# Patent

## Title
Recombinant murine leukemia virus reverse transcriptases, the genes encoding and the method for expressing it

## Abstract

This invention provides recombinant murine leukemia virus reverse transcriptases, genes encoding these proteins and expression methods. The said murine leukemia virus reverse transcriptases are a series of MLV-RT proteins wherein the 84th amino acid residue (Q84) from the N-terminus is replaced with amino acid X, which is an amino acid with a side chain shorter than glutamine. The said murine leukemia virus reverse transcriptases have higher enzyme activity and processivity than the wild type enzyme, and are expected to be widely used in the field of biotechnology for cDNA synthesis.

## Background
TECHNOLOGY CATEGORY
This invention involves recombinant reverse transcriptases, their coding genes and expression methods in the field of biotechnology. This invention particularly involves recombinant murine leukemia virus reverse transcriptases, their coding genes and expression methods.
BACKGROUND OF THE INVENTION
Reverse transcriptase (RT) is a kind of DNA polymerase encoded by retroviruses, which can synthesize DNA using DNA or RNA as template. Reverse transcriptase, which can convert RNA into cDNA, is widely used in molecular biology, including constructing cDNA libraries and analyzing the amount of RNA in biological samples by RT-PCR. Nowadays the dominant RT in the market is murine leukemia virus reverse transcriptase (MLV-RT).
MLV-RT is composed of two functional domains, a DNA polymerase domain at the N-terminus and a ribonuclease (RNase) H domain at the C-terminus. These two domains can be expressed separately without affecting the function of each other. The first generation of recombinant MLV-RT used in cDNA synthesis contained only the DNA polymerase domain, with the RNase H domain deleted. Though the enzymatic activity is similar to that of full-length MLV-RT, its processivity is relatively poor, resulting in short cDNAs. It was found that although the RNase H domain does not affect the DNA polymerase activity of MLV-RT, it affects the enzyme's processivity. The reason is that the RNase H domain binds to the template/primer complex, and thereby increases the enzyme's affinity for the template/primer. When the 524th Asp, a key residue in the RNase H active site, is replaced with Asn by site-directed mutagensis, the resulting mutant enzyme, MLV-RT-D524N, does not demonstrate any RNase H activity, but retains the DNA polymerase activity and high affinity for the template/primer. This mutant enzyme, which is patented by Invitrogen under trade mark Superscript II, is currently widely used. However, this mutant enzyme is still not perfect; its processivity is still not ideal and the most common problem encountered is the formation of short, less than full-length products.
SUMMARY OF THE INVENTION
This invention provides murine leukemia virus reverse transcriptases, named MLV-RT-Q84X, wherein the 84th amino acid residue (Q84) from the N-terminus (i.e., Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gin Phe Gly Pro Val Val Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu, see SEQ ID NO:9), wild type, is replaced with X, which represents a residue with a side chain shorter than that of glutamine.
The invention further provides murine leukemia virus reverse transcriptases, named MLVRT-Q84X-D524N, wherein the 524th aspartic acid residue from the N-terminus is replaced with asparagines (Asn), and the 84th amino acid residue (Q84) from the N-terminus is replaced with X, which represents a residue with a side chain shorter than that of glutamine.
The invention further provides murine leukaemia virus reverse transcriptases, MLV-RT-Q84X and MLV-RT-Q84X-D524N, wherein X is preferably chosen from alanine (Ala), serine (Ser), asparagine (Asn), or aspartic acid (Asp). Alanine is especially favoured.
The invention further provides the coding sequences of the recombinant murine leukemia virus reverse transcriptases.
The invention further provides a method of expressing the recombinant murine leukemia virus reverse transcriptases comprising: a) transforming the murine leukaemia virus reverse transcriptases expressing plasmids into Escherichia coli; b) culturing the clones to express recombinant murine leukemia virus reverse transcriptases. These recombinant murine leukemia virus reverse transcriptases are a series of MLV-RT proteins wherein the 84th amino acid residue (Q84) from N-terminus is replaced by a residue that has a side chain shorter than that of glutamine.
The invention also provides a method of expressing the recombinant murine leukemia virus reverse transcriptases comprising: a) transforming the murine leukaemia virus reverse transcriptases expressing plasmids into Escherichia coli; b) culturing the clones to express recombinant murine leukemia virus reverse transcriptases. These recombinant murine leukemia virus reverse transcriptases are a series of MLV-RT proteins wherein the 524th aspartic acid residue from the N-terminus is replaced with asparagine, and the 84th amino acid residue (Q84) from N-terminus is replaced by a residue that has a side chain shorter than that of glutamine. The 84th amino acid is preferably replaced by alalnine.
The invention further provides nucleotide sequences of plasmids pTacRT-Q84N-D524N and TacRT-Q84A-D524N as described in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. pTacRT-Q84N-D524N and TacRT-Q84A-D524N express murine leukaemia virus reverse transcriptases with amino acid sequences described in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The host cell expressing these proteins is Escherichia coli BL21. Both SEQ ID NO: 1 and SEQ ID NO: 3 are composed of 7488 nucleotides with an open reading frame between the 1515th and 3527th nucleotides. Both SEQ ID NO: 2 and SEQ ID NO: 4 are composed of 671 amino acids.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 SDS-PAGE analysis of purified MLV-RT-Q84A-D524N
FIG. 2 Kinetic analysis of MLV-RT-Q84A-D524N and MLV-RT-D524N
FIG. 3 The first strand cDNA synthesis by MLV-RT-Q84A-D524N and MLV-RT-D524N.
FIG. 4 SDS-PAGE analysis of purified MLV-RT-Q84N-D524N
FIG. 5 DNA polymerase activity assay of MLV-RT-Q84N-D524N



