Recombinant Expressed Vector pET32a (+) S Constructed by Ligation Independent Cloning

The aim of this work was to develop a new method for constructing vectors, named ligation-independent cloning (LIC) method. We constructed the S label expression vector and recombinant pET32a (+) S-phoN2 by LIC. The recombinant proteins were expressed in E. coli at a high level, and then the specificity of the recombinant proteins was identified by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody. Finally, we obtained protein Apyrase in E. coli (BL21), with a protein-only expression S tag. Collectively, our results demonstrated that LIC is effective for the construction of new vectors and recombinant plasmids. Free from the limitations of restriction enzyme sites and with a higher positive rate, LIC processes should find broad applications in molecular biology research.


Introduction
Generally, the purpose of DNA molecular cloning is the insertion of a particular fragment into a vector to construct a recombinant plasmid. It is widely used in the medicine and biology field. The traditional cloning method involves acquisition of a particular fragment, digestion of a particular fragment and vector connection, transformation and selection.
Traditional cloning methods are widely used in the construction of vectors and recombinant plasmids [1,2]. Some of them have even converted into kits, such as T-A Cloning [3] or Gateway [4,5]. These new technologies have greatly simplified the cloning process, and improved the efficiency of cloning, but some problems still exist. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. For example, the restriction enzyme sites of particular fragments and vectors are considered, the particular fragment is treated by a restriction enzyme, and the ligation process needs DNA Ligase [6]. Using traditional cloning methods increases not only the cost of experiments and experiment duration, but also the risk of self-vector connection. In many cases, because of the properties of the original restriction sites of a particular fragment, the experiment has to use some rare restriction. This greatly increases the difficulty and cost of the experiment [7]. A way which is not subject to restriction sites could solve the problem above.
Ligation independent cloning (LIC) has been developed as a new cloning approach which eliminates the use of restriction sites [8]. The principle of LIC is: first of all, the linearized vector and the insert fragment are amplified by PCR with primers containing homologous fragments; second, PCR products are treated with T4 DNA Polymerase (3'-5' exonuclease activity) to obtain the fragment containing 5'-cohesive ends; third, the intermediate is formed by denaturation-renaturation; finally, intermediate is transformed into Escherichia coli [9]. The LIC method has advantages over traditional cloning methods in vector and recombinant plasmid construction. LIC eliminates the use of restriction sites and DNA ligease [10]. Using the LIC method to construct vectors and recombinant plasmids not only shortens the experiments, but also reduces the cost of experiments. LIC uses constructed vectors [11], constructed recombinant proteins [12,13] and co-expression proteins [14,15]. LIC is particularly suitable for the construction of new vectors [16] and high molecular weight expression of recombinant proteins [17].
In this work, we report the development of an effective, inexpensive method named the LIC method to construct the expression vector pET32a (+) S and pET32a (+) S-phoN2, which can generate controllable overhangs.

Construction of a pET32a (+) S Expression Vector
In order to construct the plasmid-only expressed S tag, we used the LIC method to construct pET32a (+) S. Cloning of expression vector pET32a (+) S via LIC was performed as described in Figure 1. After vector pET32a (+) was digested (Figure 1a), it was amplified with a pair of specific primers as described in the Experimental Section. The PCR product was characterized by electrophoresis through 1% agarose gels, and the 5900 bp band appeared as expected with the same size as pET32a (+) (Figure 1b). The transformants were acquired by use of E. coli DH5a (Figure 1c), the positive clones were identified by PCR ( Figure 1d) and sequenced ( Table 1). The result showed the constructed vector pET32a (+) S was not a mutant.   TTT AGA GGC CCC AAG GGG TTA TGC TAG TTA TTG CTC AGC GGT GGC AGC  AGC CAA CTC AGC TTC CTT TCG GGC TTT GTT AGC AGC CGG ATC TCA GTG GTG GTG GTG  GTG GTG CTC GAG TGC GGC CGC AAG CTT GAC GAC GGA GCT CGA ATT CGG ATC CGA  TAT CAG CCA TGG CCT TGT CGT CGT CGT CGG TAC CCA GAT CTG GGC TGT CCA TGT GCT  GGC GTT CGA ATT TAG CAG CAG CGG TTT CTT TAT GTA TAT CTC CTT CTT AAA GTT AAA  CAA AAT TAT TTC TAG AGG GGA ATT GTT ATC CGC TCA CAA TTC CCC TAT AGT GAG TCG  TAT TAA TTT

Construction of a pET32a (+) S-phoN2 Recombinant Plasmid
The expression vector pET32a (+) S was amplified with a pair of specific primers as described in the Experimental Section 3.2. The PCR product was characterized by electrophoresis through 1% agarose gels, the 5502 bp band appeared as expected with the same size as pET32a (+) S (Figure 2a).
The quality of the complete genome from Shigella flexneri M90T was characterized by electrophoresis through 1% agarose gels. On the basis of the sequence of phoN2 in M90T, gene specific primers were designed. The PCR product was characterized by electrophoresis using 1% agarose gels, and the 741 bp band appeared as expected with the same size as phoN2 (Figure 2b).
The target fragment and linear vector were linked by T4 DNA Polymerase. The transformants were acquired by E. coli DH5a and identified by PCR (Figure 2c). The positive clones were sequenced. The result showed the constructed recombinant plasmid pET32a (+) S-phoN2 was not a mutant ( Table 2).

