T4 DNA ligase, Taq polymerase, and restriction enzymes were obtained from Takara. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was purchased from Merck. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT), Fetal Bovine Serum (FBS), P1, P5- di(adenosine—5’) pentaphosphate (Ap5A), glucose, ADP, NADP+, hexokinase and glucose-6-phosphate dehydrogenase were from Sigma. Thrombin (Restriction Grade) was obtained from Millipore.

pQE32 (Qiagen) was propagated in Escherichia coli XL1-Blue (Qiagen). pAK-T4 and pKILLIN vectors were described recently[30, 31].

pAK-TAG was essentially as described in the expression of AK-T4 DNA ligase fusion protein [30]. Thrombin cleavage site was then inserted into pAK-TAG at the end of the AK coding region before the multiple cloning sites using a linker composed of L-thrombin (5’ -GCTGGTGCCGCGTGGCAGCGGTAC-3’) and R-thrombin (5’-CGCTGCCACGCGGCACCAGCGACG-3’) primers. The resulting vector was designated as pAK-TAG-T. For the expression of AK-TNFα fusion proteins, DNA fragment encoding the mature human TNFα was amplified by PCR using L-TNFα (5’-GGGGTACCGTCAGATCATCTTCTCG-3’) and R-TNFα (5’-GGGAAGCTTTCACAGGGCAATGATCC-3’) and cloned into pAK-TAG and pAK-TAG-T vectors at KpnⅠand HindIII sites. All plasmid constructs were verified by DNA sequence analysis.

AK enzyme assay was carried out as previously described [26, 27]. The reaction mixture (50μl) consists of 10 mM glucose, 2 mM ADP, 0.5 mM NADP+, 1.2 units of hexokinase and 0.6 units of glucose-6-phosphate dehydrogenase (Sigma). The AK activity was calculated by the rate of increase in absorbance at 340 nm. The formation of 1μmol of ATP in one minute is defined as one unit of AK enzyme activity. Protein concentration was measured by BCA assay kit (Pierce), according to the manufacturer’s instructions.

Cell growth and induction of expression was carried out as previously described [30]. Briefly, bacteria cells were grown in LB medium at 37°C until reaching 0.6 at OD₆₀₀, 1mM IPTG was then added to induce the protein expression for 10 h at 30°C. After induction, cells were harvested by centrifugation and protein extracts were prepared by sonication in extraction buffer as previously described [27].

The level of a soluble AK fusion protein expression was estimated by the fold of increase in the specific activity of AK over that of the host cell XL1-blue containing pQE32.

After IPTG induction, the cells were collected by centrifugation at 4000 g for 20 min at 4°C. The cell pellet was resuspended in 20 ml of buffer A (20 mM Tris-HCl, pH 7.4, 1 mM MgCl₂) and then lysed by sonication. Cell debris was removed by centrifugation at 16900 g for 20 min at 4°C. Protein purification was carried out using an AKTA-Prime purification system (GE Healthcare). About 80 mg of cleared cell lysate was used as a starting material and passed through a 1 ml HiTrap Blue HP (Blue-Sepharose) column (GE Healthcare) pre-equilibrated with 20 Column volumes (CVs) of buffer A. After sample loading, 10 CVs buffer A was added to wash off weakly bound proteins until OD₂₈₀ detection declined to near baseline. The AK-TNFα and AK-T-TNFα fusion protein was eluted with a 20 CVs buffer B (20 mM Tris-HCl, pH 7.4, 50μM Ap5A, 1mM MgCl₂). The flow rate was maintained at 1 ml/min throughout the purification [26, 27].

AK-T-TNFα eluted from Blue-Sepharose column was passed through a Ni-NTA column (GE Healthcare) which was pre-equilibrated with 15 CVs of buffer C (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 5 mM imidazole). After washing off weakly bound contaminating proteins with 15 CVs of buffer C until OD₂₈₀ declined to near the baseline, another 15 CVs of buffer D (20 mM Tris-HCl, pH 8.4, 150 mM NaCl, 2.5 mM CaCl₂) was passed through the column. In-column thrombin digestion was initiated after passing 1.5 ml thrombin (9U in buffer D) and kept at the room temperature for 20 h. The column was washed with 10 CVs of buffer D followed by elution of the cleaved human TNFα with 10 CVs of buffer C. The His-tagged AK was finally eluted with a 15 CVs linear-gradient of 5–500 mM imidazole in buffer E (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 500 mM imidazole).

The purity of purified AK-TNFα and TNFα was evaluated on an Agilent 1260 HPLC using Size-Exclusion Chromatography (SEC-HPLC) with an analytic TSK G3000SWXL column (Tosoh). Phosphate Buffered Saline (PBS) was used as the mobile phase with OD₂₈₀ nm detection over a 20 min period at a flow rate of 1 ml/min. The protein molecular weight was estimated based on retention time using the protein size standard (BioRad).

The biological activity of TNFα was measured in vitro according to established method [32]. Briefly 1×10⁴ cells of L929 cells (ATCC) was seeded into each well of a 96-well cell plate with RPMI-1640 (Hyclone) containing 10% FBS. After 24 h, actinomycin D was added to the medium at 1 μg/ ml together with various concentrations of either TNFα (R&D), cleaved TNFα or AK-TNFα. After 18 hours of cell culture at 37°C, 0.5 mg/mL MTT was added to each well followed by a 4 h of incubation at 37°C. Then the media in the wells were carefully discarded and 100μl of dimethylsulfoxide (DMSO) was added into each well to dissolve Formazan crystal.The absorbance at 490 nm was recorded.

