JP2002534125A - Biologically active reverse transcriptase - Google Patents

Abstract

  (57) [Summary] The present invention provides modified reverse transcriptase polypeptides (types I, II and III), polynucleotides encoding the polypeptides, vectors carrying the polynucleotides, and host cells transformed with these polynucleotides. . These modified reverse transcriptases typically exhibit improved stability and / or improved solubility as compared to naturally occurring reverse transcriptase. These modified reverse transcriptases have been found to take a variety of forms, including monomers and both homo- and hetero-multimers. These modified reverse transcriptases can be used in one or more known methods utilizing reverse transcriptase activity, such as cDNA synthesis and amplification techniques such as PCR and RAMP.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of molecular biology. In particular, the invention relates to reverse transcriptase.

[0002]

Problems to be solved by the prior art and the invention

The activity that defines reverse transcriptase (RT) is its ability to synthesize a cDNA strand using an RNA template. This activity has been exploited in a wide variety of technologies that underlie progress in academia and industry. For example, reverse transcriptase is useful for the generation of cDNA molecules and cDNA libraries, sequence-specific probes with various labels and sequencing techniques, and also for some amplification techniques. These amplification techniques include the reverse transcription polymerase chain reaction (RT-PCR: Myers et al., Biochemistry 30: 7661-
7666 (1991); U.S. Pat. Nos. 5,310,652 and 5,407,800);
ic Acid Sequence-Based Amplification) (Kievits et al., J. Viol. Methods 35: 2
73-286 (1991), U.S. Patent Nos. 5,130,238 and 5,409,818), 3SR method (Self-
Sustained Sequence Replication) (Guatelli et al., Proc. Natl. Acad. Sci. (US
A) 87: 1874-1878, 1990) and Rapid Amplification (RAMP: PC
T / US97 / 04170). Other amplification techniques utilize, at least in part, the DNA-dependent DNA polymerase activity of some RTs. Amplification techniques belonging to this category include, for example, the polymerase chain reaction (ie, PCR: Saiki et al., Sciencec).
e 239: 487-491 (1989), U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800.
No. 159), inverse polymerase chain reaction, multiplex polymerase chain reaction, Strand Displacement Amplification (ie, SDA:
Walker et al., Proc. Natl. Acad. Sci. (USA) 89: 392-396 (1992); Walker et al., Nuc.
l. Acids Res. 20 (7): 1691-1696 (1992); U.S. Pat.
No. 455,166) and a multiplex strand displacement amplification method (Multiplex Strand Displa
cement Amplification) (U.S. Patent Nos. 5,422,252 and 5,470,723).

[0003] Reverse transcriptase is found in various retroviruses or RNA oncoviruses.

[0004] Techniques for producing RT from these natural sources require about 30 RTs per virion.
Isolation of the virus particles containing the molecule is performed. RT is released from the virion by dissolution of the virion envelope. The released natural RT can then be purified using conventional techniques. However, the steps required to produce these viruses are labor intensive and costly (1000 infected chicks produce 10-20 g of virus at 25,000-40,000 units / g-virus). A further problem associated with RT production from natural sources is the high rate of spontaneous mutation, which in part limits the host range to, for example, chicks of a particular strain.

[0005] An alternative source of RT is recombinant production, in which case the method of manufacture depends on an understanding of RT expression by various retroviruses. Broadly speaking, retroviruses bind to receptors on susceptible cells and insert retroviral core particles into the cytoplasm of their host. In the living environment of a retrovirus, two major events occur. First, the single-stranded RNA genome is converted to double-stranded DNA by reverse transcriptase.
Second, this DNA copy is inserted into the genome of the host cell (Varmus et al., Mobile DN).
A. (Ed. Berg et al., Washington DC, 1989), pp. 53-108, AM. Soc. Microbol. 972, Brown, Curr. Top. Microbiol. Immunol. 157: 19-48 (1990), Goff, Cancer
Cells 2: 172-178 (1990a), Goff, J. et al. Acquired Immune Defic. Syndr. 3: 817-3
1 (1990b), Boeke et al., Curr. Opin. Cell. Biol. 3: 502-507 (1991)). This insertion is usually an event mediated by virus-encoded integrase activity. After integration, this proviral DNA can be transcribed by host RNA polymerase to make viral RNA, which is then transported back to the cytoplasm to synthesize various viral proteins. After virus assembly occurs in the cytoplasm, the budding virus is released from the cells and a new infection cycle begins (Whitcomb et al., Ann. Rev. Cell Biol. 8: 275-306 (1992)). Defective reverse transcription or integrase function will result in defective viruses that cannot replicate. By way of example, avian myeloblastosis virus (ie, AMV) is a defective virus that requires a helper virus such as myeloblastosis-associated virus (ie, MAV) for its viral growth.

[0006] Integrases ensure stable binding of the virus to the host DNA. Integrase is site-specific for viral DNA but essentially random for the host. This finding indicates that the integrase domain contains a DNA binding region necessary for binding of viral DNA to host DNA, which occurs during the integration process in a host sequence-independent manner.

[0007] Although the integrase domain is encoded by the cognate gene, the mature MMLV-RT (ie, Moloney murine leukemia virus reverse transcriptase, type I
RT) or mature HIV-RT (ie human immunodeficiency virus reverse transcriptase, type II RT)
Not allowed within. However, the integrase domain does not
). The presence of this integrated integrase domain and thermostability are two of the hallmarks of avian RT distinguishing this type of RT from other RTs. Studies of the avian RT integrase domain have revealed that it plays a role in DNA binding and multimerization or multimerization.

[0008] Evidence for DNA binding function is obtained by alignment of the deduced amino acid sequence of retroviral integrase. Three functional domain candidates have been identified. The N-terminal region is characterized by an HHCC (histidine, cysteine) zinc finger-like domain that stabilizes the structure of the integrase (around amino acids 579-629 of SEQ ID NO: 2).
The central region of these integrases contains a catalytic domain with homology to bacterial transposases involved in cleavage and binding of nucleic acid molecules (SEQ ID NO: 2).
Amino acids around 630-807). This region contains acidic amino acid residues that have been proposed to be involved in binding the required metals (Mg ^- or Mn ⁺⁺ ). Khan et al., Nucl. Acids
Res. 19: 851-860 (1991) reports DNA binding activity in this central region. The C-terminal regions of these integrases are not conserved at the sequence level and their function is unknown (around amino acids 808-858 of SEQ ID NO: 2). However, deletion analysis indicates that this region also contains strong sequence-independent DNA binding activity.

[0009] Integrase polypeptides function as multimers or multimers.
N-terminal zinc finger-like domains and C-terminal deletion derivatives are less likely to dimerize. Hickman et al. Biol. Chem. 269: 29279-29287 (1994). Sedimentation analysis suggests that the integrase exists as a mixture of monomers, dimers and tetramers.

[0010] The retroviral genome encodes several genes, namely gag, pol, env and the cellular oncogenes tat, ars / trs, nef, rev, etc. The pol gene encodes a polypeptide having reverse transcriptase (RT) activity. RT enzymes are RNA-dependent DNA
It has several activities, such as polymerase activity, DNA-dependent DNA polymerase activity, ribonuclease (RNase H) activity, integrase activity and endonuclease activity, and possibly protease activity. In the lab, reverse transcriptase is primarily an RNA-dependent DNA that extends an oligonucleotide primer (such as tRNA) annealed to a template RNA or DNA strand to synthesize a DNA strand (cDNA) complementary to the template strand. Used to take advantage of polymerase activity (Co
peland et al. of Virology 36: 115-119 (1980); Berger et al., Biochemistry 22.
: 2365-2372 (1983)).

In general, there are three types of RTs. Moloney mouse leukemia virus (MMLV)
) Is a monomeric RT, and HIV-RT and avian RT are heterodimers. The HIV-RT heterodimer is composed of a 66 kDa β polypeptide and a 51 kDa α polypeptide.
The avian RT heterodimer is composed of a larger 95 kDa β polypeptide and a 63 kDa α polypeptide. The α-polypeptides of HIV-RT and avian RT differ in that the HIV-RTα polypeptide has no RNase H activity. The HIV-RT β polypeptide differs from the avian RT β polypeptide in that the HIV-RTβ polypeptide does not have the avian RTβ polypeptide integrase activity.

[0012] AMV-RT exists in multiple molecular forms in nature, including monomers, homodimers and heterodimers. However, the major active native form is a heterodimer of two structurally related polypeptide chains (ie, a 63 kDa α subunit and a 95 kDa β subunit). These mature subunits contain a 180 kDa precursor protein (
Gag + Pol) is the product of post-translational processing. This 180 kDa protein
Cleaved into 95 kDa β subunit. This β subunit has an additional 63 kDa α
It can be cleaved into subunits and a 32 kDa endonuclease subunit. The α and β subunits have the same N-terminus (Roth et al., J. Biol. Che.
m. 260: 9326-9335 (1985), Gerard et al., DNA 5: 271-279 (1986)).

[0013] In addition to morphological differences (monomeric or heterodimeric), avian RT also has MMLV-R
Different from T. In contrast to MMLV-RT, avian reverse transcriptase has high continuous mobility (
processivity) and yield, as well as biological activity (eg, polynucleotide polymerase activity) over a broader temperature range up to at least 70 ° C.
The ability to conduct polymerization at elevated temperatures is useful for working with RNA templates that have secondary structures. This temperature stability is used in amplification technologies such as NASBA and RAMP. Non-avian RT, including those with RNase H activity, has relatively low continuous mobility and yield. For example, it has been estimated that cDNA synthesis requires about 50 times more MMLV RT than AMV-RT.

Avian retroviruses include avian sarcoma leukemia virus (ASLV), rous sarcoma virus (RSV), avian sarcoma virus (ASV), avian oncovirus (ATV), and avian myeloblastosis virus For example, MAV, avian sarcoma helper virus UR2AVRT, Rouss-associated virus (RAV) and the like. Homology between avian reverse transcriptases is 90-98% at the DNA level, and 95-100% at the amino acid level.

[0015] The nucleotide sequences of many avian viruses are known (Schwartz et al., Ce).
ll 32: 853-869 (1983), GenBank Accession No. M24159, M37980, J02342, J02
See also 021 and J02343), cloning and expression of an active and stable RT in industrially useful amounts has not yet been achieved.

When the DNA sequences of the pol genes of AMV and MAV were compared, about 111 bp at the 3 ′ end of MAV contained AM.
V was found to be replaced by the host DNA sequence. Kan et al., Virology
145: 323-329 (1985). RNA- and DNA-dependent DNA polymerase activity and RNase
The rest of the DNA encoding H activity was intact. This deletion contains the coding region for the integrase domain of the β polypeptide, which renders AMV growth deficient, so that the production of infectious progeny virus requires helper virus MAV. Therefore, the integrase domain is essential for the production of infectious particles. However, both avian retroviruses and their helper viruses encode a reverse transcriptase with RNA- and DNA-dependent polymerase activity and RNase H activity.

AMV reverse transcriptase (ie, AMV-RT) has been characterized and conditions for synthesizing full-length cDNA products have been studied. Berger et al., Biochemistry 22: 2365-2372 (1983)
). However, the length and yield of cDNA generated by AMV-RT has been limited by nucleases or contaminants that form part of AMV-RT. See U.S. Patent No. 5,017,492. In an effort to maximize cDNA length and yield, MMLV-RT has regained attention. MMLV-RT is a reverse transcriptase with relatively high heat sensitivity and relatively low reverse transcriptase activity. Efforts to improve the stability (and thus the activity) of MMLV-RT are due to the C-terminal truncation of MMLV-RT
Has been reported to have had some success. U.S. Patent No. 5,017,492.

See also, US Pat. Nos. 5,244,797, 5,405,776 and 5,668,005. In addition to these modifications, the '492 patent reports that several C-terminal amino acid mutations enhanced MMLV-RT activity (at the cost of reduced continuous mobility). Despite these improvements, MMLV-RT is relatively temperature sensitive and inefficient in catalyzing cDNA synthesis.

Avian RT is structurally different from MMLV-RT. At the primary structure level, avian RT (eg, AMV-RT) has no more than 28% amino acid sequence similarity to MMLV-RT (
Similarity at the polynucleotide level does not exceed 50%). Besides, natural AMV-RT
Is a heterodimer consisting of a 63 kDa α peptide and a 95 kDa β peptide, but MMLV
-RT is an 80 kDa monomer. It is not surprising that these enzymes differ in their thermostability. Thermophilic AMV-RT is active over a wide temperature range reaching at least 70 ° C. Therefore, these bird RTs can often copy RNA templates that can form relatively strong secondary structures.
In contrast, MMLV-RT is a mesophilic enzyme. About 50 times more MMLV-RT is required for cDNA synthesis than AMV-RT. Furthermore, AMV-RT and MMLV-RT differ in other properties, such as continuous mobility, metal cofactor requirement, error rate (ie, the rate of incorporation of the wrong nucleotide), and tRNA primer selectivity. These shortcomings of using MMLV-RT result in increased costs for using MMLV-RT effectively. Therefore, it can be manufactured economically in the art,
Require reverse transcriptase that demonstrates one or more improvements in continuous mobility, stability, solubility, and temperature range to increase the length and yield of polynucleotide products while minimizing reverse transcriptase costs It has been continued.

[0020]

[Means for Solving the Problems]

The present invention provides, for example, a reverse transcriptase polypeptide that has been modified, such as by altering an existing integrase domain or by adding a modified integrase domain per se, having activity, stability and solubility. It relates to the discovery that it features one or more improved properties, including increased and increased ease and freedom in producing those polypeptides. The reverse transcriptase polypeptide of the present invention comprises Moloney murine leukemia virus (type I reverse transcriptase or type I RT),
It can be from any source, including but not limited to HIV (type II RT) and avian retrovirus (type III RT). One aspect of the present invention is internal and / or
Or RNA-dependent D, a feature that defines the reverse transcriptase, with a truncated C-terminus
It is directed to RT polypeptides that still retain NA polymerase activity. Truncated polypeptides can also have DNA-dependent DNA polymerase activity,
It preferably has dependent DNA polymerase activity. Preferred polypeptides of the invention exhibit RNase H activity. Full-length reverse transcriptase with integrated integrase activity (eg, avian retrovirus RT or native type I and type II RT)
In contrast to these truncated polypeptides corresponding to modified type I and type II RTs with an integrase domain, the truncation extends into the integrase domain,
Preferably, the integrase activity is effectively removed from the truncated polypeptide. Such truncated polypeptides exhibit improvements in one or more of the following properties as compared to the corresponding full-length form: RNA-dependent DNA polymerase activity,
Expression level, stability and solubility. These improvements result in cost-effective RTs for use in a wide variety of DNA synthesis, amplification and sequencing techniques.

The present invention also provides a chimeric RT polypeptide obtained by effectively adding a protein domain to the C-terminus of a truncated RT into a non-natural chimeric polypeptide (ie, a polypeptide not found in nature). Also provide. These protein domains provide (and preferably provide some ability to) DNA binding, metal binding, structural stabilization, or multimerization (ie, multimerization) capabilities. The chimeric polypeptides of the invention having these added (or enhanced) capabilities include RNA
Dependent DNA polymerase activity, protein expression level, protein stability and / or
Alternatively, they show an improvement in protein solubility, and chimeric proteins of the invention often show improvements in all four of these properties. Preferred protein domains include a number of histidine residues (ie, His tag) and the N-terminal domain of the integrase region of native RT (giving the ability to bind DNA (preferably provided by a zinc finger (zinc finger) domain)) or C-terminal domain, which provides a multimerization domain.

More specifically, the present invention provides reverse transcriptase polypeptide fragments (ie, portions of a full-length RT polypeptide), modified reverse transcriptase polypeptides and analogs and variants thereof. . The polypeptides of the invention have improved RNA- and DNA-dependence resulting in increased length and yield of the synthesized polynucleotide product
It is preferably a heat-resistant bird RT having DNA polymerase activity. The polypeptides of the present invention typically comprise the C-terminal region of a full-length polypeptide (eg, nucleotides 1719-2571 of SEQ ID NO: 1 (type III), nucleotides 2464-3012 of SEQ ID NO: 40 (type I), And lacks the catalytic activity of the integrase domain conferred by nucleotides 1840-2708 of SEQ ID NO: 42 (type II). It is expected that the absence of the catalytic activity conferred by the integrase domain will result in polypeptides having higher solubility and higher levels of expression. Therefore, such polypeptides are easy to purify economically in industrially useful amounts. In addition to this advantage, the chimeric polypeptides of the present invention can bind to nucleic acids or multimerize (homo- or hetero-multimerization) (and preferably both activities).
It is expected to help. This improvement in RT performance has resulted in improvements in many techniques that depend on RT activity, such as cDNA production and cDNA library preparation and various polynucleotide amplification and sequencing techniques. These amplification techniques include RT-PCR, NASBA, 3SR and RAMP. The improved DNA-dependent DNA polymerase activity of the polypeptide of the present invention is useful for, for example, PCR, inverse polymerase chain reaction, multiplex polymerase chain reaction, SDA, and multiplex SDA. Sequencing techniques include many variations of Sanger dideoxy sequencing.