EXPERIMENTAL DETAILS
Experiment 1: Preparation of MLV-RT-Q84A-D524N
1, Construction of Plasmid pTacRT-Q84A-D524N
The Q84A substitution was introduced into MLV-RT-D524N to generate MLV-RT-Q84A-D524N.
The Q84A mutation in the MLV-RT-D524N (Blain, S. W & Goff, S. P. (1995) J. Virol. 69, 4440-4452.) backbone was constructed by replacing the AflII-MfeI fragment of pTacRT-D524N (nt1467-2058) with two PCR-derived fragments AflII-EcoRIandEcoRI-Mfel. The 300 bp AflII-EcoRI fragment was generated using forward primer
(5′GTGGAATTGTGAGCCGA)(SEQ ID NO: 5) and a mutation specific reverse primer Q84A-AP (5′CGGAATTCCCGCGTCCAACAGTCTCTGTA)(SEQ ID NO: 6) bearing silent mutations creating a restriction site; the 300 bp EcoRI-MfeI fragment was generated using reverse primer
(5′TGGGAGTCTGGTCCAGG)SEQ ID NO: 7) and a mutation specific forward primer Q84A-SP (5′CGGAATTCTGGTACCCTGCCAGTC)(SEQ ID NO: 8) bearing silent mutations creating the same restriction site as created in the 5′ fragment. The codon for alanine was built in the mutation specific primers. The restriction sites built in the primers are underlined. The AflII-EcoRI fragment (nt1467-1770) and EcoRI-MfeI fragment (nt1770-2058) were inserted into a 6.9 kb vector, pTacRT-D524N, which was digested with AflII/-MfeI. The ligation mixture was transformed into Escherichia coli Top 10 and pTacRT-Q84A-D524N clones were picked based on restriction enzyme deigestion analysis. The result of nucleotide sequencing showed that the sequence of pTacRT-Q84A-D524N was the same as the sequence in SEQ ID NO: 1.
2, Expression of Recombinant Reverse Transcriptases in Escherichia coli 
Escherichia coli BL21 cells transformed with pTacRT-Q84A-D524N were inoculated in LB medium containing 100 μg/ml ampicillin at 37° C. When the cells were grown to a density of OD600 0.5, IPTG was added to the medium at the final concentration of 0.5 mM to induce RT expression. The cells were cultured for additional 2-3 hours at 37° C. At the end of the induction, the cells were harvested by centrifugation and washed once with 50 mM ice-cold Tris-HCl (pH 7.5) for further RT purification.
3, Purification of the RT Proteins
The cells were resuspended in buffer A (20 mM sodium phosphate (pH 7.4), 0.5 M NaCl) containing lysozyme at a final concentration of 0.5 mg/ml and incubated on ice for 30 min. The suspension was briefly sonicated and then cleared of debris by centrifugation. After HiTrap chelating HP column (Pharmacia) purification, the purity of RT was higher than 80%, as analyzed by SDS-PAGE. The enzyme was purified to near homogeneity by MonoS (Pharmacia) fast protein liquid chromatography (FPLC), as analyzed by SDS-PAGE. MLV-RT-Q84A-D524N is a nearly homogenous band of 76 kD in the gel after Coomasia Brilliant Blue staining (FIG. 1). FIG. 1: M, Molecular Weight Standard; 1, 5/g protein; 2, 2/g protein.
4, Homopolymer Assays and Kinetic Analysis
Typical assays were performed at 37° C. using 10 ng of RT in 50 μl of reaction containing 60 mM Tris.HCl (pH 8.0), 75 mM NaCl, 0.7 mM MnCl2, 5 mM DTT, 12 μg/ml poly(rA) template, 6 μg/ml oligo(dT)18 primer, 10 μCi/ml 32P-labeled dTTP (1 Ci=37 GBq) and 12 μM unlabeled dTTP. At each time point, 4/1 of the reaction was removed and spotted on DE81 paper (Whatman). The paper was washed twice with 2× standard saline citrate (SSC), followed by scintillation counting.
To measure the kinetic parameters, the enzyme was added to the reaction mix to initiate the reaction. The radioactivity retained on the paper, in comparison with the total radioactivity in each sample, was used to determine the amount of dTTP incorporated into the product. The kinetic parameters were determined by double reciprocal plot (FIG. 2). While MLV-RT-Q84A-D524N and MLV-RT-D524N displayed comparable affinities (Km) for dTTP (11.04 μM and 12.94 μM, respectively), the catalytic activity (Vmax) for dTTP of MLV-RT-Q84A-D524N was 3.2 times the level of MLV-RT-D524N (0.41 μmol·min−1·ng−1 and 0.13 μmol·min−1·ng−1, respectively) (Table. 1).