The full sequence data of pAK-TAG and pAK-TAG-T expression vectors have been deposited in GenBank under the accession numbers, KX147098 and KX147099, respectively.

We have previously described the expression of DNA ligase from bacteria phage T4 in soluble form as an AK fusion protein using pAK-TAG cloning vector we developed [30] (Fig 1A). pAK-TAG expression vector contains multiple cloning sites at the C-terminus of AK followed by a stop codon for convenient in-frame fusion of any recombinant protein. Under the control of a strong T5 promoter and the lac operator, the N-terminal His-tagged AK alone or any AK fusion proteins may be expressed at high level upon IPTG induction (Fig 1A) such as AK-T4 DNA ligase. To further demonstrate that the same goal can be attained for secreted proteins from mammalian cells, we have constructed several AK fusion proteins from human TNF family of cytokines. These fusion proteins, including AK-TNFα and AK-Trail as shown in Fig 1B, like T4 DNA ligase, were also expressed at high level in soluble forms upon IPTG induction. AK alone has a molecular weight at 27 kDa and both monomeric human TNFα and Trail have molecular weights around 16–17 kDa. The predicted molecular weights for AK-TNFα and AK-Trail thus would be around 43–44 kDa, which were consistent with our data presented (Fig 1B).

The original concept for using AK as an affinity tag was intended to take the advantage of its high-level bacterial expression, sensitive detection by enzyme assay and easy purification. However, the first AK fusion protein (AK-T4 DNA ligase) we expressed failed to come off the Blue-Sepharose column upon Ap5A elution due to the ability of T4 DNA ligase to bind to nucleotide-like dye moiety from the chromatographic matrix on its own [30]. We next chose proteins from mammalian cell origins that are known not to contain nucleotide or DNA binding properties, which are responsible for Blue-Sepharose binding. We showed that the AK-TNFα fusion protein behaved similarly to AK itself [26, 27] and was able to bind to Blue-Sepharose beads with little flow through. Upon complete washing off of any unbound proteins, AK-TNFα was efficiently and specifically eluted off the column by Ap5A with little contaminating proteins (Fig 2A). The specific binding of AK-TNFα to Blue-Sepharose and subsequent affinity elution with Ap5A was easily and quantitatively monitored in near real-time by AK enzyme activity during the purification (Fig 2B).

To ensure that a cleaved recombinant protein could be recovered from its AK-fusion form, a thrombin cleavage site was introduced in-frame into the pAK-TAG expression vector before the multiple cloning sites. The resulting expression vector was designated as pAK-TAG-T (Fig 3A).

When human TNFα was cloned into pAK-TAG-T vector and transformed into XL1-blue, the resulting AK-T-TNFα fusion protein with a thrombin cleavage site in-between was expressed at high levels in soluble form upon IPTG induction, in comparison to AK expression from the pAK-TAG-T vector alone (Fig 3B). The level of AK-T-TNFα was easily measured via AK activity. Based on the increase in the specific activity of AK, AK-T-TNFα expression level was estimated to be over 50 times of endogenous AK from the host cells (Fig 3C). Due to the high copy number of the pAK-TAG-T vector and strong T5 promoter, there were leaky expression of AK-T and AK-T fusion proteins without the inducer.

The one-step purifications of AK-TNFα and AK-T-TNFα via Ap5A affinity elution were easily monitored by the AK activity of the fusion proteins. The yield of recovery was estimated to be around 50% with over 7-fold purification for both AK-TNFα (Fig 2) and AK-T-TNFα (Fig 3) fusion proteins based on the increase in specific activity of AK (Table 1). SDS-PAGE analysis followed by densitometry quantification indicated that about 95% purity was achieved after Ap5A affinity elution. In terms of the yield of purified AK fusion proteins based on bacterial culture, it translated to about 18 and 17 mg/L for AK-TNFα and AK-T-TNFα, respectively.

Like described above, the AK-T-TNFα fusion protein with a thrombin cleavage site was first purified to near homogeneity via Blue-Sepharose with Ap5A elution (Fig 3D). To release the cleaved TNFα from AK, the purified AK-T-TNFα fusion protein was then loaded onto a Ni-NTA column. Because the AK was N-terminal His-tagged, the AK-T-TNFα was captured by the Ni-NTA. Then thrombin was added to allow in-column cleavage of the fusion protein overnight. The cleaved human TNFα was then released from the Ni-NTA column with the elution buffer with low concentration of imidazole, while the bound AK moiety was later eluted with a linear-gradient of 5–500 mM imidazole (Fig 3D). The in-column thrombin release of TNFα was estimated to be 93% in step-yield based on the protein concentration and molecular mass ratio of TNFα in AK-T-TNFα, which gave rise to an overall yield of recovery over 44% (Table 1) with about 6.3 mg/L of TNFα from bacteria cell culture.

TNFα family of cytokines are all trimeric in structure which is required for their full biological functions. To determine the conformation of AK-TNFα fusion protein and the cleaved TNFα purified with our AK-TAG method, we analyzed them on a size exclusion HPLC (SEC-HPLC). Both purified AK-TNFα and the cleaved TNFα showed as single peaks with estimated molecular weight consistent with being trimers (Fig 4A). Functionally, the AK-TNFα fusion protein had not only the AK activity but also retained TNFα biological function as determined by its cytotoxicity to L929 cells [32](Fig 4B). However, its potency was over 13 times less than that of the cleaved TNFα upon being released from thrombin cleavage (Fig 4B). The cleaved TNFα generated with our AK-TAG method exhibited the comparable potency in IC50 in L929 bioassay to TNFα from R&D systems (Table 2).