One aspect of the present invention is an isolated polynucleotide encoding a polypeptide of the present invention. Broadly, the invention encompasses polynucleotides that encode polypeptides having RT activity, typically lacking approximately 200 to 1,122 bp at the 3 'end of the corresponding native RT gene. For example, full-length bird RT
The gene (ie, MAV pol) is 2,692 bp (SEQ ID NO: 1), and the present invention contemplates a polynucleotide derived from MAV having a length of about 1,570 to 2,492 bp. More generally, polynucleotides of the present invention can be obtained by truncation on RT-encoding polynucleotides from any source, including AMV, MAV, RSV, ASLV, ATV, MMLV and HIV. In particular, in the present invention, an isolated polynucleotide encoding a polypeptide having any one of the following sequences, which is a polypeptide having an RNA-dependent DNA polymerase activity, is considered. Amino acid sequence beginning with amino acid 1 of SEQ ID NO: 1 and ending with any one of amino acids 428 to 857, amino acid sequence starting with amino acid 1 of SEQ ID NO: 39 and ending with any one of amino acids 428 to 1,054, SEQ ID NO: 41 An amino acid sequence starting with amino acid 1 and ending with any one of amino acids 548 to 1,198, an amino acid sequence starting with amino acid 1 of SEQ ID NO: 43 and ending with any one of amino acids 428 to 901, and RNA-dependent DNA polymerase activity. A variant, analog or fragment of any of the above polypeptides. Wherein said polypeptide (ie, the polypeptide and its variants, analogs and fragments)
Optionally has an N-terminal methionine. An exemplary polynucleotide is SEQ ID NO:
It has the sequence described in any one of 1, 7, 9, 38, 40 and 42. Preferably, the polynucleotide contains an initiation codon at its 5 'end that designates methionine. Other truncated polynucleotides of the invention have internal deletions, preferably those that remove at least a portion of the integrase domain. For example, the polynucleotide of the present invention comprises the sequence of SEQ ID NO: 40 in which some or all of nucleotides 2464 to 3012 have been deleted, or the polynucleotide of SEQ ID NO: in which some or all of nucleotides 1840 to 2708 have been deleted. The sequence of SEQ ID NO: 1 comprising the sequence of SEQ ID NO: 42 or deleting part or all of nucleotides 1719 to 2571 (for example, nucleotides 1860 to 2310, 1920 to 2310, or 1980 to 2310 of SEQ ID NO: 1) Deletion). Such a polynucleotide encodes a polypeptide that lacks effective integrase activity, in that the polypeptide does not promote incorporation of the detectable polynucleotide.

Other polynucleotides of the invention encode chimeric polypeptides, such polynucleotides encode a polynucleotide encoding a polypeptide having RNA-dependent DNA polymerase activity and a terminal modification of the polypeptide. And thus encodes a chimeric polypeptide. Preferred polynucleotides are those of the polypeptide having RNA-dependent DNA polymerase activity.
Encodes a chimeric polypeptide having one or more amino acids attached to its terminus. Such polynucleotides may contain one of the above coding regions fused (in the same reading frame) at its 3 'end to a region encoding one or more amino acids. For example, the 3 'end of a coding region can be fused to one or more codons specifying a charged amino acid such as histidine, lysine, arginine, aspartic acid or glutamic acid. Alternatively, the 3 ′ end of the coding region is a polypeptide (preferably having 4 to 50 (eg, 6) amino acids,
Preferably, it may be fused to a region encoding a polypeptide containing a domain selected from the group consisting of a DNA binding domain, an RNA binding domain, a metal binding domain, a multimerization domain and a structure stabilizing domain. Examples of such domains include cysteine residues that form disulfide bonds, zinc finger domains,
Acidic amino acid domain, basic amino acid domain, bulky amino acid domain (
For example, W or WH, one letter amino acid notation, PPG domain, GPRP or PRPG (
Ie, reverse GPRP) domain, leucine zipper motif or domain and
Examples include, but are not limited to, NS1 binding sites. Examples of suitable domains include, but are not limited to, the N-terminal domain of the MAV-RT integrase region that provides a DNA binding domain, and the C-terminal domain of the integrase region that provides a multimerization domain. In addition, a polynucleotide encoding a chimeric polypeptide having multiple C-terminal amino acids may encode the same amino acid multiple times. Such polynucleotides have a basic amino acid at the C-terminus (eg,
Histidine). Further, a polynucleotide having a stop codon (for example, TAA, TAG or TGA) at the 3 ′ end of the coding region of the chimera of the present invention is also preferable.

[0025] Representative polynucleotides encoding the chimeric polypeptides include SEQ ID NOs: 11-
19. A sequence selected from the group consisting of the sequences described in any one of 19.

Still another polynucleotide of the present invention is characterized in that one or more amino acids are RNA-dependent.
Encodes a chimeric polypeptide linked to the N-terminus of a polypeptide having DNA polymerase activity. The present invention also contemplates polynucleotides encoding two or more modifications, such as N-terminal peptide addition and C-terminal peptide addition or C-terminal peptide addition in combination with at least a partial internal deletion of integrase.

[0027] The present invention also provides a vector comprising any of the above polynucleotides. Preferred vectors include a polynucleotide operably linked to a promoter.

Another aspect of the present invention is directed to a host cell such as a prokaryotic cell (eg, Escherichia coli) or a eukaryotic cell (eg, insect cell) transformed with the polynucleotide of the present invention.
In a related aspect, the invention is a method of transforming a host cell, comprising introducing the vector of the invention into a host cell, incubating the host cell, and identifying a host cell containing the vector. A method comprising the steps of identifying a transformed host cell.

[0029] Yet another aspect of the invention is a method for producing an isolated reverse transcriptase polypeptide, comprising transforming a host cell with a vector as described above and expressing the host cell with the polypeptide. And then recovering the polypeptide to produce the isolated reverse transcriptase polypeptide of the present invention.

In another aspect, the present invention provides a polypeptide encoded by the above-described polynucleotide. These polypeptides include polypeptide fragments as described above (eg, a βRT fragment that contains some but not all of the C-terminal integrase domain) and chimeric polypeptides and variants and analogs thereof. You. Broadly, the present invention relates to MMLV-RT, HIV-1-RT and avian R
Includes all types of reverse transcriptase fragments and chimeras (and their variants and analogs), including but not limited to the three RTs exemplified by T. Representative chimeric polypeptides contain an N-terminal methionine or a C-terminal peptide that provides a useful function (eg, expression enhancement, nucleic acid binding domain, metal binding domain, structural stabilization domain or multimerization domain). Other chimeric polypeptides of the invention may be, for example, by modification of RT from the following sources of types I-III: ASLV, ATV, MMLV, HIV-1 and HIV-2. A preferred addition to RT is a C-terminal peptide comprising a number of amino acids (such as basic amino acids), a nucleic acid binding domain, a metal binding domain or a multimerization domain. The C-terminal peptide preferably provides more than one functionally significant domain. Also preferred are one or more C-terminal cysteine residues that confer at least the ability to induce homo or hetero multimerization (such as dimerization) of the polypeptide. Typical polypeptides of the invention are relatively soluble and capable of high levels of expression, resulting in relatively high levels of RT activity expected to facilitate economical purification.

Yet another aspect of the present invention is an improvement in a method for copying a target nucleic acid by extending a primer bound to the target nucleic acid, the improvement comprising combining the target nucleic acid and the primer with a polypeptide of the present invention. Contact. The method preferably produces one or more copies of the target nucleic acid, and the polypeptide may be multimeric. All methods for copying a target nucleic acid using a polymerase include, for example, cDNA synthesis, polymerase chain reaction, polymerase chain reaction reverse transcription,
Inverse polymerase chain reaction, multiplex polymerase chain reaction, strand displacement amplification,
The present invention includes, but is not limited to, the multiple strand displacement amplification method, the NASBA method, the 3SR method, the rapid amplification method, and the like.

Another aspect of the invention is directed to an improved method for sequencing a target nucleic acid by extending a primer bound to the target nucleic acid, the improvement comprising:
Contacting a target nucleic acid and a primer with a polypeptide of the invention.

[0033] Yet another aspect of the present invention is a kit for copying a target nucleic acid comprising one or more nucleotides and a polypeptide of the present invention. Preferred polypeptides include SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15. , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 38, SEQ ID NO:
40, SEQ ID NO: 42 and polypeptides encoded by a polynucleotide having a sequence selected from the group consisting of the C-terminal amino acid or a polypeptide derivative thereof encoding the polypeptide at the 3 'terminus.

[0034] Numerous other aspects and advantages of the present invention will become apparent from a review of the accompanying drawings and the following detailed description.

[0035]

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides truncated reverse transcriptase polypeptides (ie, fragments) and analogs and variants thereof. Preferably, these polypeptides exhibit improved levels of RNA-dependent DNA polymerase activity over a wide temperature range, often reaching 70 ° C. or higher. Internal or truncated polypeptides having sequences compatible with increased expression levels are also preferred. Preferred polypeptides of the invention are 45-55
It has a temperature optimum condition of ° C. Also preferred are polypeptides consisting of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43. Some of these polypeptides correspond to truncated C-terminal forms of avian reverse transcriptase, such as, for example, Myelogenetic Avian Virus reverse transcriptase (ie, MAV-RT). Preferred polypeptides of the invention lack effective integrase catalytic activity and are expressed at increased levels to provide a source of soluble, recoverable, active form of the polypeptide. As a typical integrase domain, a type I domain (nucleotide 24 of SEQ ID NO: 40)
64-3012), type II domains (nucleotides 1840-2708 of SEQ ID NO: 42) and II
Includes type I domains (nucleotides 1734 to 2571 of SEQ ID NO: 1), any of which can be modified by internal or terminal deletions or substitutions or chemical modifications. Since integrase and RT function continuously in the viral living environment, RT
And integrase can act as a complex. Thus, while not wishing to be bound by theory, the additional nucleic acid binding and multimerization functions provided by the avian RT integrase domain increase their continuous mobility and their superior performance. It may be bringing. Thus, the non-naturally occurring chimeric polypeptides of the present invention further comprise a C-terminal addition of a multimerization domain (eg, a number of the same or different amino acids). A non-naturally occurring chimeric polypeptide is defined herein as a polypeptide that is not found in nature. Thus, when parts of the chimeras are found in nature, they are not found in the same relationship as they occur in the non-natural chimeric polypeptide. Preferred C-terminal amino acid additions are with basic amino acids such as histidine, lysine and arginine. These preferred C-terminal additions can promote multimerization by, for example, metal chelation. Also, these basic amino acids may confer nucleic acid binding ability to the polypeptide or may enhance the nucleic acid binding ability of the polypeptide. The preferred number of C-terminal amino acid additions is 4 to 50, more preferably 6 amino acids.

As an alternative to a number of basic amino acids, one or more cysteine residues may be added to the C-terminus of the polypeptide. A 4 to 50 amino acid C-terminal peptide having multimerization ability or DNA binding ability (preferably both) is another possible option. The polypeptide may also have a DNA-dependent DNA polymerase activity or an RNase H activity in addition to the RNA-dependent DNA polymerase activity.

The invention also encompasses polypeptide variants having substantially the same amino acid sequence as one of the above-described polypeptides. “Substantially the same” means that the sequence of the polypeptide is identical to one of the sequences disclosed herein by any method known in the art (eg, DNASIS, Hitachi Sofware Engineering America, Ltd., CA) (San Bruno) means that the sequences can be aligned to be at least 90%, preferably 95% or 98% similar over the aligned region. For example, in the present invention, a conservative substitution of aspartic acid with asparagine at any one or more of amino acid positions 450, 505 or 564 of SEQ ID NO: 2 to make a MAV-RT polyprotein lacking RNase H activity It is conceivable to make peptide variants. The same substitution at any one or more of amino acid positions 497, 552 or 603 of SEQ ID NO: 43 results in a variant of the HIV-RT polypeptide that lacks RNase H activity. By altering with conservative substitutions, RNase H of MAV-RT
-Other residues capable of generating variants include amino acid positions 484, 549 and 572 of SEQ ID NO: 2. More generally, the invention encompasses polypeptides having substantially the same amino acid sequence, regardless of whether the differences involve conservative substitutions. For example, the residues listed above may be changed in a non-conservative manner. Also, other residues known to be involved in RNase H activity can be modified by substitution or deletion. These residues include positions 441-578 of SEQ ID NO: 2 (see also AMV-RT and MAV-RT, RSV-RT), SEQ ID NO: 39 (HIV-2-RT)
Positions 427 to 1055 of SEQ ID NO: 41 (MMLV-RT), positions 625 to 911 of SEQ ID NO: 43 (HIV-1-
RT) positions 427 to 902, but are not limited thereto. Also, the present invention
Also encompasses polypeptide analogs. A polypeptide analog is defined herein as 1
Contains known equivalents in place of one or more conventional amino acids, is derivatized by art-recognized methods (eg, glycosylation, PEGylation, phosphorylation, etc.), or Both are defined as polypeptides.

[0038] Another aspect of the present invention is directed to a polynucleotide encoding the above-described polypeptide. Preferred polynucleotides are SEQ ID NO: 1, SEQ ID NO: 6
, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 38, SEQ ID NO: 40 or It consists of the sequence set forth in SEQ ID NO: 42. The invention also contemplates polynucleotides that are substantially the same as the polynucleotides having one of the sequences listed above. "Substantially the same" with respect to a polynucleotide means a polynucleotide having a sequence that is at least 90% homologous to one of the above polynucleotides.

In addition to polynucleotides, the present invention relates to at least one of these polynucleotides
And a vector containing the two. Further, these vectors may function in prokaryotic cells, eukaryotic cells, or both types of cells. A preferred vector is pBacPak9
(Clontech Inc., Palo Alto, CA). The present invention also provides prokaryotic and eukaryotic host cells transformed with the above-described polynucleotides. Preferred host cells are Sf9 insect cells transformed with a baculovirus-based recombinant molecule of the present invention. Other insect cell lines such as SF21 HighFive may be used.

In another aspect, the present invention provides a method for producing an RT of the present invention using the present polynucleotide. Specifically, the polynucleotide is transformed into a prokaryotic or eukaryotic host cell under conditions that allow expression of the encoded RT polypeptide,
After an incubation period, the polypeptide is isolated.

As a further aspect of the present invention, there is provided a method of using the present RT polypeptide. These methods are based on the speed and yield obtained by using highly active and thermostable RT polypeptides to copy target nucleic acids (eg, cDNA synthesis, cDNA library construction), amplify or sequence target nucleic acids. It realizes the advantage of. Suitable amplification methods include, but are not limited to, PCR, RT-PCR, reverse PCR, multiplex PCR, SDA, multiplex SDA, NASBA, 3SR and RAMP. As a suitable sequencing method,
The original enzyme sequencing technique disclosed by Sanger and co-workers or numerous variations of this technique developed since its disclosure.

[0042]

【Example】

Various aspects of the invention are described in the following examples. In the first embodiment, the full-length MAV-
The cloning of the coding region encoding RT will be described. Example 2 describes the sequencing of the full-length MAV pol gene encoding reverse transcriptase. Example 3 discloses the cloning of selected polynucleotides of the invention. Example 4 details large-scale purification of expressed recombinant RT. Example 5 describes SDS-PAGE and Western blot analysis of expressed proteins. Example 6
Now disclose an assay for RNA-dependent DNA polymerase activity. Example 7
Here, an assay characterizing natural reverse transcriptase (nRT) and recombinant reverse transcriptase (rRT) for optimal conditions with respect to temperature, pH, MgCl ₂ and other divalent cations is illustrated.
Example 8 discloses the use of RT in a method for copying and / or amplifying a target nucleic acid. In Example 9, DNA dependent D used to characterize nRT and rRT
The NA polymerase assay is described. Example 10 reports a comparison of the RNase H activity of nRT and rRT. Example 11 describes further cloning and expression of a polynucleotide of the present invention.

Example 1 The pol gene of MAV encoding the full-length RT precursor polypeptide was converted to pMAV (POL of MAV, gag
And a pBR322 derivative containing the partial env gene). The data obtained from the partial restriction map of the pMAV insert is shown in Table 1. Based on the data in the map, the pol coding region was cut out along with some 5 'and 3' non-coding sequences and ligated to some prokaryotes and eukaryotes, as described below. Some recombinants were obtained from these vectors. RT expression was analyzed using an anti-RT monoclonal antibody (see Example 5).

