A Novel Approach to Cloning and Expression of Human Thymidylate Synthase

Thymidylate synthase (TS, E 2.1.1.45), consisting of two identical subunits with molecular weight of 36.0kDa, catalyzes the reductive methylation of dUMP to produce dTMP (Carrers et al., 1995). This process is the only de novo source of dTMP, which is subsequently metabolised to thymidine triphosphate (dTTP), exclusively for incorporation into DNA during synthesis and repair (Ackland et al., 2006). In the rapid proliferative cells, it can result in apoptosis to decrease the level of TS. This phenomenon is exploited in therapeutic protocols utilizing TS inhibitors, such as raltitrexed, pemetrexed, or prodrugs such as 5-fluorouracil and 5-fluorodeoxyuridine that are metabolized to TS inhibitors. Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs. Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TSinhibiting agents such as pemetrexed. Other antifolate TS inhibitors, currently tested in clinical studies, that show encouraging anticancer activities are Plevitrexed, GW7904L and Nolatrexed. A new prospect among antifolates, demonstrating a very desirable pattern of pharmacological properties, is BGC 945 that showed promising antitumor activities and has been nominated for clinical development (Gibson et al., 2011). In order


Introduction
Thymidylate synthase (TS,E 2.1.1.45),consisting of two identical subunits with molecular weight of 36.0kDa,catalyzes the reductive methylation of dUMP to produce dTMP (Carrers et al., 1995).This process is the only de novo source of dTMP, which is subsequently metabolised to thymidine triphosphate (dTTP), exclusively for incorporation into DNA during synthesis and repair (Ackland et al., 2006).In the rapid proliferative cells, it can result in apoptosis to decrease the level of TS.This phenomenon is exploited in therapeutic protocols utilizing TS inhibitors, such as raltitrexed, pemetrexed, or prodrugs such as 5-fluorouracil and 5-fluorodeoxyuridine that are metabolized to TS inhibitors.Two antifolates, Raltitrexed and Pemetrexed, are licensed anticancer drugs.Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TSinhibiting agents such as pemetrexed.Other antifolate TS inhibitors, currently tested in clinical studies, that show encouraging anticancer activities are Plevitrexed, GW7904L and Nolatrexed.A new prospect among antifolates, demonstrating a very desirable pattern of pharmacological properties, is BGC 945 that showed promising antitumor activities and has been nominated for clinical development (Gibson et al., 2011).In order

A Novel Approach to Cloning and Expression of Human Thymidylate Synthase
Ying-Tao Lv, Pei-Juan Du, Qiao-Yan Wang, Yuan Tan, Zong-Bin Sun, Zhong-Liang Su, Cong-Min Kang* to exploit new TS inhibitors, it's important to study the structure and properties of TS and also the crystal structures of hTS.It is reported that a new crystal form of human TS (hTS) which allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site.Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate in high-throughput analysis of hTS complexes with drug candidates.Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand (Jarmula 2010).Steadman et al. (1998) utilized site-directed mutagenesis to create 12 mutant proteins with substitutions at residue 216.The result proved that a serine residue corresponding to Ser216 in human TS is an essential site to binding dUMP.Phan et al. (2001) found that TS functions as an RNA binding protein that regulates the expression of its own mRNA translation, the emergence of resistance to the treatment is often related to the increased level of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding.During the early stage, TS was obtained from human cells (Lockshin et al., 1979;Dolnick et al., 1977).With the development of gene recombination and prokaryotic expression system, it is feasible to expression human TS in E. coli.Davisson et al. (1989) isolated a full-length cDNA clone of human TS and firstly expressed it in Escherichia coli.Since wild-type human TS cDNA is eukaryote gene (Joan et al., 1997), and have many rare codons of E. coli, the yield of this enzyme was only 1.6% of the E. coli protein.Joan (Hori et al., 1990) converted purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product, which helped to lighten the effect of rare codon on the expression level in E. coli, and the yields of this enzyme could be improved to 25%-30% of the E. coli protein.
Although site-directed mutagenesis can helpe to lighten the effect of rare codon on the expression level in E. coli, that needs large amount of experimental operations.In this paper, we describe a novel method for improving the expression of wild-tpye human TS cDNA in E. coli.In the experiment, wild-type TS gene was cloned into expression vector pET-28b (+), and respectively tansformed into expression host BL21 (DE3) and Rosseta (DE3), which has been added the rare codons of E. coli.The results showed that the yields of TS in Rosseta (DE3) was more than the BL21 (DE3).By optimizing the expression conditions, the yields of TS in Rosseta (DE3) was up to about 40% of the E. coli protein.

