Site-selective protein conjugation at histidine

Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology.


Design and preparation of the truncated N-terminal His-tagged IFN variants
The interferon α2-a (IFN) variants with four N-terminal truncated His 2 -tags are listed below:    The N-terminal His-tagged IFN genes were then cloned into the pET SUMO vector 2 .
The PCR product (insert, I) and vector (vector, V) were mixed using a 1:1 molar ratio and the T4 DNA ligase was used for the ligation reaction as shown in Table S4. The resulting ligation reaction was incubated overnight at 20°C, followed by ligation reaction transformation into the TOP10 chemically competent E. coli cells.

Quantity [µL]
PCR product (25 ng/mL) 3 µL (120 ng) h and was inducted with 1 mM IPTG. The bacterial pellet was next subjected to lysis and following conformation of all of the pET SUMO-IFN variants, the scale-up fermentation using the SUMO fusion system was as described for interferon consensus 2 .

Preparation of the site-selectively engineered IFN variants
The internal His-tags, or PEG-tags, were selected based on information  The mutagenic oligonucleotide primers were designed for incorporation of the desired mutations within the IFN sequence. Both mutagenic primers (forward and reverse) were designed to contain the desired mutation and anneal to the same sequence on opposite strands of the pET SUMO-IFN template plasmid. Primers were designed using Translate accessed at ExPasy and were purchased at Invitrogen (primers listed in Table S6).

IFN variant
Primer Nucleotide sequence   Table S8. Yields obtained during fermentation of SUMO fusion of the N-terminally tagged IFN variants and the site-selectively modified IFN variants.

Purification of the SUMO-IFN variants
Typically 3 g of bacterial pellet was re-suspended in 25 mL of lysis buffer (1× PBS, lyzozyme (0.4 mg/mL), protease inhibitor cocktail (1:500 dilution), DNase (0.02 mg/mL)) and then vortexed until a homogeneous mixture was obtained. This mixture was incubated for 1 h at 4°C with gentle rocking. The solution was then sonicated (three times 1 min sonication at 50%, 0.5 cycles) and an equivalent volume of PBST (1× PBS, 1% Triton×-100, 25 mL) was added to the sample. The sample was then incubated for an additional 1 h at 4°C with gentle rocking.
The sample was centrifuged at 3,500 g for 45 min at 4°C to pellet the cellular debris and separate the supernatant.  Elution of the IFN variants from the HiTrap™ QHP column was conducted by applying a gradient elution conducted with buffer B (20 mM Tris, pH 8.0, 1 M NaCl). The target protein was eluted using a gradient elution from 0% to 30% of buffer B run over 30 min at 1 mL/min.
The column was then washed with 100% of buffer B and fractions collected in this purification process were analysed by SDS-PAGE.

Characterisation of the His-tag IFN variants
Following protein preparation, the His-tagged IFN variants were characterised by (i) RP-HPLC, (ii) SDS-PAGE and (iii) A549/EMCV antiviral assay. The antiviral assay was conducted using the NIBSC control for IFN and protein activity was expressed in IU/mg. On the next day a 2-fold dilution of the tested samples were added. were analysed using GraphPad Prism 5 software. The potency was expressed as the concentration of the test substance that achieves 50% effective dose (ED 50 ), which in this assay corresponded to the protection against viral infection.

Optimisation of N-terminal His-tag PEGylation -small scale studies
Initial PEGylation studies were performed on a small scale (5 g of protein) and was performed   Table S13. Small-scale PEGylation reactions using IFN to examine the influence of protein concentration and the stoichiometry of the PEG mono-sulfone 3. Table S14. Preparation of a small-scale PEGylation studies conducted on the N-terminally his-tagged IFN variants: HHGGGG-IFN, HGHGHG-IFN and HGHGGG-IFN.

Characterisation of the PEG conjugates of the His-tagged IFN conjugates
The PEG conjugates of (i) 5(HGH)-IFN, (ii) 106(HGHG)-IFN and (iii) 120(HHH)-IFN were characterised to assess (i) sample purity by SDS-PAGE and RP-HPLC analysis, (ii) sample stability at 4°C for 4 weeks and (iii) bioactivity by A549/EMCV-based antiviral assay. Activity studies were conducted in parallel with the NIBSC standard for IFN. The antiviral activity of the PEGylated conjugates was then expressed as specific activity calculated against the NIBSC standard. In addition, the percentage of the retained activity in respect to parental protein was determined.
Stability studies were performed in 50 mM sodium phosphate, 150 mM NaCl, pH 7.4 buffer with PEG-IFN conjugates at 0.2 mg/mL. The stability studies were performed for 4 weeks at 4°C during which samples were investigated weekly. All of the samples were supplemented with 1 mM sodium azide to inhibit bacterial growth and 0.2 mg/mL protease inhibitor to avoid the risk of protein degradation by proteases. At each time point 10 L of the sample was drawn and stored at -80°C until all time points were collected and then analysed by SDS-PAGE followed by protein stain conducted with colloidal blue, PEG stain and anti-IFN western blot 2 .