[0044]

[Table 1]

All non-coding 5 ′ (ie, upstream) nucleotides were removed to increase RT expression. In addition, the open reading frame of the natural RT gene is “ACT” (Th
Starting at r), this is not a frequently used start codon in prokaryotes.
The most frequently used codon is "ATG" (Met). "ATG" can serve as an initiation codon for the full expression of RT in both prokaryotes and eukaryotes. Therefore, "ATD" was added 5 'to the natural "ACT" start codon to allow for efficient expression of the protein in prokaryotes and eukaryotes (ATG ACT GTT GCG CTA CAT
CTG GCT ATT CCG CTC AAA TGG AAG CCA AAC CAC ACG CCT GTG TGG ATT TTC CAG
TGG CCC etc .: Compare the sequences provided in SEQ ID NOs: 2 and 3).

The pH Construction of prokaryotic recombinant vector contains the Amp ^r for powerful and closely regulated lambda P _R promoter, temperature-sensitive λcI repressor, the E. coli origin of replication and selection. This vector is
No special E. coli strain was required for regulation of expression as it encodes a temperature-sensitive repressor.

As characterized by the restriction map data in Table 1, the entire coding region of MAV-RT (EcoRI-XhoI obtained by restriction digestion or PCR with appropriate primer pairs)
Fragment) was inserted into the multiple cloning site (MCS) at pH. In short, vectors are
Restricted with EcoI and SalI. The 1: 1 ratio of insert to vector was determined using ligation buffer (100 mM Tris-HCl, pH 7.6, 10 mM MgCl ₂ , 20 mM) using standard protocols.
(M DTT) and T4 DNA ligase in the presence of 1 mM ATP. Sambrook et al., I
n Molecular Cloning: A Laboratory Manual, Cold Spring Harbot Laboratory
Press, Cold Spring Harbot NY (24th edition, 1989). The ligation mixture was incubated at 16 ° C for 2-4 hours. The ligation mixture was transformed into electro-competent E. coli cells in a 1 mm cuvette using a BioRad electroporator at 1.8 KeV and 200 ohms. Transformed cells were plated on LB-ampicillin plates and single colonies were picked for overnight growth and mini-prep analysis. The recombinants were then confirmed by sequence analysis. Subsequently, the 5 'uncloned region of the selected recombinant was removed, if appropriate, by site-directed mutagenesis. The pH vector containing the full length RT gene was named pHSEM1, and the vector lacking the 5 'non-cloning region was called pHSEMUE33 (ie, pHRT). The ET protein was expressed, analyzed by SDS-PAGE, and subjected to Rt assays as described in Examples 5 and 6. Other prokaryotic vectors have also been used successfully (eg, pET21d and pTZ18U, which have T7 and lacZ promoters, respectively).

Construction of Eukaryotic Recombinant Transfer Vector Using a transfer vector, a baculovirus expression system consisting of a wild-type virus AcMNPV (Autographa californica nucleopolyhedrovirus) or a derivative of ACMNPV (ie, BacPak6 (CLontech Inc.)). Thus, a recombinant transfer vector containing the RT gene was obtained.

The AcMNPV genome is a 134 kb double-stranded circular DNA. The size of the viral marker makes it difficult to manipulate the viral genome itself. Therefore, the transfer vector pB
Using acPak9, pMBacRT, pBacMIBA, pBacMIKA, pBacMIBAhis and pBacMIKAhi
s (see below), a recombinant molecule according to the invention was obtained. Using these recombinant molecules, which contain the exogenous and typically exogenous RT coding regions, sequences were introduced into the viral genome for expression and propagation. Vector pBacPak9
Has a strong polyhedron promoter that is introduced into insect cells late in the viral replication cycle. Thus, exogenous genes, including lethal genes, expressed by the promoters described below are not toxic to growing cells. The polyhedron gene is not required for the maintenance of the virus, and therefore exogenous genes (eg,
MAV-RT pol gene).

The 2.81 kb PstI fragment from pHSEMI containing the full length RT gene was ligated to the PstI of the MCS of pBacPak9.
The recombinant was called pBpHPC3.4 (ie, pBacRT). The gene was inserted and confirmed by miniprep analysis and sequencing. The 5 'non-coding region (see SEQ ID NO: 1) was removed by site-directed mutagenesis as described by Sambrook et al. (1989). PB
It was called pHPCM10,11,17 (ie, pMBacRT). In pMBacRT, the RT gene is
It is flanked in close proximity by the viral DNA sequence of BacPak6 (a derivative of AcMNPV). pMBa
When cRT is introduced into insect cells together with BacPak6 DNA, the plasmid is recombined with BacPak6 DNA and the recombinant (infectious progeny virus containing the RT gene (M1-5 and
M1-6, collectively M-5,6)) were obtained.

In general, when SF9 tissue culture cells are infected with a recombinant virus, virus particles enter the cells and the viral DNA is uncoated in the nucleus. Viral DNA replication is
Occurs approximately 6 to 24 hours after infection. During the late phase of viral infection, approximately 48-72 hours after viral infection, all transcription is turned off except for the polyhedron (which is transcribed at very high levels) and the p10 promoter. Thus, the RT gene under the control of the polyhedron promoter in the recombinant virus was expressed at very high levels late in the infection cycle. This recombinant AcMNPV grew only in bud morphology.

Example 2 The sequence of the NAV-RT pol gene was confirmed using a primer walking sequencing strategy implementing the Sanger enzymatic sequencing technique. Sambrook et al. (1989). The sequencing template was the insert of pHSEM1. Primers were designed to be uniform or complementary to the ends of previously determined sequences. These primers were then used to gradually expand the identification of the pol gene sequence until the entire coding region was determined.

The polynucleotide sequence of the MAV-RT gene and flanking sequence is SEQ ID NO: 1
It describes in. The amino acid sequence encoded thereby is set forth in SEQ ID NO: 2. Of the 3,155 bp shown in SEQ ID NO: 1, 1,498 bp is the beta fragment of MAV-RT
(Nucleotides 253-2751 of SEQ ID NO: 1); the alpha fragment of MAV-RT is encoded by nucleotides 253-1990 of SEQ ID NO: 1. These coding regions include amino acids 1-895 of SEQ ID NO: 2 (full length RT; see also SEQ ID NO: 3), amino acids 1-833 of SEQ ID NO: 2 (β-like polypeptide; reference),
And amino acids 1-597 of SEQ ID NO: 2 (α-like polypeptide;
(See residues 1-578). The β-like polypeptide is a fragment of a native MAV-RT β polypeptide. The α-like polypeptide is larger than the native MAV-RT α polypeptide, smaller than the native MAV-RT β polypeptide, and the native α polypeptide sequence is extended from the N-terminus of the α-like polypeptide. Briefly, α-like and β-like polypeptides are referred to as α and β polypeptides, respectively.

Example 3 As described in Example 1, the plasmids pHSEM1 and pBacRT were each 2.95.
kb and 2.81 kb inserts. These fragments contained the entire reverse transcriptase gene with 5 'and 3' non-coding regions. The 5 'of each construct was then determined by site-directed mutagenesis, a technique well known in the art.
Removed non-coding. In particular, primer FSDRT (5'-TGTACTAAGGAGGTGTTCA
PHRT (pHSEMUE33) was obtained using TGACTGTTGCGCTACAT-3 '; see SEQ ID NO: 20) with pHSEM1 as a template. Primer RSDBAC2 (5'-GCCAGATGTAGCGCAACAGTCATA
Using TTTATAGGTTTTTTTATTAC-3 ′; SEQ ID NO: 21) with pBacRT as a template, p
MBAcRT (pBPHPC3M10, pBPHPC3M11, pBPHPC3M17, or, respectively, pMBac10, pMBac11
And pMBac17).

In constructing deletion derivatives lacking the 3 ′ end of the MAV-RT coding region to various extents, the full length RT coding region was used as a starting material. Full length gene (MI-5.6
(See below), a 3 '(terminal) deletion extending to the KpnI site (MIKA) increased RT expression levels as shown by SDS-PAGE. For the full-length gene (MI-5,6), deletion of the region extending from the BglII site to the 3 ′ end (MIBA) resulted in RT expression levels, activity and activity as shown by SDS-PAGE and activity assays (see below). Increased solubility. In contrast to the alpha fragment of MAV-RT, the beta fragment has at the C-terminus an additional 254 amino acids that provide integrase activity. This region in the polypeptide contributes to the insolubility of the polypeptide, and the (+) integrase form of RT (eg, MIKA gene product, see below) compared to the (−) integrase form (eg, MIBA gene product) ) Reduces its recovery from cell extracts as indicated by the relative insolubility of This region was deleted in MIBA (fragment equivalent) because the integrase domain is only required for the retroviral life cycle and not for RNA- or DNA-dependent DNA polymerase activity. MIBA alpha fragment (
Amino acids 1-578 of SEQ ID NO: 2 are naturally occurring α fragments of MAV-RT (SEQ ID NO: 2).
2 amino acids 1-573). Without wishing to be bound by theory, it was predicted that this deletion would result in increased solubility of the protein and thus recovery.

Using the full-length RT recombinant, additional clones were constructed to express a polypeptide with a C-terminal deletion, increase the level of expression, and increase RT activity (RNA-dependent DNA polymerase activity). Stabilized. BglII (spanning nucleotides 1,986-1,991 of SEQ ID NO: 1) and KnpI (spanning nucleotides 2,745-2 of SEQ ID NO: 1)
750) to eliminate the 3 'end of the coding region of the RT gene (see Table 1). The 3 'deletion derivatives encoding the RT polymerase fragment with the C-terminal deletion were pMBacRT and pHRT (BglI in the MAV-RT coding region, respectively).
I and KpnI sites; PstI site in vector) obtained by BglII-PstI or KpnI-PstI restriction. Recombinant molecules containing a BglII-PstI 3'-terminal deletion were cloned into pBacMIBA
And the recombinant molecule containing the KpnI-PstI deletion named pBac (pH33ΔBP6) and pBac
Named MIKA and pHKRT (pH33ΔKP5). Deletion derivatives pBacMIBA and pBacMIKA
Had about 1.2 and 0.4 kb deletions from the 3 'end of the full length gene, respectively (SEQ ID NO:
1). Using the fragment ligated at its 3 ′ end bounded by the BglII site (see SEQ ID NO: 6), the alpha fragment equivalent of RT is expressed and the fragment bounded by the KpnI site (SEQ ID NO: 8) is Used to express a beta fragment equivalent of RT (a β fragment equivalent of MIKA contained amino acids 1-832 of SEQ ID NO: 2; native MAV-RTβ contained amino acids 1-858 of SEQ ID NO: 2) I let it.

Minipreps and sequencing analyzes were performed to confirm the identity of the recombinant clones described above. Recombinant viruses obtained from co-transfection with the virus BacPal6 and the transfer vector pBacMIBA or pBacMIKA are MIBA and
Called MIKA.

While not intending to be bound by recombinant theory encoding the 3 ′ terminal amino acid tag, constructs that lack the integrase domain of RT, such as MIBA and pBacMIBA, are DNA that is attributable to the integrase domain.
It was not expected to possess binding, structural stabilization, and polymorphic functions. To reintroduce these functions without the deleterious impact on solubility and host cell viability associated with the native integrase domain, a codon specifying the amino acid (His) was added 3 'to the modified RT coding region. . The basic nature of the added amino acids would be responsible for the increased binding to negatively charged nucleic acids, which would enhance the stability of the polypeptide. This time, the increased binding would have been responsible for the increased activity found with his-tagged RT on their untagged counterparts. In addition, his tag, perhaps, Ni ^- such through the his- mediated chelation of metal ions, would have contributed to the trend of his--tagged RT of the present invention to form the polymer. Superose 12HR10 / 30 (1-300 kDa spanning range; Pharmacia-
His-tagged RT (MIBAhis), as measured using native PAGE and molecular sieving chromatography on Upjohn), is a homopolymer form (molecular weight greater than 200 kDa).
). Thus, the present invention includes RT polypeptides that lack an effective integrase domain, but have the ability to bind and / or polymerize DNA.
These additional functions include known DNA binding domains, zinc-finger or zinc-finger-like domains, polymerization domains, acidic amino acids, basic amino acids,
Alternatively, a structure such as one or more cysteines can be provided, preferably by adding to the C-terminus of the modified RT. Such modified RT can ultimately be obtained from avian or non-avian sources.

The addition of a His-tag to the C-terminus of the RT polypeptide was achieved by recombinant expression of the coding region that fused the RT coding region to the His codon. In particular,
The fusion can be performed by adding an oligonucleotide containing six histidine codons to the 3 'end of the RT gene using ligase, as in the construction of pBacMIKAhis, or as in the construction of pBacMIBAhis, by adding six histidines. Constructed by PCR amplification with codon specific oligonucleotides.

The construction of pBacMIKAhis was accomplished with oligonucleotides FNhis (SEQ ID NO: 33) and RNhis (SEQ ID NO: 34), each containing an internal histidine codon and a compatible NotI restriction site at each end. Following their normal synthesis, the oligonucleotides were annealed and ligated to the 3 'end of RT in pBacMIKA cut with NotI. For construction of pBacMIBAhis or pBacMIKAhis using PCR, primers FRT (SEQ ID NO: 22) and either MIBARSDhis (SEQ ID NO: 23) or MIKARSD (SEQ ID NO: 24) were used with pHSEM1 as a template. The blunt-ended and phosphorylated PCR products containing the 3 'deletion and the histidine tag-coding region were purified from Sm in the MCS of pBacPak9.
Inserted at the aI site. The his-tag derivatives of the transfer vector are pBacMIBAhis and pBa
The viruses obtained by co-transfection of Sf9 cells with the transfer vector and BacPal6 were called MIBAhis ((-) integrase) and MIKAhis ((+) integrase), respectively. Incorporation of His codons led to increased activity of the encoded polypeptide in eukaryotes as measured by SDS-polyacrylamide gel electrophoresis analysis and RT assay (see below). As shown below, his-tagging increased stability (perhaps by providing a DNA binding site), activity, polymerization ability, and ease of purification of RT, such as MIBAhis.

The 5 ′ end of the MAV pol gene was also modified. Beyond the deletion of the 5 'non-coding sequence of pol (see pHRT and pMBac10 above), the widely recognized Met start codon ("ATG") is replaced by the native start codon of the MAV pol gene (SEQ ID NO: 1). At nucleotide 253
-255 just upstream of the Thr codon "ACT").

In general, the cloning strategy described above reflected efforts to eliminate the integrase domain of avian RT, thereby avoiding the insolubility and lethal problems associated with its protein domain. By terminating the coding region at the KpnI site (Table 1), a 192 bp deletion from the 3 'end of the full length MAV-RT gene (SEQ ID NO: 1) resulted in the "MIKA" clone series. These clones encoded a β polypeptide smaller than the naturally occurring β polypeptide. These clones exhibited enhanced RT expression, and the expressed polypeptide exhibited enhanced activity levels (see below, M1-5,6 [full length] compared to MIKA β [β polypeptide]). )
. Using the convenient BglII site, a larger deletion extending from the 3 ′ end of the full-length MAV-RT gene was constructed to generate a series of MIBA clones. These clones encoded the alpha subunit of RT larger than the naturally occurring alpha polypeptide. MIBA
Clones exhibited increased expression and activity compared to the expression and activity of full-length MAV-RT; furthermore, MIBA was more soluble than naturally occurring MAV-RT.

The present invention also provides certain advantages of integrase, such as DNA binding and polymerization, to avian RT without reintroducing the deleterious (ie, insoluble and lethal) features of the avian RT integrase domain. Polynucleotides and polypeptides derived from the recognition that they can be reintroduced are contemplated. One approach is to attach RT integrase domains or non-RT integrase domains known in the art to the (-) integrase polypeptide, or add the coding region of these domains to these (-) integrase polypeptides. Attaching to a polynucleotide encoding a Rase polypeptide. Another approach is to attach an amino acid tag to the (-) integrase RT polypeptide (or to attach a corresponding codon to the (-) integrase polynucleotide), as disclosed herein. Preferred tags are basic amino acid tags such as the His tag. As disclosed below, a His at the C-terminus of the α polypeptide equivalent
Tags were attached (MIBAhis). This clone exhibited relatively high levels of expression, activity and solubility. Thus, the present invention provides an avian RT that is improved in terms of the level of expression and activity, and in terms of solubility and ease of purification, while retaining the processability and thermostability characteristics of the avian RT.