TABLE 1







Comparason of Kinetic Patameters between


MLV-RT-Q84A-D524N and MLV-RT-D524N












Vmax




Enzyme
(μmol · min−1 · ng−1)
Km (μM)







RT-D524N
0.13 ± 0.04
12.94 ± 2.08



RT-Q84A-D524N
0.41 ± 0.04
11.05 ± 0.72










5, First Strand cDNA Synthesis:

Here we show the difference in first strand cDNA synthesis between MLV-RT-Q84A-D524N and MLV-RT-D524N.
The total RNA of Rat-2 cells was isolated using the RNeasy kit (Qiagen) following the manufacturer's instructions. The first strand cDNA synthesis was performed in 20 μl of reaction containing 50 mM Tris.HCl (pH 8.0), 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 500 μM dNTPs, 20 u RNasin, 10 μCi α-32P-dCTP (1 Ci=37 GBq), 1 μg of total RNA template. The reaction mixture was preheated at 42° C. for 2 min before 0.5 μg RT was added to initiate DNA synthesis. The reaction was carried out at 42° C. for 1 hour and then stopped by heating at 70° C. for 15 min. The products were analyzed by electrophoresis on a 1.4% alkaline agarose gel, followed by autoradiography. As shown in FIG. 3, RT-Q84A-D524N synthesizes longer and more cDNAs than RT-D524N.
Example 2
Preparation of MLV-RT-Q84N-D524N
1, Construction of Plasmid pTacRT-Q84N-D524N
The of Q84N substitution was introduced into MLV-RT-D524N to generate MLV-RT-Q84N-D524N.
The Q84N mutation in the MLV-RT-D524N (Blain, S. W & Goff, S. P. (1995) J. Virol. 69, 4440-4452) backbone was constructed by replacing the AflII-MfeI fragment of pTacRT-D524N (nt1467-2058) with two PCR-derived fragments Afl-EcoRI and EcoRI-MfeI. The 300 bp AflII-EcoRI fragment was generated using forward primer
(5′GTGGAATTGTGAGCCGA)(SEQ ID NO: 5) and a mutation specific reverse primer Q84N-AP (5′CGGGATCCCGTTGTCCAACAGTCTCTGTA)(SEQ ID NO:6) bearing silent mutations creating a restriction site; the 300 bp EcoRI-MfeI fragment was generated using reverse primer
(5′TGGGAGTCTGGTCCAGG)(SEQ ID NO: 7) and a mutation specific forward primer Q84N-SP (5′CGGGATCCTGGTACCCTGCCAGTC)(SEQ ID NO: 8) bearing silent mutations creating the same restriction site as created in the 5′ fragment. The codon for asparagine was built in the mutation specific primers. The restriction sites built in the primers are underlined. The AflII-BamHI fragment (nt1467-1770) and BamHI-MfeI fragment (nt1770-2058) were inserted into a 6.9 kb vector, pTacRT-D524N, which was digested with AflII and MfeI. The result of nucleotide sequencing showed that the sequence of pTacRT-Q84N-D524N was the same as the sequence in SEQ ID NO: 3.
2, Expression of Recombinant Reverse Transcriptases in E. coli 
Escherichia coli BL21 cells transformed with pTacRT-Q84N-D524N were inoculated in LB medium containing 100 μg/ml ampicillin at 37° C. When the cells were grown to a density of OD600 0.5, IPTG was added to the medium at the final concentration of 0.5 mM to induce RT expression. The cells were cultured for additional 2-3 hours at 37° C. At the end of the induction, the cells were harvested by centrifugation and washed once with 50 mM ice-cold Tris-HCl (pH 7.5) for further RT purification.
3, Purification of Recombinant Reverse Transcriptase