Enzymes and chemicals
DNA polymerase, restriction enzymes, and T4 DNA ligase were purchased from TaKaRa Biotechnology (Dalian, China).All other chemicals were from Sigma.

Bacterial strains, plasmids and culture medium
Escherichia coli DH5α was utilized for subcloning propagation of plasmids, BL21 (DE3) (Meck, China) and Rosetta (DE3) (Meck, China) was utilized for host of gene expression.pMD18-T (TaKaRa, Dalian) and pET-28b (+) (Meck, China) were respectively used for subclone vector and expression vector.pcDNA3.1zeo-TSwas gifted by Dr. Maria Zajac-Kaye (Basic Research Laboratory and Molecular Therapeutic Program, National Institute of Health).The strains were maintained on LB agar plates and grown at 37 °C unless otherwise.

Construction of subclone vector for amplification of human TS cDNA
According to the sequence of TS gene (GenBank ID: NM_001071), The primers were designed as the following, upstream primer: 5'-ATCAT ATGTA CCGCG CCATG CCTGT G-3' (NdeI) and downstream primer: 5'-GCCTC GAGAA CCCTA AACAG CCATT TCCAT TT-3' (XhoI).The PCR product was purified by DNA gel extraction kit (Sangon, Shanghai).Specific fragments about 1kb were connected to pMD18-T vector, then transformed into competence cells of E. coli DH5α.By blue-white spot screening, identification of plasmid PCR (Figure 1), the positive strain was sequenced in the DNA sequencing department of Biosune systems Biology, Shanghai.The subclone vector was named as TS-pMD18-T (Figure 1A).

Construction of E. coli expression vector for synthesis of human TS
The full length of human TS cDNA in TS-pMD18-T was isolated by a double digestion of NdeI, XhoI and cloned into the multiple cloning site (MCS) downstream of the expression vector pET-28b (+) that was previously digested with the same two enzymes.The recombinant plasmid was transformed into E. coli DH5α, identified by PCR, double enzymes digestion (Figure 3) and named TS-pET-28b (+) (Figure 1B).

Expression and expression form analysis of recombinant human TS
E. coli BL21 (DE3) and Rosetta (DE3) with TS-pET-28b (+) was activated in LB broth, containing 50μg/ml kanamycin, and 34µg/ml chloromycetin at 37℃ with 200 rpm shaking.500 μl of activated culture was transferred into 10ml of fresh LB broth with the mentioned antibiotics under the same condition for about 1h.When OD600 reached 0.5-0.7,IPTG (final concentration = 1.5 mM) was added to induce the synthesis of human TS.After 8h of induction at 37℃, E. coli was harvested by 15min of centrifugation at 8000 rpm and resuspended in 3 mL of buffer A (50 mM phosphate buffer, pH 8.0).The samples were taken out and analyzed by 15% SDS-PAGE (Figure .4A), The rest were sonicated (50 cycles with 10s on/15s off) in ice using a sonicator JY92-11 system (Scientz Biotechnology Co, Ltd, Ningbo) and then centrifuged at 13000rpm for 15min.The supernatant and resuspended precipitation were analyzed by 15% SDS-PAGE (Figure 4a) and West-blotting.

Purification and refolding of recombinant human TS with 6×His-tag
Because there is 6×His oligonucleotide sequence between the promoter and MCS, human TS had a 6×Histag in the N-terminal.A nickel affinity column was utilized for purification of the recombinant human TS.