Thus, the present invention contemplates the construction of similar polynucleotides and recombinant molecules encoding non-natural length RT polypeptides from other sources, such as MMLV, HIV, RSV, ASLV, ATV and others. . Further extended to polynucleotides encoding these RTs of modified length or full length RT, provided that the polynucleotides additionally encode at their 3 'end a polymerization or nucleic acid binding domain, preferably both domains. Shall be. Examples of polynucleotides encoding non-natural length non-avian RTs include the following SEQ ID NO: 39, position 1-765 (HIV-2 RT sequence), SEQ ID NO: 41, position 1-800 (MMLV-RT sequence) And a polynucleotide encoding an RT portion or fragment having the amino acid sequence set forth in any one of positions 1-625 (HIV-1 RT sequence) of SEQ ID NO: 43. These polynucleotide sequences contain the MAV-derived MIBA
It is expected to have some relevance to the sequence of the polynucleotide encoding the polypeptide, and to function similarly to the polynucleotide encoding MIBA. Of course, with the 3 'terminal sequence encoding the polymerization and / or nucleic acid binding domain, encodes the full length β polypeptide of HIV-2 (SEQ ID NO: 38), or an equivalent polypeptide from MMLV or HIV-1 (each, Polynucleotides encoding SEQ ID NO: 40 or SEQ ID NO: 42) are also contemplated by the present invention.

With respect to polypeptides, the invention includes polypeptides encoded by said polynucleotides, as well as polypeptides having a C-terminal polymerization and / or nucleic acid binding domain that have been added by means other than expression. For example, RT polypeptides having a Cys or His residue attached to the C-terminus by chemical considerations are within the scope of the invention. In addition, effective elimination of the integrase domain, as found in avian RT, alters the appropriate coding region by inserting, deleting, or replacing (transition and / or transversion) one or more nucleotides. You can't do that. Thus,
The invention includes RT polypeptides that are the same length as the naturally occurring RT. these
An RT polypeptide can have the same amino acid sequence as a naturally occurring RT. However, the RTs of the present invention shall have a polymerization and / or nucleic acid binding domain at their C-terminal. Alternatively, an RT of the same length as a naturally occurring RT can have a different sequence than the native RT, thereby effectively eliminating integrase and gender. Also, the RT of the present invention may be shorter or longer than the naturally occurring RT polypeptide. The shorter RT polypeptides of the present invention eliminate some or all of the C-terminus of naturally occurring RT containing an integrase domain in the case of avian RT. RTs of the invention that are longer than the naturally-occurring RT polypeptide contain the sequence of the naturally-occurring RT, and in addition, contain sequences of flanking peptide regions.
In addition, these polypeptides of non-natural length can have a polymerization and / or nucleic acid binding domain added to their C-terminus.

Example 4 The RT construct described in Example 3 was transformed into prokaryotic and eukaryotic host cells and analyzed for expression of the RT polypeptide. Prokaryotic host cells E. coli DH5αF 'were transformed with pHRT, pHBRT or pHKRT using techniques standard in the art. Cells subjected to the transformation protocol were transferred to 50 μg / cell for selection of transformed host cells.
LB plate containing ml ampicillin (10 g tryptone, 1 liter total volume,
Plated on 5 g yeast extract, 5 g NaCl, 1 ml 1N NaOH, 1.5 g agar, ddH ₂ O). Single colonies were picked, grown in small cultures (ie, 5 ml), episomal DNA was rapidly isolated from aliquots of cells, and purified DNA was analyzed for the presence of recombinant molecules of the expected size. Dideoxynucleotide-based sequencing of these DNAs confirmed that the first ATG (ie, the start codon) was in frame with the rest of the RT coding region.

Another aliquot of those small cultures containing cells transformed with pHRT, pHBRT, or pHKRT was used to inoculate a flask containing 10 ml of LB-ampicillin and reach 30 at an OD ₆₀₀ of 0.6. Grow at ℃. Then, the flask containing these cells were shifted to 42 ° C., de suppress .lambda.P _R promoter was expressed recombinant protein. After 1 hour at 42 ° C., cells were pelleted and analyzed for protein expression by SDS-PAGE, Western blot analysis, and RT activity assay as described below.

In general, about 10% of the expressed protein was recovered in soluble form and 90% of the expressed protein, as revealed by pelleting cells lysed at 12,000 × g for 5-15 minutes. % Was found in the inclusion bodies. Also, the total length or C
-PTZ18U and pET21, containing similar insert fragments encoding truncated MAV-RT
RT activity was found when both full length and deleted derivatives of the MAV pol coding region from other recombinant vectors were expressed, such as d.

Eukaryotic host cells suitable for use in practicing the present invention are Sf9 insect cells.

Several polynucleotides were separately introduced into Sf9 cells using the baculovirus expression system. O'Reilly et al., In Baculovirus Expression Vectors: A Labor
atory Manual, Oxford University Press (1994). The polynucleotides (ie, pMBacRT, pBacMIBA, pBacMIBAhis, pBacMIKA and pBacMIKAhis) are
Purified by standard alkaline lysis methods as described by ok et al. (1989). The DNA was then transferred to a CHROMA SPIN + Te-400 column (Clonte
ch Laboratories, Inc., Palo Alto, CA) at 500 xg for 7 minutes.
(NH-SH centrifuge tube from IEC. Inc.).

[0071] Sf9 insect host cells were prepared for transformation using established techniques.
Using a hemocytometer, Sf9 cells from exponentially growing cell cultures were first counted and 10% fetal bovine serum (FBS) and antibiotics (50 units / ml nystatin, 50 units / ml penicillin, And 50μg / ml of streptomycin)
TNM-FM insect cell culture medium (product number T-1032; Sigma Chemical Co., St. Louise, MO)
Diluted to 5 × 10 ⁶ cells / ml. Subsequently, 1.5 ml of this medium was added to each well of several 12-well tissue culture dishes. Cells were allowed to attach to the plate for 1 hour.

Next, the medium covering the cells was removed and 2 ml of TNM-FH medium without serum was added. The serum-free medium was agitated on the cells and the medium was removed again. This process was repeated one more time to remove any trace fetal bovine serum (ie, FBS) and antibiotics. The cells were then incubated for 30 minutes in TNM-FH medium while preparing the transfection mixture.

50 μl of the transfection mixture contains 500 ng of DNA, 500 ng of Bsu36I-digested Bac
It contained Pak6 viral DNA and ddH2O. This mixture is gently mixed with 50 μl of transfection reagent (Clontech, Inc.) and incubated for 15 minutes at room temperature to allow the transfection reagent to complex with the DNA as recommended by the transfection reagent supplier. The body was formed.

The medium covering the Sf9 cells was removed and 300-500 μl of TNM-FH medium was added to each well. While the plate is gently rotated in this medium, the transfection reagent-DN
The A mixture was added dropwise. The cells were then incubated for 5 minutes at 27 ° C., after which 2 ml of TNM-FH medium containing 10% FBS and the antibiotic was added. DNA-
Cell contact was continued at 27 ° C. for 60-72 hours. The media from these plates was then collected and used as the primary virus stock.

Subsequently, King et al., In The Baculovirus Expression System: A Laboratory
Primary virus stocks were subjected to plaque purification to obtain clone stocks by standard methods, as described in the Guide (Ed. Chapman and Hall, N.Y., 1992). The clone stock was amplified using a 1: 1 ratio of virus to insect cells to yield large amounts of recombinant virus.

The virus from the clonal stock was used to infect insect cells and finally analyzed for RT expression in a eukaryotic environment. Infection was set up using a ratio of 5 viruses per Sf9 cell based on the titers obtained from the plaque assay. Sixty hours later, media and cells were collected. Pellet cells and add cell lysis buffer (10 mM Tris H
Cl, pH 8.0, 50 mM NaCl, 5% glycerol, 0.5% Triton X-100, and protease inhibitor (50 μg / ml benzamidine HCl, 0.1 mM 4- (2-aminoethyl) -benzenesulfonyl fluoride, and 1 μg / ml pepstatin A)) and dissolved by sonication. Subsequently, these samples were subjected to SDS-PAGE, Western blot and RT activity assay.

For large-scale expression experiments, Sf9 cells were first grown in T25 tissue culture flasks under the conditions described above. Mildly detach Sf9 cells attached to T25 tissue culture flask, Ki
ng et al., 1992, adapted to suspension culture. These suspension cultures were grown in spinner flasks to a volume of 1-3 liters. When insect cells reached a density of 1 × 10 ⁶ cells / ml, they were infected with a concentrated stock of recombinant virus at a ratio of 5: 1 virus per insect cell. These cells were infected using a variation of the standard protocol. Large volumes of the amplified virus stocks (MIBA, MIKA, MIBAhis and MIKAhis, or M1-5,6) were concentrated using 1/2 volume of 40% PEG8000 and 1/6 volume of 5M NaCl. Precipitated virus was collected at 12,000 xg for 30 minutes using a Sorvall RC5C centrifuge tube (Supont, Newtown, CT). The pelleted virus was diluted with 1 × PBS (10 mM K ・ PO ₄ ,
pH 7.5 and 150 mM NaCl) and stored at -20 ° C. Before infection 0.2
Filtered through a μ filter.

After 48 hours of infection, 1 ml aliquots of insect cells were collected for RT assay to monitor RT expression levels. Cells were harvested at the peak of RT, judging from previous studies (generally 60 hours post-infection). Cells were pelleted at 5,000 xg for 30 minutes and stored at -80 ° C.

The polypeptide expressed in insect cells was also characterized by SDS-PAGE and Western blot analysis. Anti-RT monoclonal antibody 1D8, 2E10, 6F1, 4C
The results of the Western blot assay using a mixture of 4, 9H10 and 9C2 are shown in FIG. 1 (lane 1: 123 kDa, 90 kDa, 64 kFa, 50 kDa, and 38 kDa prestained molecular weight markers; lane 2: native AMV-RT (nRT) (Lane 2); lane 3: MIBAhis, and lane 4: MIKAhis). Further experiments on the antigenic properties of MIBAhis and native RT revealed that monoclonal antibody 6F1 recognized native RT but did not recognize MIBAhis polypeptide. Thus, at least one epitope found in native RT is not found in MIBAhis, indicating structural differences between proteins.

The results further indicate that both MIBA and MIKA expressed 10 times more RT than M1-5,6, which encodes full-length RT. When the cell pellet was assayed for RNA-dependent DNA polymerase activity, MIBA was found to be 10 per liter of insect cell culture.
MIKA and M1-5,6 were expressed at 1,000 units per liter of insect cell culture. When analyzed by Western blot, MIKA
In addition, MIBA was expressed, but MIKA recovered from the cell pellet was 10 times less than MIBA. Most of the expressed MIKA remained insoluble in the pellet. As revealed by Western blotting, the corresponding his-tagged proteins (MIBAhis and MIKAhis) were expressed at similar levels as their MIBA and MIKA counterparts, but the activity of his-tagged proteins was higher. Was. MIKAhis was expressed at 2,000 units per liter of insect cell culture and MIBAhis was expressed at 200,000-400,000 units per liter of insect cell culture.

The baculovirus system is preferred for expression of RT and fragments thereof. Relative comparisons of RT expression in prokaryotes and eukaryotes, as measured by reverse transcriptase assays of purified recombinant and crude proteins, show that His-tagged polypeptides from eukaryotic insect cells are the most It was found to be active and stable, while the untagged polypeptide expressed in prokaryotic cells was the least active and not stable.

The recombinantly expressed polypeptides of the invention were purified using conventional protocols with metal-affinity chromatography included for the isolation of His-tagged polypeptides. A recombinant molecule encoding RT or a fragment thereof (ie, M1-5,6,
Centrifuge the host cells containing MIBA, MIKA, MIBAhis and MIKAhis) and pellet the cell pellet in 20 ml lysis buffer (20 mM Tris HCl, pH 8.0, 150 mM NaCl, 0.5% Triton X, and 5% glycerol). Solubilized. The resuspended cells were sonicated on ice at 50% power with five 30 second bursts, cooling for 30 seconds between each round of sonication. The sonicated cells were then incubated at 4 ° C. for 1 hour,
Cell lysis was completed by stirring at low speed with a magnetic stirrer. Dissolve the sample
Centrifuged at 12,000 × g for 30 minutes. The pellet was discarded and the supernatant was subjected to column chromatography.

RT lacking the his tag was purified according to standard protocols including removal of cellular contaminants by centrifugation and subjecting the supernatant to chromatographic purification known in the art. The soluble extract containing his-tagged RT is mixed with a commercially available Ni ⁺⁺ affinity column (Ni-NTA resin from Qiagen, Inc., Chatsworth, CA), thereby reducing the his tag to a metal Used for known purposes to facilitate purification via affinity chromatography. The extract and affinity resin were rocked gently in ice in a 50 ml plastic test tube for 1 hour. Next, the resin is packed in a column,
Two column volumes of wash buffer (20 mM Tris HCl, pH 8.0, 250 mM NaCl, 0.5% Triton X-100, and 5% glycerol) and two column volumes of Buffer A (20 mM Tris-
HCl, pH 8.0, 250 mM NaCl, 0.5% Triton X-100, 5% glycerol and 50 mM imidazole). (Of course, as would be understood in the art, the extract could be applied to a preformed affinity column and could have been purified using conventional column chromatography). From buffer A to buffer B (20 mM Tris-HCl, pH 8.0,
(250mM NaCl, 0.5% Triton-100, 5% glycerol and 250mM imidazole)
The protein bound to the column was eluted by setting a linear gradient to.

As shown in FIG. 2, fractions from a nickel affinity column having RT activity were analyzed by SDS-PAGE to determine protein purity. FIG. 2 shows an electrophoresis image of an 8% SDS-PAGE gel stained with Coomassie blue. The lanes of the gel shown in FIG. 2 contain: 94 kDa, 64 kDa, 43 kDa, 30 kDa and 20 kDa (lane 1) molecular weight markers and aliquots of the fractions obtained from the nickel affinity column (lanes 2 to 4). Fractions greater than 95% pure are pooled and stored in buffer (200 mM KPi, pH 7.2, 5 mM DTT, 0.2% Triton X-100 and 50% glycerol).
Dialyzed against. In addition, as will be understood in the art, conventional purification steps can be incorporated into the protocol to achieve greater purity.

The protein concentration was determined by the Bradford protein assay (BioRad Laboratories, Inc.
, Hercules, CA). Generally, the inactivity of rRT (MIBAhis) is almost
It was calculated to be 30,000-100,000 units / mg, similar to the inactivity of nRT (30-100,000 units / mg).

Example 5 Purified rRT prepared from a culture expressing MIBAhis in a 400,000 unit / liter culture (well above commercially viable production limits) was purified by electrophoretic fractionation using 10% SDS-PAgE. Judged to be greater than 95% purity. The apparent molecular weight of the monomer is 60 kDa, which is more comparable to the calculated molecular weight of approximately 59.5 kDa. Recombinant proteins were analyzed on a 12.5% polyacrylamide non-denaturing gel for the presence of monomers and polymers (eg, dimers) using the Pharmacia Phasr System. Protein samples were prepared in one of two ways. One aliquot was added to the processing buffer (0.125 mM Tris-HCl, pH 6.5, 4% SDS, 2
It was completely denatured by heating in 0% glycerol, 10% β-mercaptoethanol) at 100 ° C. for 3 minutes. Another aliquot was partially denatured at 70 ° C. in processing buffer without 2-mercaptoethanol. Under completely denaturing conditions, it was observed to migrate at approximately 66 kDa (BSA marker), with the partially denatured sample having an additional band in the range of 60-200 kDa, indicating that rRT formed the polymer . The protein size was determined by the Superose 12HR10 / 30 (1-300 kDa
Of the rRT, which revealed that most of the rRT eluted between beta amylose (approximately 200 kDa) and apoferritin (443 kDa). Thus, rRT was predominantly in polymeric form. Without wishing to be bound by theory, the addition of a C-terminal histidine is attributed to the deleted integrase domain, perhaps providing polymerization capacity by complexation via metal (e.g., nickel) chelation. It was able to replace that loss of ability. Thus, the present invention contemplates RT polypeptides having a C-terminal attachment in the form of a compound capable of promoting polymer formation. Suitable compounds will include, but are not limited to, a plurality of basic or acidic amino acids, as well as Cys residues capable of forming disulfide bonds.

[0087] The expressed rRT was also characterized immunochemically. Monoclonal antibodies against AMV reverse transcriptase were prepared using techniques well known in the art. Harlow
Et al., Antibodies: A Laboratory Manual, Cold Spring Harbor laboratory, Cold
See Spring Harbor, New York (1988). Briefly, spleen cells from mice immunized with RT were fused with mouse myeloid cells to create hybridomas. These hybridomas were grown to colonies in 96-well plates: the supernatants from these wells were then tested to find those that appeared to make anti-RT antibodies. Further testing confirmed these results.

