresed by 10% SDS-PAGE. Approximately 38 ml of the fraction containing the target protein was recovered and then loaded onto a HisTrap^TM FF column (5 ml) equilibrated with 20 mM HEPES buffer (pH 7.9) containing 500 mM KCl. After eluting the bound protein with an imidazole 0-500 mM gradient, each fraction was electrophoresed by 10% SDS-PAGE. Then, a fraction containing the target protein was recovered from the column.

The concentration of Taq DNA polymerase expressed above was determined to be about 2 mg/ml according to the Bradford method and the Epoch microplate spectrophotometer method. It indicates that approximately 50 mg of purified Taq DNA polymerase was finally recovered from 800 ml culture medium (OD₆₀₀ = 15.6/ml/total wet cell: 21 g). This yield is expected to be further increased when the transformed P. pastoris is cultured for 36 hours or more.

The results demonstrate that a significant amount of Taq DNA polymerase can be obtained from P. pastoris according to the method of the present disclosure.

<2-6> Confirmation of amplification of E. coli gene using Taq DNA polymerase

PCR methods for detecting E. coli typically have used primers for uidA gene encoding β-D-glucuronidase (Bej A. K. et al. Appl. Environ. Microbiol. 1991, 307-14; Molina, F, et al. BMC Biotechnology 2015, 48).

In this Example, it was investigated whether the Taq DNA polymerase obtained according to the method of the present disclosure can amplify the nucleic acid of target E. coli without contamination of non-target E. coli genomic DNA.

For this purpose, a PCR reaction solution was added with primers (SEQ ID NOs: 10 and 11) for uidA gene, and subjected to denaturation at 95℃ for 3 minutes, then 35 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 1 minute. Amplification products corresponding to the 162 bp uidA gene was detected by 1.5% agarose gel electrophoresis.

The results are shown in Fig. 7. In the figure, the leftmost lane represents a 1 kb ladder as a molecular weight marker; 1^st lane shows the amplification result for E. coli DH5α genomic DNA using Taq DNA polymerase according to the present disclosure; and 2^nd lane shows the amplification result using only the Taq DNA polymerase in the absence of E. coli DH5α genomic DNA.

As shown in Fig. 7, in the case of a sample containing E. coli DH5α genomic DNA, the amplification product corresponding to the 162 bp uidA gene was clearly visible, while in the case of a sample not containing E. coli DH5α genomic DNA, any amplification product was not visible.

The results demonstrate that the Taq DNA polymerase according to the present disclosure can amplify the target E. coli gene without contamination of any non-target E. coli genomic DNA.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

A method for preparing Taq DNA polymerase, comprising:

(a) providing an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding the Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide;

(b) transforming Pichia pastoris with the expression vector to obtain a transformed P. pastoris;

(c) culturing the transformed P. pastoris in a culture medium under conditions permitting intracellular expression of the Taq DNA polymerase; and

(d) lysing the cultured P. pastoris and purifying the intracellularly expressed Taq DNA polymerase from the lysate.
The method of claim 1, wherein the polynucleotide encoding the Taq DNA polymerase is codon-optimized for P. pastoris.
The method of claim 2, wherein the polynucleotide encoding the Taq DNA polymerase comprises a sequence of SEQ ID NO: 1 or a sequence having at least 95% sequence identity thereto.
The method of claim 1, wherein the promoter is a glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.
The method of claim 1, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a histidine tag, which is linked to the polynucleotide encoding the Taq DNA polymerase.
The method of claim 5, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a (GSG)n linker between the polynucleotide encoding the Taq DNA polymerase and the nucleic acid sequence encoding the histidine tag, wherein n is an integer from 1 to 5, G denotes glycine, and S denotes serine.
The method of claim 1, wherein the expression vector further comprises an antibiotic resistance gene as a selectable marker.
The method of claim 7, wherein the antibiotic resistance gene is a bleomycin resistance gene, a phleomycin resistance gene, or a zeocin resistance gene.
The method of claim 1, wherein the expression vector comprises a plurality of the nucleic acid constructs.
The method of claim 9, wherein the plurality of the nucleic acid constructs is 6 to 10 in total.
The method of claim 1, further comprising linearizing the expression vector prior to the step (b).
The method of claim 1, wherein the step (b) induces homologous recombination between the expression vector and the genome of P. pastoris.
The method of claim 12, wherein the genome of P. pastoris after homologous recombination has 1-10 copies of the nucleic acid constructs integrated thereinto.
The method of claim 1, wherein the purified, intracellularly expressed Taq DNA polymerase is produced in an amount of 6 to 15 mg per 100 ml of the culture medium.
The method of claim 1, wherein the purified, intracellularly expressed Taq DNA polymerase does not comprise a secretion signal peptide.
The method of claim 1, wherein the purified, intracellularly expressed Taq DNA polymerase is free of nucleic acids derived from E. coli.
The method of claim 16, wherein the purified, intracellularly expressed Taq DNA polymerase does not lead to any false positives from a non-target E. coli in an amplification reaction.
Taq DNA polymerase, prepared by the method of any one of claims 1 to 17.
The Taq DNA polymerase of claim 18, which is free of E. coli-derived nucleic acids.
The Taq DNA polymerase of claim 19, which does not lead to any false positives from a non-target E. coli in an amplification reaction.
A method of amplifying one or more target nucleic acids in a sample using the Taq DNA polymerase of claim 18.
The method of claim 21, wherein at least one of the one or more target nucleic acids is from E. coli.
A transformed Pichia pastoris, prepared by transforming P. pastoris with an expression vector having a nucleic acid construct therein, the nucleic acid construct comprising: (i) a polynucleotide encoding a Taq DNA polymerase, and (ii) a promoter operably linked thereto, wherein the expression vector lacks a polynucleotide encoding a secretion signal peptide.