Bioactivity assay of recombinant human TS
Bioactivity assay was carried out on the basis of Williams's method (Steadman et al., 1998).Enzyme The absorbance change at 340nm due to the conversion of CH2H4folate to H2folate (ε340=6.4 mM -1 cm -1 (Wahba et al., 1961) was monitored for 1-10min.One unit of enzyme activity is defined as the amount of enzyme required to synthesize 1μmol of dTMP per minute under these conditions.

The subclone and expression vector construction of human TS cDNA
After constructed the subclone vector TS-pMD18-T, we carried on preliminary identification using PCR.The product was analyzed by 1% agarose gel electrophoresis, and the result showed that there was a band about 1kb (Figure 1), proved human TS cDNA had insert into pMD18-T.We sent DH5α with TS-pMD18-T to sequence analysis; The result showed that there was no nucleotide mutation.
Since TS-pET-28b (+) contain NdeI, XhoI site.We digested it with NdeI, XhoI (Figure 3a), and identified with PCR (Figure 3b).The result of restriction enzyme digesting showed that there were two DNA bands about 1kb, 5kb, which were similar as human TS cDNA and pET-28b (+).Expression vector construction of human TS cDNA was constructed.
When total cells of TS-pET-28b (+)/BL21 (DE3) and TS-pET-28b (+)/Rosetta (DE3) after inducing were analyzed by SDS-PAGE (Figure 4a) and West-blotting (Figure 4b), was a significantly protein band on the TS-pET-28b (+)/Rosetta (DE3) track at 36.0kDa which matched well with human TS relative molecular mass (Carrers et al., 1995), however, the TS-pET-28b (+)/BL21 (DE3) was not significantly.The reason is that human TS gene has many E. coli rare codons, and E. coli rare codons have significant effect on the expression of human TS (Hori et al., 1990;Joan et al., 1997).Rosetta (DE3) has T7-promoter and have added the rare codon tRNA.In order to lighten the effect of rare codon to the expression of human TS, we selected Rosetta (DE3) as the expression host.By location analysis (Figure 4b), human TS expressing in Rosetta (DE3) was mainly on inclusion body.

Purification and refolding of recombinant human TS with 6×His-tag
Human TS was found to be expressed in Rosetta (DE3) as inclusion bodies from the SDS-PAGE analysis (Figure 4b).Attempts to solubilize human TS from these pellets with Buffer failed, unless cells were lysed by ultrasonication.Human TS became soluble under these conditions and could be subjected to further purification using Ni-NTA cartridge.The use of successive stepwise elution on the gel allowed the best elution (Buffer B containing 200mM imidazole) and finally purification of protein to 89% (Figure 5).

Bioactivity assay of recombinant human TS
Under room temperature conditions, determination of enzyme activity employed ultraviolet spectrophotometry.Experimental results see Table 1.The linear equations obtained is Y=0.0145X+0.054(R=99.98%),including the reaction time X and the amount of the resultant dTMP Y. Then the specific activity of human TS after renaturation calculated was SP=0.0186/0.15=0.124U/mg (protein concentration was 0.15 mg/ml).The result is similar with the standard specific activity of human TS (Steadman 1998).

Discussion
In the early stage of this experiment, we transformed TS-pET-28b (+) into the general expression host BL21 (DE3), the result (Figure 4a) showed that the yield of human TS was low.We thought that the conditions (temperature, inducing time, and IPTG concentration) may be not the optimal, and carried out the orthogonal experiments to optimizing the expression conditions.But the expression level was never improved.We thought the reason may be that codon preference in E. coli and eukaryotic organisms is different, and exists many rare codon of E. coli in human TS cDNA.Grosjean (Zhang et al., 1989) found that there are eight types rare codons (AGA, AGC, AUA, CCG, CCT, CTC, CGA, et al), and lack of corresponding tRNA in E.cloi.Kane (Fu et al., 2007) proved the codons (CUA, CGA, CCC, GGA) had

Figure 3 .
Figure 1.Construction of Subclone Vector and Expression Vector

Table 2 . The Rare Codon Analysis of Human TS cDNA
The codon usage frequency in Escherichia coli B [gbbct]: 11 originates from GenBank Codon Usage Database