To prepare spleen cells for hybridoma production, BALB / C mice were injected by several intraperitoneal injections with AMV-RT (Molecular Biology Resources, Inc.) using a routine immunization schedule. (Female, 10 weeks old, Harlan Sprague Dawley, Madiso
n, WI). To prepare RT for injection, the storage buffer was diluted by diluting the enzyme in phosphate buffered saline (PBS) and reconstituting it using a Centricon 30 concentrator (Amicon Corp.). Was removed from purified RT. The concentrated RT was then diluted again in PBS and emulsified with an equal volume of adjuvant. For the first injection, complete Freund's adjuvant (Sigma Chemical Co.); for the booster injection, the Ribi Adjuvant System (Rivi Immunochem Research, Inc, Hamilton,
VT). The dose of RT was approximately 20 micrograms per injection. Injections are given at consecutive intervals of 5, 4, 3, 11, 8 and 3 weeks,
Performed over an eight month period. Fusion was performed 5 days after the first boost.

In fusion experiments, spleen cells were excised and fused with myeloma cells (P3X63-AG8.653, ATCC CRL1580) using techniques well known in the art. Har
See low et al. In particular, cells were fused in 50% polyethylene glycol, resuspended in selective medium (ie, medium with HAT), and distributed into wells of 14 96-well plates. After three weeks of growth, approximately 350 wells contained hybridoma colonies.

[0090] Hybridomas producing anti-R antibodies were identified by ELISA. In this technique,
The wells of a 96-well polystyrene ELISA plate were first purified with purified RT (100 mM Tris
-HCl, pH 8.5, 2 micrograms RT / ml in 0.05% NaN ₃ : overnight incubation at room temperature) and coated with TBST (Tris-buffered saline, pH 8.5, 0.05% Triton X-10).
Washing was performed at 0) to remove excess RT. In the assay itself, wells were filled with 95 microliters of% TBST + 5 microliters of hybridoma culture supernatant. Plates were incubated at room temperature for 2 hours and then washed with TBST to remove unbound immunoglobulin. To detect wells with anti-RT antibody,
Peroxidase-conjugated goat anti-mouse IgG (heavy chain specific: Jackson ImmunoResearch
, West Grove, PA) was diluted 5000-fold in TBST and added to the wells of the ELISA plate. After incubating the wells for 1 hour at room temperature, the unbound peroxidase conjugate was removed by thoroughly washing the plate with TBST. Following the addition of the substrate 3-methyl-2-benzothiazolinone hydrazone / 3-dimethylaminobenzoic acid / hydrogen peroxide, RT2-positive wells were visualized colorimetrically to detect immobilized HRT. Hybridomas from positive wells were repeatedly cloned by limiting dilution until all growing wells were ELISA-positive.

Supernatants from wells that tested positive by ELISA were further screened by immunoprecipitation of RT using techniques well known in the art. Harl
See ow et al. This immunoprecipitation assay was performed using Staphylicoccus aureus cells (SAC, Sigma
Rely on the presence of Protein A (binding to IgG) on the surface of Chemical Co.). Since protein A does not bind strongly to mouse IgG, the centrifuged SAC cell pellet was first treated with rabbit anti-mouse IgG antibody. The pellet from 10 microliters of a 10% suspension of these cells was then incubated with 50 microliters of hybridoma culture supernatant for 2 hours at room temperature. The resulting SAC cells were centrifuged, washed and resuspended in diluted RT. The RT cell suspension was incubated at 4 ° C. for 3 hours and centrifuged. The resulting supernatant was removed and tested for RT activity depletion using a standard radiochemical assay.

The six hybridoma lines scored positive in both the ELISA and the immunoprecipitation assays. These lines were named 1D8, 210, 4C4, 6F1, 9C2 and 9H10. All six monoclonal antibodies had γ-1, κ isotypes.

The form of the active rRT (ie, monomer or polymer) was confirmed using a sandwich-type ELISA with an anti-RT monoclonal antibody. First, the monoclonal antibody was immobilized in a DNA binding plate (Costar Corp., Cambridge MA). next,
Plates were blocked with BSA to prevent non-specific binding. The wells were then incubated with purified rRT (ie, MIBAhis). Excess or unbound protein was removed by washing with phosphate buffered saline.

The wells were then incubated with the same monoclonal antibody conjugated to biotin for detection. If rRT is present as a monomer, the biotin-linked monoclonal antibody should not bind to it. However, the biotin-monoclonal antibody did not bind to rRT, indicating that rRT formed a polymer.

To determine the purity of a sample containing reverse transcriptase, either solubilized cell pellets or protein fractions from different chromatography columns expressed from each of the various clones (eg, MIBAhis) and used for purification Was subjected to SDS-PAGE. Samples were subjected to electrophoresis on an 8% polyacrylamide gel containing a 6% stacking gel, followed by standard protocols (Sambroo
k et al., 1989). The recombinant protein was found to be greater than 95% pure.

Using the Pharmacia Phast System, recombinant (MIBAhis) and native reverse transcriptase, appropriate standards supplied to the system (ie, IEF 3-9) were subjected to isoelectric focusing. (Pharmacia-Upjohn, Piscataway, NJ). Experimentally measured pI for rRT and rRT
Was 6.0. The theoretical pI of Rrt calculated from the amino acid sequence was 6.8.

For analysis of total expression, several recombinant DNAs (ie, pMDacRT, pBacMIBA,
Host cells containing one of pBacMIKA, pBacMIBAhis and pBacMIKAhis) were induced to express the recombinant protein. The induced cells were pelleted at 12,000 × g for 5 minutes. The cell pellet was then washed with SDS sample buffer (Sambrook
1989) or cell resuspension buffer (20 mM Tris HCl, pH 8.0, 250 mM NaCl, 0.5%
(Triton H-100 and 5% glycerol) to evaluate protein solubility. Resuspend cells for 20 seconds each (500 mM Tris HCl, pH 6.5, 14% SDS, 30
% Glycerol, 9.3% DTT and 0.012% glomophenol blue), 3 settings
(Virsonic 100 from Virtis Company, Inc., Gardiner, NY) was pulse-sonicated three times, and a small aliquot of the sample in SDS sample buffer was loaded on a duplicate gel and subjected to electrophoresis. One of the duplicate gels was stained with Coomassie blue, and the other gel was used to transfer proteins to a 0.2μ nitrocellulose membrane using a Bio-Rad transfector for Western blot analysis. Bio Rad Laboratories, Inc., Hercules,
CA. Detection of expressed protein in the fractionated crude lysate was performed using a specific monoclonal anti-RT antibody (monoclonal antibody 4C4, 1) for detecting recombinant protein.
Mixture of D8, 2E10, 6F1, 9H10 and 9C2; Molecular Biology Resources, Inc.,
Milwaukee, WI).

In practice, the nitrocellulose membrane containing the transferred proteins was treated with a blocking buffer (5% casein hydrolyzate, 150 mM NaCl, 10 mM Tris HCl, pH 8.0)
For 30 minutes followed by incubation with a 1: 1000 dilution of the anti-RT monoclonal antibody in blocking buffer. After overnight incubation,
The blot was rinsed in wash buffer (10 mM Tris HC1, pH 8.0, 150 mM NaCl, 0.5% Tween20) and incubated for 1 hour with a 1: 500 dilution of alkaline phosphatase-conjugated goat anti-mouse antibody in blocking buffer. Subsequently, the blot was rinsed three times with wash buffer and once with AP buffer (100 mM Tris HC1, pH 9.5, 5 mM MgCl ₂ and 100 mM NaCl). MBT (75 mg / ml in nitroblue tetrazolium: dimethylformamide) and BCIP (5-bromo-4-chloroform) that form a blue precipitate when dephosphorylated by any immunologically immobilized phosphatase. RT was detected indirectly by performing a colorimetric phosphatase assay using a standard substrate mixture of -3-indolyl phosphate, 50 mg / ml in dimethylformamide). The anti-RT antibody recognizes two bands, one at approximately 61 kDa and one at 92 kDa, in the lane containing native RT. Recombinant His-Taqd RT expressed from MIBAhis (alpha fragment equivalent)
A single band at approximately 60 kDa was found: in the lane containing recombinant His-tagged RT (beta fragment equivalent) expressed from MIKAhis, a single 91
A kDa band was found.

Assays were also performed to measure endogenous / exogenous exonuclease, endonuclease (ie, nicking) DNase and RNase activity of rRT. Three
Assays for '→ 5' exonuclease activity were performed using a radiolabeled Taq1 fragment of λDNA as a substrate. The 3 'end (265 μg) of the Taq1-digested lambda DNA fragment was prepared using a standard labeling reaction using 40 units of the exo-Klenow fragment of DNA polymerase, 60 μCi [ ³ H] -dCTP (57.4 μCi / mmol) and 60 μCi [ ³ H ] -dGTP (8.9 μCi). Sambrok et al., (1989). This 3 '→ exonuclease assay yields 5
Performed in a final volume of 10 μl containing 0 mM Tris HC1, pH 7.6, 10 mM MgCl ₂ , 1 mM DTT, 0.015 μg of labeled Taq1 fragment of λ DNA, and 2.5 or 10 units of RT enzyme.

One unit of RT enzyme is the amount of enzyme required to incorporate 1 nanomolar dTTP into the acid-insoluble form within 10 minutes at 37 ° C. under the indicated assay conditions (see Example 6). reference
). Each sample was incubated at 37 ° C. for 1 hour. 50 μl yeast tRNA and
The reaction was stopped by adding 200 μl of 10% trichloroacetic acid. After a 10 minute incubation on ice, the samples were centrifuged in a microcentrifuge tube for 7 minutes. The supernatant (200 μl) containing the released label was removed, added to 6 ml of scintillation fluid and counted in a scintillation counter. The result is that rRT is 0.1
Release 3%, which was an acceptably low level of 3 '→ 5' exonuclease activity.

In addition, a 5 ′ → 3 ′ exonuclease assay was performed using a radiolabeled HaeIII fragment of λDNA. In the usual ^{manner, 60μCi [γ- 33 P] dATP} (2,000Ci / mmol)
The λ fragment was labeled at the 5 ′ end using 40 units of T4 polynuclease kinase. Sambrook et al., (1989). This assay was performed as described above for the 3 ′ → 5 ′ exonuclease assay, except that the 5′-end labeled HaeIII fragment was used as a substrate. Purified rRT releases 0.36% of the label in acid-soluble form, which
The '→ 3' exonuclease activity was at an acceptably low level.

Again, double-stranded and single-stranded DNAse assays were performed using this 3 ′ → 5 ′ exonuclease assay protocol, except for the type of labeled substrate used. In each of the DNAse assays, intact lambda DNA (0.5 μg) was converted to 30 μCi [α- ³³ P] dATP (2,000 Ci /
Mmol). Each assay had 0.015 μg of labeled λ DNA.
In the single-stranded DNase assay, the labeled λ DNA fragment was further subjected to thermal denaturation (3 min at 100 ° C., immediately followed by cooling on ice) to prepare a substrate. Again, with the exception of the type of substrate used, each of the DNase assays was performed as described above in the sense of a d '→ 5' exonuclease assay. rRT is 0.5% of the label in the double-stranded DNase assay.
% Released: In a single-stranded DNase assay, 0.02% of the label was released. Both results showed acceptably low levels of DNase activity. Also, purified r
RT was subjected to endonuclease, or nicking (assay by examining the extent to which rRT was converted to a supercoiled substrate in the form of pBR322, as visualized by agarose gel electrophoresis by fractionation). Assays for endonuclease activity were performed in a final volume of 10 μl containing 50 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM β-mercaptoethanol, 0.5 μg pBR322 and 2.5, 5 or 10 units of enzyme. . Each sample was incubated at 37 ° C. for 1 hour. 2 microliters of 0.25% bromophenol blue, 1 mM EDTA and 4
The reaction was stopped by adding 0% sucrose. After a brief centrifugation, 6 μl of the sample was subjected to electrophoresis on a 1.0% agarose gel in 1 × TBE. Sambrook et al., (1989
). The results showed that less than 10% of the supercoiled substrate was converted to a relaxed form, with acceptable low nicking.

[0103] rRT was also characterized in terms of its RNase activity. In particular, the assay was designed to measure general RNase activity but not RNase H activity. The run-off transcription from a T7 promoter substrates were prepared by using in the presence of the ^"alpha-33 P" UTP. In particular, plasmid pPV2 (ColE1 origin; ampicillin selectable marker; T7
, T3 and lac promoters; and a pTZ-based vector containing a 695 bp insert from plum pox virus) were linearized with PvuII. The runoff transcription reaction, using conventional techniques, the linearized pPV2,30μCi of 1μg ^"alpha-33 P 'dATP (2,00
0 Ci / mmol) and 10 units of T7 RNA polymerase. Then
RNase assays were performed in the presence of single-stranded RNA substrate (0.15 μg) and rRT (2.5, 5 or 10 units). Using the TCA precipitation technique described above, the released label was again acid-
Collected as soluble material. Scintillation counting indicates that 1% of the released label is released, indicating an acceptably low level of RNase activity.

Example 6 The RNA-dependent DNA polymerase activity (purified expression product of MIBAhis) of native RT and recombinant RT was compared. One unit of the enzyme was converted to poly rA: dT12-18 (20
: 1) or mRNA for RT assays. The amount of product was measured by either glass filter precipitation or binding to a DE52 filter; the amount of product was monitored by autoradiography on a 1.2% TBE agarose gel containing the fractionated reaction products.

The reverse transcriptase activity of native and recombinant proteins was measured by Meyers et al., Biochemistry 3
0: 7661-7666 (1991) using a modification of the procedure described. The reaction mixture was 1 × reaction buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 10 mM MgCl ₂ )
, 1 mM DTT, 0.4 mM poly rA: dT18, 0.5 μCi `` α ³² P '' TTP (3,000 Ci-mmol), 0.5 mM
dTTP, 1 unit of enzyme (1 unit of RT enzyme is at 37 ° C under the indicated assay conditions)
Is the amount of enzyme required to incorporate 1 nmole of dTTP into the acid-insoluble form within 10 minutes), and ddH ₂ O to a total volume of 50 μl. The reaction mixture without enzyme was preincubated for 1 minute at 37 ° C. prior to addition of the enzyme. The reaction was then incubated at 37 ° C. for 20 minutes and stopped by adding 2 μl of 0.5 M EDTA, followed by applying 40 μl of each reaction mixture to a separate DE52 filtration membrane. The filters were washed three times with 5% Na ₂ HPO ₄ for 5 minutes each, then rinsed with ddH ₂ O, followed by 95% ethanol.

The filter was sealed, placed in scintillation fluid, and the immobilized radioactivity was quantified. A variation of the filter assay was used to compare the quantity and quality of the reaction products. Messenger RNA, 891 bp control and 7.5 kb mRNA are from GIBCO BRL, Gaithersbur
g, obtained from MO. The following substitutions were made in the assay described above: poly rA:
1μg of mRNA originating from the 0.5mM oligo dT12-18 primer instead of DT18; and ^"alpha-32 P" dGTP of mixed dNTPs (each 0.5μM instead of dTTP, dATP, TTP and dCTP
, And 0.02 μCi “α- ³² P” -dATP (6,000 Ci / mmol)). The reaction was started by adding 5 units of RT to the reaction mixture. After 1 hour of incubation at 37 ° C., a 20 μl sample was removed and mixed with 5 μl of stop solution (95% deionized formamide, 10 mM EDTA, 0.05% xylene cyanol FF and 0.05% gromphenol blue) and a 1 kb ladder standard (Chimerx, Madison, WI) on a 1.2% TBE agarose gel. The gel sample was subjected to electrophoresis at 100 volts for approximately 2 hours and dried. The dried gel was subjected to autoradiography at -70 ° C for 3 days and developed to visualize the band. The results are shown in FIG. 3 and represent autoradiographic data showing size-fractionated reverse transcriptase products using poly A-tailed mRNA as template and oligo dT50 primer. In particular, the mold is 891
Nucleotides (lanes 2 and 4) or 7,5,000 nucleotides (lanes 1 and 3)
nRT was used in reactions analyzed in lanes 3 and 4, while rRT was used in reactions analyzed in lanes 1 and 2. Both native and recombinant RT yielded 891 bp and 7.5 kb products, depending on the size of the mRNA template.

Example 7 The properties of native MAV-RT and recombinant RT were compared. In particular, optimal values of temperature, pH, magnesium ion concentration, and other divalent cation (ie, calcium, copper, manganese and zinc) concentrations were determined. a) Optimal temperature The RNA-dependent DNA polymerase activity of native and recombinant MAV-RT (ie MIBAhis) was compared in RT assays performed at different temperatures.