The cells were resuspended in buffer A (20 mM sodium phosphate (pH 7.4), 0.5 M NaCl) containing lysozyme at a final concentration of 0.5 mg/ml and incubated on ice for 30 min. The suspension was briefly sonicated and then cleared of debris by centrifugation. After HiTrap chelating HP column (Pharmacia) purification, the purity of RT was higher than 80%, as analyzed by SDS-PAGE. The enzyme was purified to near homogeneity by MonoS (Pharmacia) fast protein liquid chromatography (FPLC), as analyzed by SDS-PAC-E. MLV-RT-Q84N-D524N is a nearly homogenous band of 76 kD in the gel after Coomasia Brilliant Blue staining (FIG. 4). FIG. 4: M, Molecular Weight Standard; 1, 1 μg protein; 2, 2 μg protein; 3, 5 μg protein.
5, Homopolymer Substrate Assays
Typical assays were performed at 37° C. using 10 ng of RT in 50 μl of reaction containing 60 mM Tris.HCl (pH 8.0), 75 mM NaCl, 0.7 mM MnCl2, 5 mM DTT, 12 μg/ml poly(rA) template, 6 μg/ml oligo(dT)18 primer, 10 μCi/ml 32P-labeled dTTP (1 Ci=37 GBq) and 12 μM unlabeled dTTP. At each time point, 4 μl of the reaction was removed and spotted on DE81 paper (Whatman). The paper was washed twice with 2× standard saline citrate (SSC), followed by autoradiography.
MLV-RT-Q84N-D524N manifested higher activities than RT-WT-H but had a similar activity to MLV-RT-Q84A-D524N (FIG. 5).
Application
Based on the high-resolution structure of a catalytically active fragment of MLV-RT, we discovered that the 84th amino acid residue of this enzyme, glutamine (Q84), which is located in the active site, regulates the catalytic activity of the enzyme. The long side chain of this residue presumably blocks the elongation of the products and affects the DNA polymerase activity and processivity. By replacing Q84 with an amino acid residue with a side chain shorter than that of glutamine, we generated a new enzyme, MLV-RT-Q84X, wherein X is an amino acid with a side chain shorter than that of glutamine, like alanine (Ala), serine (Ser), asparagine (Asn), or aspartic acid (Asp) and so on. In this invention the Q84X substitution was also introduced into MLV-RT-D524N, to generate MLV-RT-Q84X-D524N. These recombinant murine leukemia virus reverse transcriptases demonstrate higher enzyme activity and processivity than MLV-RT-D524N (Superscript II). These new enzymes are expected to be widely used in the field of biotechnology, particularly for cDNA synthesis.

## Claims

1. A variant of the Moloney murine leukemia virus reverse transcriptase of SEQ ID NO:2, wherein said variant has the amino acid sequence of SEQ ID NO:2 except having the amino acid X at position 84, which is an amino acid with a side chain shorter than that of glutamine, and said variant has reverse transcriptase activity.

2. The variant of claim 1, except having the amino acid asparagine at position 524 replaced with the amino acid aspartic acid.

3. The variant of claim 1, wherein the amino acid X is alanine, serine, aspartic acid or asparagine.

4. The variant of claim 3, wherein the amino acid X is alanine.

5. The variant of claim 2, wherein the amino acid X is alanine, serine, aspartic acid, or asparagine.

6. The variant of claim 5, wherein the amino acid is alanine.

7. A variant of the wild type Moloney murine leukemia virus reverse transcriptase having the amino acid sequence of SEQ ID NO:9, wherein said variant has the amino acid sequence of SEQ ID NO:9 except having the amino acid mutation at position 84, such that the glutamine is replaced with amino acid X, wherein said amino acid X is an amino acid with side chain shorter than that of glutamine, and said variant has reverse transcriptase activity.