Recombinant Protein Expression
The pET32a (+) S-Apyrase/BL21 was cultivated and OD600 was measured every hour. An S-type bacterial growth curve was observed, pET32a (+) S-Apyrase/BL21 was induced at 3 h when they were in the bacterial logarithmic growth phase (Figure 3).
To determine the solubility of recombinant protein, pET32a (+) S-Apyrase/BL21 without induction, whole protein of pET32a (+) S-Apyrase/BL21 after induction, the supernatant of pET32a (+) S-Apyrase/BL21 and the pellet of pET32a (+) S-Apyrase/BL21 were analyzed by SDS-PAGE (Figure 4). The recombinant protein was identified in the induced bacteria but not the control. Comparing the content of ultrasound supernatant and ultrasound pellet, most of the fusion proteins were present in the supernatant, meaning that the fusion protein Apyrase-S was a soluble protein. Lastly, in order to check the activity of recombinant apyrase expression, Apyrase-S protein was purified ( Figure 5).

Detecting of the Specificity of Expressing Bacteria
The induced bacteria were lysed, and the specificity of expressing bacteria was analyzed by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody ( Figure 6). This means the expression of fusion protein pET32a (+) S-Apyrase/BL21 only contains S tag. The molecular weight of S tag is only 1.7 kDa, which is too small to affect the structure of the target protein. Using the S tag, we can affinity purify the target protein and combine it with other tags for pull-down experiments. However, pET32a (+) contains two other tags in addition to the S tag. It was very inconvenient to construct a recombinant protein which only expressed S tag, so we constructed pET32a (+) S by LIC, which made the next experiment convenient.

Vector Construction
Construction of the expression vector pET32a (+) S is described in Figure 7. First of all, vector pET32a (+) was treated with MscI. LIC-specific primers were designed as shown below. The primers include homologous fragments. In the process of amplification, the 294-692 bp (Trx tag site) of pET32a (+) was removed: SF: 5' GGTTTCTTTCATATGTATATCTCCTTCTTAAAGTTAAACA 3' SR: 5' ATGAAAGAAACCGCTGCTGCTAAATTCG 3' Figure 7. Vector construct.
The PCR was performed in 50 μL reaction mixes using the following cycles: initial denaturation at 98 °C for 30 s; 25 cycles of denaturation at 98 °C for 30 s and extension at 68 °C for 6 min and 30 s; and a final extension at 68 °C for 7 min. PCR products were analyzed by electrophoresis and purified with PCR cleanup kit (Beijing, China).

Construction of pET32a (+) S-Apyrase (Figure 8)
Apyrase was amplified by PCR using AF (5'GCGGATCCATGAAAACCAAAAAC3') and AR (5'CTCGAGTTATGGGGTCAGTTCATTG3') primers. PCR products were analyzed by electrophoresis and purified with a PCR cleanup kit. The linear vector pET32a (+) S was amplified by primers SAF and SAR. The sequences are shown below; the upstream primers and downstream primers included homologous fragments of phoN2: SAF: 5' GAAAGTTTTTGGTTTTCATGGATCCGATATCAGCCATGG 3' SAR: 5' CCAATGAACTGACCCCATAACTCGAGCACCACCACCACCA 3' The cloning reaction was performed by T4 DNA Polymerase. The annealing product was transformed into E. coli DH5α, and the bacteria were identified by primers AF and AR.

Recombinant Protein Expression
The recombinant pET32a (+) S-Ayrase was transformed into E. coli BL21 (DE3) and selected on LB agar (ampicillin) at 37 °C overnight. The bacterial colonies were transferred into LB medium and the bacteria were cultured at 37 °C, 200 rpm overnight. In order to prepare the growth curve of the bacteria, the bacteria were sampled at 1, 2, 3, 4, 5, 6, 7 and 8 h. Then OD600 of each sample was measured. To determine the fusion of recombinant proteins, the bacteria were cultured at 37 °C, 200 rpm for 4 h, until OD600 was 0.6. Then the bacteria were dissolved in PBS and lysed by sonication. The different samples were tested by SDS-PAGE.

Purification of Proteins
The pS-Apyrase protein was purified using S agarose and insoluble material was removed by centrifugation (12,000 r/min, 15 min, 4 °C). Supernatants were filtered through 0.2 μm sterile Acrodisc ® syringe filters with membrane onto S agarose and rotated on a rocking platform at 4 °C for 4 h. Beads were collected by centrifugation (3000 r/min, 5 min, 4 °C) and washed three times in a wash buffer (20 mM Tris-HCl, 150 mM NaCl, 1%Triton × (v/v) − 100). The fusion proteins were stored in S agarose beads at 4 °C.

Conclusions
We have successfully constructed the expression vector pET32a (+) S and recombinant pET32a (+) S-Apyrase by LIC in just one week. Using LIC to construct vectors not only has a higher success rate, but also takes less time. The new method of construction of vectors and recombinant proteins provides an efficient and cost-effective parallel method and thus is applicable for the construction of different vectors, tags and proteins.