[0108] The relative RT activity of the enzymes was compared between 37 ° C and 70 ° C at pH 8.0. The activity assay was 50 mM Tris HCl, pH 8, 40 mM KCl, 10 mM MgCl ₂ , 1.34% trehalose, 2% maltitol, 1 mM DTT, 0.5 mM poly rA: dT18, 0.5 mCi “ ³ H” -dTTP (70-90
50 μl containing 0.5 mM dTTP, 5 U enzyme (rRT or nRT) and ddH ₂ O
In the reaction mixture. Duplicate reactions were incubated at each temperature for 10 minutes.
It was quantified product by measuring the ^"3 H" -dTTP captured using a scintillation counter. The results are shown in FIG.4A (black: rRT (i.e.
BAhis); hatching: nRT), expressed as counts per minute as a function of temperature in ° C. These results demonstrate that the optimal temperature for both nRT and rRT in the RT assay is 55 ° C.

The temperature profiles of nRT and rRT (ie, MIBAhis) in the RAMP assay were also measured. RAMP reactions were performed as described in PCT / US97 / 04170, which is incorporated herein by reference. In particular, the target nucleic acid to be amplified was Cryptosporidium mRNA from one oocyte. As described in detail in Example 8, this mRNA target was reverse transcribed into cDNA at different temperatures using 20 units of natural RT or 15 units of recombinant RT. The results are expressed as absorbance at 450 nm as a function of temperature in ° C. as shown in FIG. 4C (hatched line: standard; filled circle: rRT (ie, MIBAhis)). Actual absorbance values at various temperatures are shown below the figure (
Upper row: standard; lower row: rRT). b) Optimal pH The relative RT activities of nRT and rRT in reactions at different pH values were compared. Two sets of comparative reactions were designed: one set was incubated at a normal temperature of 37 ° C, and the other set was incubated at a temperature of 60 ° C, which was suitable only for thermostable enzymes.

The pH value of the selected buffer was adjusted at room temperature. The activity assay was 40 mM KCl, 10
mM MgCl ₂ , 1 mM DTT, 0.5 mM poly rA: dT18, 0.5 mCi [ ³ H] -dTTP (70-90 Ci / mmol),
Performed in a 50 μl reaction mixture (pH 6, 7, 8, 8.3, 9 or 9.5) containing 0.5 mM dTTP, 5 units enzyme, ddH ₂ O and 50 mM and -HCl. The reaction is performed at 37 ° C or 60 ° C for 10
Incubated for minutes. Using a scintillation counter, activity diagrams quantified product by measuring the ^"3 H" -dTTP incorporated as counts per minute, nRT and rRT under various pH conditions (i.e., MIBAhis) 4D and 4E
(Black: rRT (ie, MIBAhis); gray hatching: nRT). The data in the figures establish that the optimal pH for nRT and rRT is pH8. c) to modify the RP assay described optimal Mg ⁺⁺ ions Example 7 (b), to determine the effect of MgCl ₂ concentration on the activity of native and recombinant RT. The reaction buffer was 50 mM Tris-HCl, pH 8.3,
And MgCl ₂ ranging in concentration from 0-100 mM; all other reaction components were as described in Example 7 (b). The reaction was incubated at 37 ° C.

The “ ³ H” -dTTP incorporated was measured by scintillation counting and the results are expressed as counts per minute. The optimal MgCl ₂ concentration is as shown in FIG.4F (black: rRT
(Ie MIBAhis); gray hatching: nRT) was found to be 5 mM for both nRT and rRT. d) Requirements for other divalent cations The reaction described above was modified in the sense of measuring the Mg ⁺⁺ concentration optimum to determine the effect of different divalent cations on RT activity. The reaction buffer contained 50 mM Tris-HCl, pH 8.3 and 10 mM chloride salt of divalent cation (MgCl ₂ , CuCl ₂ , MnCl ₂ , ZnCl ₂ , or CaCl ₂ ). An independent experiment was performed to create a curve. FIG. 4G shows the activity of the enzyme as an incorporated count as a function of the cation used in the reaction (black: rRT (ie MIBAhis): gray hatching: nRT). As shown in FIG.4G, nRT and rRT (
That is, maximal activity of both MIBAhis) was achieved using magnesium as the divalent cation.

Example 8 Conceptually, RT-PCR consists of a preamplification reaction followed by an amplification reaction. The pre-amplification reaction is performed using CAT (i.e., chloramphenicol acetyltransferase) mRNA.
Using reverse transcriptase to synthesize the first strand of cDNA using as a template.

This CAT mRNA was provided in the Superscritp kit from GIBCO-BRL and the reactions were performed according to the supplier's recommendations. Following this reaction, RNA from the RNA-DNA hybrid was removed by RNase H, releasing the first strand used as a template in the polymerase chain reaction (PCR).

The pre-amplification reaction mixture initially contained 50 ng of control mRNA (ie, CAT mRNA), 500 ng
Of oligo dT12-18 and ddH ₂ O, making the mixture a total volume of 12 μl. This mixture was incubated at 70 ° C. for 1 minute. Subsequently, 2 μl of 10 × PCR
Buffer, 2 μl of 25 mM MgCl ₂ , 1 μl of dNPT from a combined stock solution containing 10 mM each of dGTP, dATP, TTP and dCTP, and 2 μl of 0.1 M DTT were added to the mRNA / oligo dT mixture. One set of reactions was incubated with 20 U of nRT and the other set of reactions was incubated with 20 U of rRT (ie, MIBAhis).

Incubate one tube from each set at one of several temperatures,
Each reaction was allowed to proceed for one hour. The reaction was stopped by incubation at 90 ° C. for 2 minutes. Then, it was cooled on ice, 1 μl of RNase H was added to each tube, and incubated at 37 ° C. for 20 minutes.

For the amplification reaction, 5 μl of 10 × PCR buffer supplied in the Superscript kit, 3
1 μl dNTP from a combined stock solution containing μl 25 mM MgCl ₂ , 10 mM dGTP, dATP, TTP and dCTP each, 1 μl amplification primer 1 at 10 μM each and
10 μl amplification primer 2, 1 μl Taq DNA polymerase and Pyrococcus woes
ii (i.e., Pwo) DNA polymerase mix (Bochringer Mannheim Corp., Ind.
ianapolis, IN), each reaction mixture was assembled in a thin-walled tube containing ₂ μl of the cDNA mixture from the first strand synthesis reaction and up to 50 μl total volume of ddH ₂ O. 1.2% TBE
The fractions were subjected to a 5 μl reaction on an agarose gel, followed by a DC40 camera and Kodac
Ng DNA using an imager equipped with Digital Sciences 1DTM software
The reaction products were analyzed by measuring the intensity of the band represented by. The amount of DNA synthesized by rRT was comparable to the amount synthesized by nRT.

The results show that the optimal temperature for RT-PCR is 60 ° C. using either nRT or rRT, as shown in FIG. 4B (results were generated PCR as a function of temperature in ° C.
Expressed as ng of product, open squares indicate rRT (ie, MIBAhis) and solid squares indicate nRT). The amount of gene-specific product is 60
It was large at ° C. The optimal temperature for RNA-dependent DNA polymerase activity for both nRT and rRT was 55 ° C. (see Example 7a and FIG. 4A). Differences in optimal temperature are probably due to DNA-dependence (with different temperature optimalities) in RT-PCR
May be due to the need for both DNA polymerase and RNase H activity.

Rapid amplification (ie, RAMP) is an amplification technique disclosed in International Application No. PCT / US97 / 04170. Also, the RAMP reaction can be performed with the nicking enzyme (ie, BsiHKCl) and Bst DNA polymerase together with RT according to the invention and Cryptospori as a template.
This was performed using the first strand of cDNA from dium oocyte mRNA. This Bst DNA polymerase provides both polynucleotide synthesis activity and strand displacement activity.

This reaction was performed at 35 mM K · PO ₄ , 0.7 mM Tris-HCl, pH 7.9, 1.4 mM dCTP, 0.5 mM each.
Of dATP, dGTP and dTTP, 35 mg of bovine serum albumin, 10.2mM MgCl _2, 3.4mM KC
1, 0.7 mM DTT, 2% maltitol, 1.34% trehalose, 0.5 mM amplification primer (5′-ACCCCATCCAATGCATGTCTCGGGTCGTAGTCTTAACCAT-3 ′: SEQ ID NO: 1) and amplification primer 2 (5′-CGATTCCGCTCCAGACTTCTCGGGTGCTGAAGGAGTAAGG-3 ′: SEQ ID NO: 1) :
32) and 1% glycerol. 10 μl total volume for each reaction
With 36 units of Bst DNA polymerase and 250 units of BstHKCl in 15
Units of rRT (ie, MIBAhis) or 20 units of nRT were added.

The amount of product synthesized in each reaction was determined by a plate assay. This pute assay uses a gene-specific capture primer (5'-AAACTATGCCAACTAGAGATT) that binds to the wells of a microtiter plate and is used to capture the product.
GGAGGTTGTTT-3 ′: SEQ ID NO: 30). The product captured by the oligonucleotide (HRP-conjugated P2 Comp; SEQ ID NO: 37) bound to horseradish peroxidase was then detected. HRP combined
Was detected by colorimetric assays standard in the art.

The amount of product synthesized by rRT was twice as high as that synthesized by nRT at temperatures between 55 ° C. and 64 ° C., as shown in FIG. 4C. The difference in the temperature optimum between the RT assay and the amplification is due, in part, to the relative RNase at the assessed temperature.
May be due to differences in H activity. Lowest RNase H activity was observed between 60-65 ° C. which resulted in longer cDNA products and larger template amplification.

Example 9 In addition to RNA-dependent DNA polymerase activity, MAV-RT has additional enzymatic activities, such as DNA-dependent DNA polymerase activity. This DNA-dependent DNA polymerase activity was measured using a single-stranded M13mp18 DNA template and a sequence-specific [λ ³² P] -labeled primer (ie, forward sequencing primer or FSP, 5′-CGCCAGGGTTTTCCCAGTCACCA-3 ′;
It investigated using SEQ ID NO: 29). 10 μl of the reaction mixture is 50 mM Tris HCl, pH 8.3
, 40 mM KCl, 10 mM MgCl ₂ , 20 μM of each convenient dNTP, 0.24 pmol of sequence-specific primer FSP and 800 ng of single-stranded M13mp17 DNA template. 4 units of r
RT (ie, MIBAhis) and 5 units of nRT were compared to a commercially available DNA polymerase (Sequitherm: 5 units) using the buffer provided in the kit. (S
(equitherm Cycle Sequencing kit, Epicenter Technologies, Madison, WI)
. The DNA-dependent DNA polymerase activities of nRT and rRT were almost equivalent.

In addition, DNA-dependent DNA polymerase activity was measured at different temperatures.
In these reactions, the incorporated [α- ³² P] -dTTP served as a label and non-radioactive primers were used. The reaction was performed with 200 ng of single-stranded M13mp18 DNA, 1 in a total volume of 24 μl.
.5 pmol FSP, 50 mM _{Tris-HCl, pH8.3,40mM KCl, 10mM MgCl 2} , 1mM DTT, 0.
6 μCi of [α- ³² P] -dTTP (3,000 Ci / mmol) and 20 μM each of dATP, dGTP, dC
It consisted of TP and dTTP. Use the usual protocol for the reaction (Sambrook
(1989)), the reaction was stopped by adding 2 μl of 10 mM EDTA (0.8 mM final concentration). The incorporated [α- ³² P] -dTTP was detected using DE52 membrane and scintillation counting as described above. The results shown in FIG. 5 show that the optimal temperature for DNA-dependent DNA polymerase activity for rRT is 45 ° C.-50 ° C .; for nRT, the temperature optimal was 55 ° C. The DNA-dependent DNA polymerase activity of the RT of the present invention extends the range of applications in which RT can be used. In addition to copying DNA as well as RNA, the enzyme can be used in any of the various amplification techniques described above known in the art. In addition, using the polypeptide of the present invention,
The RNA or DNA target can be sequenced using the originally disclosed Sanger enzymatic approach, or any one of a variety of variations of the technology that have been developed since then.

Example 10 rRT according to the present invention (ie, MIBAhis) was subjected to an RNase H assay using protocols known in the art. Hillenbran et al., Nucl. Acids Res. 10: 833 (1982
). The reaction (25 μl) was performed at 20 mM HEPES-KOH, pH 8.0 (23 ° C.), 10 mM MgCl ₂ , 50 mM KCl,
It contained 1 mM DTT, 0.24 mM [α- ³² P] poly (A) -poly (dT) (1: 2; 15 μCi / ml), and 4 μl of the diluted enzyme purified from M1BAhis as described above.

In a control reaction, a standard stock of RNase H with known activity (Molecular Biology
Resources, Inc. Milwaukee, WI) was assayed in the range of 0.05 to 0.5 units / reaction (1 unit of activity was determined from acid from [α- ³² P] polyA-poly (dT) within 20 minutes at 37 ° C. (Defined as the amount of enzyme required to produce 1 nanomol of soluble ribonucleotide). Two reactions were performed without the enzyme serving as a negative control.

A reaction mixture, a smaller amount of the enzyme was prepared, and the reaction was started by adding the enzyme.
After 20 minutes of incubation at 37 ° C., 25 μl of cold yeast tRNA (
The reaction was stopped by adding 10 mg / ml in 0.5 M acetic acid, pH 5.0), followed by the addition of 200 μl of 10% trichloroacetic acid. The samples were then placed on ice for at least 10 minutes. Mix the mixture in Eppenborf microcentrifuge tubes (Brinkman Instruments, Westburg,
(NY) at 16,000 × g for 7 minutes, and 200 μl of supernatant fluid was removed and counted in 5 ml of scintillation fluid.

The RNase H activity of rRT was tested at different temperatures using the reaction mixture described above.
The results are as shown in Figure 6A (black: rRT (i.e.MIBAhis); gray patching: nRT)
Expressed as counts per minute of radiolabeled ribonucleotide released for each of the two tests. The data shows that rRT had RNase H activity comparable to that of native RT. In addition, rRT activity was evaluated at various temperatures, and the results depicted in FIG. 6B indicated that rRT was active over a wide temperature range. The optimal RNase H activity for rRT was 50 ° C. In contrast, RNase H activity was 37 ° C, 60
It was relatively low at temperatures of ° C and 65 ° C. Due to differences in temperature optima for RT RNase H activity and other RT activities, such as RNA- and DNA-dependent DNA polymerase activities, adjusting the temperature to achieve the desired mix of activities can reduce RT activity. The various methods that rely on can be optimized. For example, methods involving the use of RNase H activity can be performed at temperatures relatively close to the optimal 55 ° C. temperature for rRT RNase H activity. Methods that are convenient from reduced RNase H activity such as RT-PCR and RAMP can be performed at 60-65 ° C. to maintain low levels of RNase H activity.

Example 11 Various polynucleotides encoding modified RT fragments were constructed. These modified RTs delete the naturally-occurring terminal region of the peptide, resulting in RNA-dependent
Includes α and β polynucleotides that have been terminally modified by generating α-like and β-like fragments that retain DNA polymerase activity. Other modified RTs according to the present invention may comprise one or more peptides at the N-terminus, C-terminus, or both termini (these peptides may be simple homo-oligomeric peptides, preferably DNA-bound, metal-bound, Stabilization and polymerization [including, for example, charged or bulky polypeptides that include useful functions such as zinc finger domain, leucine zipper motif, NS1 binding site, GPRP (single letter amino acid identification) or its inverse PRPG] ability, among others. Α-like or β-like fragments attached to crabs. Still other modified RTs according to the present invention include fragments that lack sequences found internally in one of the native polypeptides α or β.

The techniques used to construct polynucleotides encoding these modified RTs are known in the art and are described in Examples 1 and 3 above.
Generally, the strategy is to construct the desired polynucleotide using PCR,
This, in turn, results in a cloned, expressed and encoded modified RT.
This expression experiment was generally performed as described in Example 4.

As a result of the expression of eukaryotic genes in prokaryotes, misincorporated, truncated and / or insoluble proteins (misfolded) due to the presence of rare codons in those eukaryotic genes Can be produced. Translation of these rare codons is limited by the regulated expression of the tRNA corresponding to these rare codons. Thus, expression of eukaryotic genes with abundant rare codons sometimes results in incorrect uptake, truncation and / or misfolding. One approach to minimizing such problems is to clone tRNAs corresponding to these rare codons and express the clones in E. coli to enhance eukaryotic gene expression. Since the arginine codons (AGG, AGA, CGA and CGG) represent the largest number of rare codons in AMV-RT, we cloned and expressed ArgU tRNA. AMV-RT and ArgU co-expression expresses AMV-RT (i.e., activity level)
Is expected to improve. Also, other rare codons such as leucine (CTA) and proline (CCC) are cloned and co-expressed.

[0131] Another approach to improved expression of the modified RTs of the invention in prokaryotes is to change rare codons in the modified RT coding region to frequently used codons. Such changes can be readily made by various techniques known in the art, for example, site-directed mutagenesis using synthetic oligonucleotides. In the E. coli expression system, 90
Rare codons (38 arginine, 23 proline, 15 isoleucine, 10 leucine and 4
Serine codons), all or some of which can be advantageously changed to frequent codons. All 90 rare codon changes to the frequent codons found in abundantly expressed genes, however, can upset the metabolic balance of the host cell. To modulate the deleterious effects on host cell metabolism resulting from too high a modified RT expression level, clones were cloned using, for example, an M13-based approach to site-directed mutagenesis involving oligonucleotide primer incorporation. A library can be built. Specifically, using a pool of synthetic oligonucleotides, each oligonucleotide designed to convert one or several rare codons to frequent codons, and a template containing a modified RT coding region, A series of modified R with rare codon changes in the range of 1-90
T can be synthesized. Clones with RT activity can be isolated from this library by conventional screening techniques, such as, inter alia, binding to radioactive substrates and activity assays.

To facilitate understanding of the structure of the various polynucleotides and polypeptides disclosed in this example, Table 2 below gathers relevant information. All constructions generated by PCR used the appropriate full-length coding region as a template, such as the pol gene sequence found in M1-5,6.

[0133]

[Table 2A]

[0134]

[Table 2B]

[0135]

[Table 2C]

[0136]

[Table 2D]

[0137]

[Table 2E]

[0138]

[Table 2F]

[0139]

[Table 2G]

[0140]

[Table 2H]

[0141]

[Table 2I]

[0142]

[Table 2J]

[0143]

[Table 2K]

A. Terminus by deletion using conventional methods RT said that was deleted was truncated to full length RT coding region (e.g., Example 3). One set of deletion derivatives contains the MAV-RT coding region.
The 3 'end was missing to varying degrees. Again, for the full-length gene (SEQ ID NO: 1),
As shown by S-PAGE, a 3 '(C-terminal) deletion (MIKA) extending to the KpnI site (see Example 8) reduced RT expression levels. For the full length gene (SEQ ID NO: 1), BglI as shown by SDS-PAGE and activity assay (see below).
Deletion of the region extending from the I site to the 3 'end (MIBA: see SEQ ID NO: 6) increased RT expression and activity. C-terminally truncated RTs (MIKA and MIBA) have lengths that fall between those of the native α and β polypeptides. In contrast to the alpha fragment of MAV-RT, the beta fragment has an additional 254 amino acids at the C-terminus, which provides integrase activity. (-) RT compared to integrase form (eg, M1BA gene product)
+) This region of the polypeptide contributes to the insolubility of the polypeptide, as indicated by the relative insolubility of the integrase form (eg, the M1KA gene product, see below), reducing its recovery from cell extracts. Since the integrase domain was only required for the life cycle of the retrovirus and not for RNA- or DNA-dependent DNA polymerase activity, this region was deleted with MIBA (α-like fragment). The MIBA α-like fragment (amino acids 1-578 of SEQ ID NO: 2) is larger than the naturally occurring α fragment of MAV-RT (amino acids 1-573 of SEQ ID NO: 2). Without wishing to be bound by theory, it was predicted that this deletion would result in increased protein solubility and thus recovery.

A series of clones were constructed to increase expression levels and to modify the M1BA of modified RT with a C-terminal deletion to stabilize RT activity (RNA-dependent DNA polymerase activity)
And M1KA were expressed. Convenient restriction sites for full length clones such as PMBacRT and pHRT, for example, BglII (spanning nucleotide 1, SEQ ID NO: 1,
986-1,991) and KpnI (spanning nucleotides 2,745-2,750 of SEQ ID NO: 1)
Was used to eliminate the 3 'end of the coding region of the RT gene (see Table 1). C-
The 3 'deletion derivatives encoding the RT polypeptide fragment with terminal deletions are each pMBa
Obtained by BglII-PstI or KpnI-PstI restriction of cRT and pHRT (BglII and KpnI sites in MAV-RT coding region; PstI site in vector). BglII-P
Recombinant molecules containing the stI 3 ′ terminal deletion were named pDacMIBA and pHBRT (pH33ΔBP6), and the recombinant molecules containing the KpnI-PstI deletion were named pBacMIKA and pHKRT (pH33ΔKP5
). The deletion derivatives pBacMIBA and pBacMIKA had approximately 1.17 and 0.4 kb deletions, respectively, from the 3 'end of the full length gene (see, eg, SEQ ID NO: 1). Using a fragment (SEQ ID NO: 6) bordered at the BglII site and its 3 'end, the alpha-like RT fragment (the α-like fragment M1BA contains amino acids 1-578 of SEQ ID NO: 2; native MAV- RTα contains amino acids 1-572 of SEQ ID NO: 2) and expresses Kp
Using the fragment bordering the nI site (SEQ ID NO: 8), a beta-like RT fragment (the β-like fragment MIKA contains amino acids 1-832 of SEQ ID NO: 2; the native MAV-RT β Is SEQ ID NO: 2
Containing amino acids 1-858).

Miniprep and sequencing analyzes were performed to confirm the identity of the recombinant clones described above. Recombinant virus obtained from co-transfection with the virus BacPak6 and the transfer vector pBacMIBA or pBacMIKA was used for M1BA and M1KA, respectively.
I called.

B. Alpha-like recombinant encoding a non-natural-terminal peptide 1. One category of simple peptide tag α-fragment modifications was designed to mimic one or more properties of the integrase domain found in the β-fragment but missing in the α-fragment of type III RT. Partial mimicry of the integrase domain without the deleterious impact on solubility and host cell viability associated with the native integrase domain is a polynucleotide sequence encoding a His tag at the 3 'end of the modified RT coding region Was achieved by adding

The addition of a His tag to the C-terminus of the RT polynucleotide was fused in frame to the His codon.
Achieved by recombinant expression of a polynucleotide containing the RT coding region. In particular, the fusion uses ligase to ligate an oligonucleotide containing six histidine codons 3 'of the RT gene, as in the case of the construction of pBacMIKAhis.
By adding to the end or as in pBacMIBAhis, 6
Constructed by PCR amplification with oligonucleotides specifying two histidine codons.

The basic nature of the added His amino acids was predicted to increase binding to negatively charged nucleic acids and increase the stability of the polypeptide. The increased stability
This time it was expected to result in increased activity of amino acid tagged RT on their untagged counterparts. In addition, the His tag was predicted to chelate to metal ions (eg, Ni ⁺⁺ ), thereby enhancing the polymerization of the modified RT.

Native PAGE and Superose 12HR10 / 30 (separation range of 1-300 kDa; Pharmacia-Upjohn
), His-tagged RT (MIBAhis) was found in homopolymeric form (molecular weight greater than 200 kDa) as determined using molecular sieve chromatography at

The expression level of the RT fragment modified by amino acid tagging indicated that the addition of a peptide tag to the C-terminus of the AMV-RT alpha fragment stabilized the structurally unstable alpha fragment. .

Another modified RT bearing a peptide at the C-terminus of the α-like fragment was generated by PCR as described above. The forward and reverse PCR primers had codons corresponding to the-and C-termini of the AMV-RT alpha fragment, with codons corresponding to the peptide tag to be added. A linearized template containing the full length RT gene (pHSEMI) was used for PCR amplification. Additional information regarding this class of modified RTs, as well as the polynucleotides that encode them, is provided in Table 2.

The PCR product was restricted with appropriate restriction enzymes and ligated into pBacPak9 digested with the appropriate enzymes. Selected recombinants were sequenced to confirm the addition of the appropriate tag.

[0154] 2. The DNA motif of a C-terminal peptide protein having DNA binding properties can have any general affinity for DNA (ie, non-specific binding) or sequence-specific affinity. Several nucleic acid binding domains have been identified and reported to play roles in important cellular functions, such as viral packaging, transcription and translation regulation, nuclear and cytoplasmic transport, splicing and stability, among others. Karaya et al., J. Bi
ol. Chem. 266: 11621-11627 (1991); Burd, et al., Science 265: 615-621 (1994);
Weiss, et al., Biopolymers 48 (2-3): 167-180 (1998); Nassal, M., J. Virol.
66 (7): 4107-16 (1992) Ritt, et al., Biochemistry 37: 2673-81 (1998). DNA binding domains with general affinity are preferred for target-specific binding domains due to the reduced substrate specificity of modified RTs having such general binding domains.

Some basic amino acids are known to enhance the affinity of proteins for nucleic acid templates. The positive charge of arginine, lysine and histidine increases the non-specific affinity of polypeptides containing such residues for nucleic acids, thereby facilitating the search for specific binding sites. Several arginine-rich and arginine-lysine-rich motifs have been identified in the nucleic acid binding domain. Arginine-lysine-rich motif ELKIKRLR
KKFAQKMLRKARRK is involved in RNA binding, which could enhance the activity of RT. In addition, lysine-rich proteins are involved in DNA in kinetoplasts and play a role in the isolation of kinetoplast DNA. Hines, Mol. And Biochem. Parasito
l. 94: 41-52 (1998). Similarly, amino acid tags have been reported to be involved in the packing of viral DNA. This packing can be mediated by metal ions that have an affinity for DNA. International application publication number WO98 / 07869. In addition, charged amino acids are present on the surface of structural proteins and can play a role in stabilizing secondary structure.

The addition of histidine, glutamic and aspartic acid tags enhanced the activity of the alpha fragment by a factor of 20 to 100. A peptide tag consisting of six arginine residues improved the activity 5-fold. However, specific arginine-rich motifs such as RNRNRQY (Arg3X4, found at the C-terminus of the GP67 envelope glycoprotein that has been proposed to be involved in baculovirus DNA packaging) have a 20- to 40-fold increase in activity. Enhanced (ie, increased or facilitated). RRDRGR
Other RNA- and DNA-binding motifs such as S are expected to produce similar results. However, six consecutive lysine residues did not increase development. Larger numbers of lysine residues or correct spacing of lysine residues may be necessary for enhanced function.

The mechanism of enhanced activity by these tags may be due to increased structural stability of the recombinant or stability derived from direct or metal-mediated nucleic acid binding. M1BA 2000-3000 units / g Insect cells M1BA his 50000-200,000 U / g M1BA arg6 15,750 U / g M1BA lys6 2050 U / g M1BA Arg3X4 57,000 U / g M1BA glu6 170,000 U / g M1BA asp6 40,000 U / g M1BA leu6 2250 -3900 U / g Nhis M1BA asp4 95,000 U / g Nhis M1BA asp5 115,250 U / g Nhis M1BA asp6 236,250 U / g Sequence-specific DNA binding proteins are mostly common basic regions and DNA
Has a sequence-specific region for binding to Zinc-finger domain (
For example, there are several sequence-specific DNA binding motifs, such as TFIIIA CX2CX12HX3H) and the basic region of the bZIP family of proteins. Similarly, TRQARRNRRRWRARQR and YGRKKRR that recognize specific RNA sequences predicted to enhance the activity of RT
There are arginine-rich domains like QRRRP. The N-terminus of the RT integrase domain has a zinc-finger-like (Hx3HX23CX2C) motif. The N-terminus binds zinc, induces proper folding of the N-terminus, and is reported to be significantly thermostable. Burke et al., J. Biol. Chem. 267: 9639-44 (1992). full length
Since the MAV-RT gene has a zinc-finger-like domain, the reverse primer used in some PCR amplifications included the region of integrase (
Table 2).

The beta-like derivative containing the zinc-finger-like motif (620 amino acids) was more active than the untagged alpha fragment (578 amino acids) and was expressed 30,000 units per gram of cell pellet. MIBK620 31,950 U / g The addition of the MIBK620 his 50,000-140,000 U / g sequence-specific zinc-finger-like motif, however, resulted in a lower level of RT activity than the His-tagged fragment. These results indicate that the general nucleic acid binding domain (His tag) can enhance RT activity to a greater extent than the sequence-specific domain (zinc-finger-like motif), and thus the sequence-specific Suggests that the zinc-finger-like motif can be replaced, leading to increased activity. Common nucleic acid binding domains enhance the stability of both 578- and 620-amino acid fragments.

[0159] 3. The disulfide bond forming domain (cysteine-) present in the C-terminal peptide-tagged immunoglobulin gene having a polymerization domain
The rich region) is involved in disulfide bond formation between the light and heavy chains. Thus, the addition of two cysteine residues to the C-terminus was predicted to promote dimer formation through disulfide bonds.

[0160] The addition of two cysteine residues enhanced the activity of the alpha-like fragment; however, six consecutive cysteine residues reduced the activity of the modified RT. M1BA 2000-3000 U / g M1BA cyst2 190,000 U / g M1BA cyst6 720 U / g GPRP (fibrin clot forming) tetrapeptide is the major polymerization pocket of the blood clotting protein fibrin. This domain is exposed to the amino terminus of fibrin monomers by proteolytic cleavage of the precursor protein. Then
Upon binding to complementary binding sites on other fibrinogen molecules, the domains polymerize to form blood clots. Since the peptide was added to the C-terminus of the α-like construct, the reverse sequence tetrapeptide PRPG was also examined.

The addition of GPRP enhanced RT activity almost 50-fold, while the addition of PRPG almost increased RT activity.
Increased by 100 times. In another embodiment, the D-isomer of the amino acid is used in the peptide tag. For example, the D-isomer is used to generate the PRPG peptide used to prepare the modified RT of the present invention. M1BA 2000-3000 U / g M1BA GPRP 107,500 U / g M1BA GRPG 243,500 U / g Histidine residues can promote metal ion-mediated dimer formation. The addition of 6 His residues to the C-terminus of α-like RT resulted in an activity of 20 to
Increased 40 times. The addition of histidine tags of different lengths is conceivable. M1BA 2000-3000 U / g M1BA his 50000-200000 U / g NS1 is a DNA-binding protein produced by mouse minute virus. The protein has replication and transcription functions. The homo-oligomerization of NS1 is required for its function, and a small region of NS1 N-VETTVTTAQETKRGRIQ
TK-C has been identified as a domain involved in oligomerization. Pugol et al., JV
irol71: 7393-7403 (1997). Addition of this peptide tag to the C-terminus of the AMV-RT fragment enhanced RT activity. M1BA 2000-3000 U / g M1BA NSI 380,000 U / g 4. C-terminal peptide tag with metal binding domain As described above, a histidine tag can be used as a metal binding domain. In addition, a modified RT with a C-terminal His tag was constructed and subjected to expression analysis. The above results indicate that a peptide tag with metal binding ability enhances RT expression.

[0162] Zinc fingers also exhibit metal binding ability and are involved in DNA binding. As described above, the N-terminus of the integrase domain of MAV-RT has a zinc-finger-like (
Hx3HX23CX2C) motif. The N-terminus is reported to bind zinc and induce proper folding of the N-terminus. Peptide tags containing one or more zinc-finger-like domains are expected to enhance the activity of the modified RT in which they are found.

[0163] 5. C-Terminal Peptide Tag with Structure-Stabilizing Domain Another embodiment of the present invention provides a structurally stable alpha-like fragment so that it no longer requires a second fragment for structural stability. Includes the addition of domains designed to Among other things, there are several motifs, all of which have been identified and shown to form specific structures, such as alpha-helix, beta-sheets and coils known in the art. The formation of a defined structure facilitates the formation of an active domain and facilitates interaction with other such domains. Beta strands and beta sheets often promote aggregation in solution and form a precipitate from solution. Desjarlais et al., Curr. Opin. IN Biotechnol. 6
: 460-466 (1995). Thus, most of the C-terminal tagging was able to form a helix or coil. The prediction of these secondary structures is based on the well-known Chou and Fassman algorithm.

The WEAAH (WH) motif, consisting of histidine and tryptophan, promotes the formation of an alpha helix or a defined structure, thereby conferring structural stability on the protein. Addition of the M1BA 2000-3000 U / g M1BA WH 104,720 U / g WH domain can extend the C-terminal helix, thereby enhancing the stability of the alpha fragment. However, for whatever reason, modified RTs containing the WH motif exhibit enhanced RT activity.

The “PPG” triple helix domain is responsible for binding interactions in the structural protein collagen. This motif is responsible for the structural stability and proper assembly of collagen. The addition of a peptide containing this motif in the generation of a modified RT according to the present invention will result in such a
It is expected to enhance the activity of RT.

The addition of tryptophan residues is expected to extend the C-terminal α-helix and enhance the stability of the alpha-like fragment. Tryptophan is a bulky amino acid and could replace the histidine tag in providing structural stability. Comparative assays showed that the domain containing the Trp residue enhanced RT activity almost 50-fold. M1BA 2000-3000 U / g M1BA Trp 96,500 U / g GPRP and PRPG motifs identified in fibrin as domains involved in interactions with other coagulation proteins enhance the activity of AMV-RT alpha-like fragments . This motif is predicted to form a coil-turn-coil structure. M1BA 2000-3000 U / g M1BA GPRP 107,500 U / g M1BA PRPG 243,500 U / g The NS1 domain mainly forms beta sheets and coils. The presence of a hydrophobic residue alone is not highly desirable. This is because it forms beta sheets, which are typically buried in the secondary structure of the protein. This can affect the natural folding of the domain. Therefore, motifs with a mixture of coils and beta sheets were selected for analysis. Addition of this domain resulted in an active α-like fragment that appeared to be stable. M1BA 2000-3000 U / g M1BA NS1 380,000 U / g The leucine zipper motif is a helix-turn-helix motif that has been reported to dimerize by coiled-coil interaction. This defined structure of leucine zippers is expected to enhance the stability of alpha-like fragments in addition to providing dimerization capabilities. M1BA 2000-3000 U / g M1BA Lzip23 7170 U / g M1BA Lzip3 1620 U / g Addition of a single heptad repeat enhanced activity 2-3 fold. Addition of two heptad repeats did not improve activity. However, the addition of 4-5 heptad repeats resulted in RT with reduced activity levels.

[0167] 6. N-Terminal Peptide Tags Several structures were formed and characterized as described in Examples 3 and 4 for N-terminal peptide tags added to modified RTs that exhibited enhanced expression. 1
One modified RT, NhisMIBA, contained a His tag attached to the N-terminus of the α-like fragment. Other RTs were modified to contain peptide tags at both ends (Nhis MIB
A asp4, Nhis MIBA asp5, Nhis MIBA asp6 and Nhis MIBA WH). Expression experiments performed as described in Example 4 led to the following results. Nhis M1BA 10,000-41,700 U / g M1BA Chis 50,000-20,000 U / g Nhis M1BA asp 4 95,000 U / g Nhis M1BA asp 5 115,250 U / g Nhis M1BA asp 6 236,250 U / g Nhis M1BA WH 86,000 U / g Expression of MIBAChis Was measured to provide a relative control for measurement of Nhis MIBA expression. The result is a RT modified with a His tag at either the N-terminus or C-terminus.
Shows increased activity against untagged RT. Other variants, such as the addition of a peptide tag to both ends of RT (eg, an N-terminal His tag coupled to a C-terminal Asp-, Glu- or Trp-His- (ie, WH) tag) are also described. Invented by the invention.
Large-scale expression experiments indicate that nearly 100,000 units / g insect cells with similar activity levels
1BA asp (N-terminal modified RT) and Nhis M1BA asp (N-terminal 6 His residues and C-
RT with 4-6 Asp residues at the end.

[0168] 7. Peptide Tagging of Other Type III RTs The strategy was also used to modify RT from other avian sources, such as Rous sarcoma and avian tumor virus. The C-terminal of the 6-histidine peptide to the alpha fragment of each of these avian RTs substantially increased RT activity relative to the untagged AMV-RTα-like fragment. M1BA 2000-3000 U / g RSV-RT 43,350 U / g ATV-RT 71,900 U / g Therefore, the modification strategy applied to AMV-RT polynucleotides and polypeptides is:
More generally applicable to dimer (ie, type II and type III) reverse transcriptase coding regions and peptides, and all of these modified RTs are within the scope of the present invention.

C. Modifications of Beta-Like Recombinant Beta RT Polynucleotides encoding various beta-like modified RTs were constructed using the technique described in Example 3, along with M1-5,6 encoding full length AMV-RT. , Using the technique described in Example 4. Full-length beta-expression resulted in low levels of highly insoluble full-length protein in both eukaryotic (insect cells) and prokaryotic (E. coli) hosts. Since expression of the full-length beta fragment results in an almost insoluble protein, the native beta polypeptide has its solubility, and thus,
Modified in an effort to increase activity. One strategy for modifying the beta fragment involves deletion of a portion of the native beta RT. The native beta coding region specifies 858 amino acids, and the full length β-like fragment disclosed herein consists of 832 amino acids.
Thus, the β-like fragment lacks the 26 C-terminal amino acids of full length native β. Full length natural β
In contrast, expression of full-length β-like polypeptide showed a 100-fold increase in expression, as shown by SGS-PAGE analysis; however, β-like polypeptide was still highly insoluble (almost 90% insoluble ), Resulting in a 5-fold increase in activity. M1KA 1000 U / liter cells M1KAhis 2,000 U / L M1-5 200 U / L Also modified RT with C-terminal between 580 and 832 amino acids (SEQ ID NO:
2) are also contemplated in the present invention. The 580- and 620-amino acid recombinants are both soluble, and the 832- and 858-amino acid recombinants are relatively insoluble, so that the C-terminus is truncated to a position between 580-832 amino acids. Deletions are expected to result in the modified β-like polypeptide being soluble. In particular embodiments, the β-like polypeptide is 237, 217, 197, 117, 77, respectively, relative to full length βRT.
And has a C-terminus at any one of positions 580-832, such as at positions 620, 640, 660, 740, 780 or 800 resulting from a deletion excluding 57 amino acids (
SEQ ID NO: 2). Construction and expression of a deletion derivative specifying a 620 amino acid modified β-like RT was accomplished as described generally in Examples 3 and 4, and the results of the expression are shown below. M1KA 2000-3000 U / g M1KA 1000 U / L M1BK 620 31,950 U / g Thus, truncated β-like RT shows considerable activity, consistent with increased solubility in full-length native βRT.

Similar modifications of other avian RTs to the corresponding β polypeptide result in similarly increased RT activity. RSV-RT 620 his 33,000 U / g In addition to the 3 ′ deletion resulting in a polynucleotide encoding a β-like polypeptide having a C-terminus in the range of positions 580-832, preferably in the range of 620-800 (sequence number:
2) In the present invention, a polynucleotide having an internal deletion relative to the natural β gene,
Also contemplated are polypeptides encoded by polynucleotides having such internal deletions. The central core region of the integrase domain is
Relevant for DNA cleavage and conjugation properties of V-RT.

The core region of the integrase domain was deleted to varying degrees (region between amino acids 620-770, 640-770 or 660-770 of SEQ ID NO: 2), for example, using standard techniques to obtain M1BK Cint Lacks amino acids 620-722 of SEQ ID NO: 2. This method involved the initial construction of a first polynucleotide fragment encoding a C-terminally truncated β-like fragment using PCR with the full length AMV-RT pol gene as a template (see Table 2). . po
l a second fragment containing 3 'ends of various lengths 3' of the end of the gene (ie 3 ')
Was also constructed using PCR. These 3 'fragments encoded the C-terminal region of the integrase domain; some 3' fragments contained part (but not all) of the core region of the integrase domain. One of skill in the art can recognize that the first polynucleotide fragment or 5 'fragment encodes a peptide tag at their 5'end; and that the 3 'fragment has the tag encoded by the 5' fragment. With or without a peptide tag (e.g., see 3 'fragment 2a in Table 2), these tag-coding fragments can be used to encode the PCR primers disclosed herein (e.g., F Cint XhoI (SEQ ID NO: 85) and R Cint830 His XhoI (SEQ ID NO: 9)
It is easily synthesized using 1)). The final step in generating constructs with internal deletions is the truncated β-, as determined by routine screening of ligation products.
Was to ligate the coding regions in the appropriate order and orientation to the 3 'fragment. In one embodiment, amino acids 620-770 were deleted, thereby removing the core region of the integrase domain. Then, the C of the integrase domain
-The terminal region was placed adjacent to the N-terminal region of the domain.

Expression of such constructs in insect cells revealed increased solubility (10-20%) and activity in full-length intact βRT, as described below. Amino acids 620-731, 64
Effective removal of some or all of the central regions of the integrase domain, such as deletions of 0-771, 640-731, 660-771, 660-731, 680-771, 680-731, and 740-771 Other deletions (SEQ ID NO: 2) are contemplated by the present invention. M1KA 1000 U / L M1-5 200 U / L Some modified β fragments have a terminal peptide tab. Thus, the present invention contemplates internal deletions and, optionally, modified RTs having a peptide tag at the N-terminus, C-terminus, or both termini. In addition, for α-like modified RTs, along with the polynucleotides that encode them, β-like modified RTs can be derived from any type II or type III RT.

[0173] Any of the modified RTs of the present invention can be produced by any of the processes disclosed herein or known in the art, such as in vivo synthesis, in vitro synthesis or chemical synthesis. . In addition, any of these processes can be used to produce various forms of active polypeptides, including monomers, homodimers or homomultimers, heterodimers and heteromultimers, all of which are included in the present invention. In particular, the expression of the modified β-like fragment MIBK620 Cint results in the expression of a heterodimeric form of RT, the beta-like fragment is processed as expected, and the α polypeptide associated with the modified β-like peptide Is suggested. Other core domain deletions (eg, amino acid 620 of SEQ ID NO: 2)
-731, 640-771, 640-731, 660-771, 660-731, 680-771, 680-731 or 740-771
Other modified RTs of the invention, such as (β-like fragments lacking
For example, it is expected to show activity in a heterodimeric form. In addition, whether the polypeptide is produced by in vivo or in vitro expression, or by chemical synthesis, heterodimers or other non-monomer forms can be modified α-like polypeptides and native β It can arise from the interaction of polypeptides or from modified α-like and modified β-like polypeptides.

While the invention has been described with respect to particular embodiments, many modifications and changes will occur to those skilled in the art in view of the preferred embodiments of the invention. Accordingly, the scope of the invention should be limited only by the claims provided in the appended claims.

[0175]

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a photograph showing the result of Western blot analysis of a reverse transcriptase expression product in insect cells.

FIG. 2 shows recombinant reverse transcriptase fragmented on an 8% SDS-PAGE gel and stained with Coomassie blue.

FIG. 3 is an autoradiograph of cDNA fragmented on a gel produced with a reverse transcriptase polypeptide according to the present invention.

FIG. 4 is a graph showing the production and temperature of cDNA using natural reverse transcriptase and recombinant reverse transcriptase (FIG. 4A), and a graph showing temperature of natural reverse transcriptase and recombinant reverse transcriptase catalyzing RT-PCR (FIG. 4A). 4B), temperature profile for reverse transcriptase-mediated RAMP (FIG. 4C), graphs for native and recombinant reverse transcriptase in pH and reverse transcriptase assays (FIGS. 4D and 4E), magnesium ion and reverse transcription. Graphs for native and recombinant reverse transcriptase in an enzyme assay (FIG. 4F) and for native and recombinant reverse transcriptase in other divalent cation and reverse transcriptase assays (FIG. 4G). Photographically shown.

FIG. 5 shows the relative activities of DNA-dependent DNA polymerases for native and recombinant reverse transcriptase.

FIG. 6 is a graph for the relative comparison of the ribonuclease activities of native and recombinant reverse transcriptase at 37 ° C. (FIG. 6A) and a temperature graph of the ribonuclease H activity of the recombinant reverse transcriptase (FIG. 6B). is there.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (Reference) 4B024 AA03 AA11 AA20 BA10 CA04 DA02 DA06 EA04 GA11 HA01 HA08 HA19 4B050 CC01 CC04 DD01 LL10 4B063 QA13 QQ41 QR07 QR62 QS25 4B065 AA26X AA90X AA97Y AB01 AC14 BA02 CA29 CA46

Claims (33)

[Claims] 1. An isolated polynucleotide that encodes a polypeptide having RNA-dependent DNA polymerase activity, ie, the polypeptide comprises: (a) beginning with the first amino acid of SEQ ID NO: 2; An amino acid sequence ending with any one of amino acids 857 to 857; (b) an amino acid sequence starting with the first amino acid of SEQ ID NO: 39 and ending with any one of amino acids 428 to 1054; An amino acid sequence beginning with the first amino acid of SEQ ID NO: 41 and ending with any one of the amino acids of positions 548 to 1198; (d) starting with the first amino acid of SEQ ID NO: 43 and having positions of 428 to 901 An amino acid sequence ending with any one of the amino acids; and (e) a variant, analogous pair or fragment of any of the subelements (a) to (e) having RNA-dependent DNA polymerase activity; Body, similar pairs and fragments has the N- terminal methionine arbitrarily. 2. The polynucleotide according to claim 1, wherein the polypeptide of the amino acid sequence (a) consists of an amino acid sequence beginning with the first amino acid of SEQ ID NO: 2 and ending with about 578 amino acids. 3. The polynucleotide according to claim 1, wherein the polypeptide of the amino acid sequence (a) comprises the amino acid sequence of SEQ ID NO: 4. 4. The polynucleotide according to claim 1, which has a sequence selected from the group consisting of the sequences set forth in any one of SEQ ID NOs: 1, 6 to 10, 38, 40 and 42. 5. The polynucleotide according to claim 1, wherein the polynucleotide is DNA. 6. The polynucleotide of claim 1, wherein said polynucleotide encodes a polypeptide lacking effective integrase activity. 7. The polynucleotide of claim 6, wherein said polynucleotide lacks at least a portion of an integrase coding region. 8. The polynucleotide of claim 1, further comprising a contiguous polynucleotide encoding at least one terminal modification of said polypeptide selected from the group consisting of an N-terminal modification and a C-terminal modification. 9. The polynucleotide of claim 8, wherein said modification is a cysteine residue adjacent to the C-terminus of said polypeptide. 10. The polynucleotide of claim 8, wherein said adjacent polynucleotide encodes a polypeptide consisting of a C-terminal modification. 11. The C-terminal polypeptide comprises 4 to 50 amino acids, and the polypeptide comprises a DNA binding domain, an RNA binding domain, a metal binding domain, a structure stabilizing binding domain, and a polymerization domain. 11. The polynucleotide according to claim 10, comprising a domain selected from the group consisting of: 12. The polypeptide, wherein the polypeptide is an acidic amino acid domain, a basic amino acid domain, a W domain, a WH domain, a zinc finger-like domain, a leucine zipper domain, a PPG domain, an NS1 domain, a GPRP domain, and a PR.
12. The polynucleotide according to claim 11, comprising a PG domain. 13. The C-terminal peptide comprises six amino acids.
3. The polynucleotide according to item 1. 14. The polynucleotide of claim 11, wherein said C-terminal peptide comprises the same amino acid. 15. The method of claim 15, wherein the C-terminal peptide comprises a basic amino acid.
12. The polynucleotide according to item 11, 16. The polynucleotide according to claim 15, wherein the basic amino acid is histidine. 17. The polynucleotide according to claim 8, having a sequence selected from the group consisting of the sequences set forth in any one of SEQ ID NOs: 11 to 19. 18. A vector comprising the polynucleotide according to claim 1. 19. The vector according to claim 18, wherein said polynucleotide is operably linked to a promoter. 20. A host cell transformed with the vector according to claim 18. 21. The host cell according to claim 20, wherein said host cell is a eukaryotic cell. 22. The host cell of claim 20, wherein said host cell is selected from the group consisting of E. coli and insect cells. 23. An isolated polypeptide encoded by the polynucleotide according to any one of claims 1 to 5. 24. An isolated polypeptide encoded by the polynucleotide according to any one of claims 6 to 17. 25. A method of transforming a host cell, comprising the steps of: (a) introducing the vector of claim 18 into the host cell; (b) incubating the host cell; (c) identifying a host cell containing the vector, thereby identifying a transformed host cell. 26. A method for producing an isolated reverse transcriptase polypeptide, comprising the steps of: (a) introducing the vector according to claim 18 into a host cell; Incubating said host cells under conditions suitable for expression, and (c) recovering said polypeptide, thereby producing an isolated reverse transcriptase polypeptide. 27. A method for copying a target nucleic acid by extending a primer to which the target nucleic acid is bound in the presence of a polymerase, wherein (a) the target nucleic acid and the primer are copied according to any one of claims 23 and 24. Contacting with a polypeptide. 28. The method of claim 27, wherein said copy produces a plurality of copies of said target nucleic acid. 29. The method of claim 27, wherein said polypeptide is in a form selected from the group consisting of a monomer and a polymer. 30. The method comprises: cDNA synthesis, polymerase chain reaction, polymerase chain reaction-reverse transcription, reverse polymerase chain reaction, multiple polymerase chain reaction, strand displacement amplification, multiple strand displacement amplification, nucleic acid sequence-based amplification, sequence specific strand. 28. The method of claim 27, wherein the method is selected from the group consisting of replication and rapid amplification. 31. A method for sequencing a target nucleic acid by extending a primer to which a target nucleic acid bond is bound, wherein (a) the target nucleic acid and the primer are the polypeptide according to any one of claims 23 and 24. An improved method, including contacting with. 32. The method of claim 31, wherein said polypeptide is in a form selected from the group consisting of a monomer and a polymer. 33. A kit for copying a target nucleic acid, comprising: (a) one or more nucleotides; and (b) SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and polynucleotide derivatives thereof that encode a C-terminal modification at their 3 'end A polypeptide encoded by a polynucleotide having a sequence selected from the group consisting of: