Expression, Purification and Refolding of the HTLV-1 Proteases

provided the plasmid encoding the HTLV-1 PR 1-116 harboring the mutations L40I and Y114N for our studies. First, the asparagine was re-mutated to tyrosine by site-directed mutagenesis to obtain the favored plasmid only containing the L40I mutation.

The plasmid encoding the full-length protein was ordered from Life Technologies/GeneArt (Germany).

2.2.1 Site-Directed Mutagenesis of the HTLV-1 PR 1-116 Plasmid by PCR

The polymerase chain reaction (PCR) is one of the most important methods in molecular biology. The PCR technique was invented and developed by Kary Mullis in the 1980s and awarded with the Nobel Prize in Chemistry in 1993.¹¹ The PCR is e.g.

used in the diagnostics of diseases, in forensic analysis, and as an important technique

36 2.In-House Establishment of a HTLV-1 Protease Technology Platform

in molecular biology research, for example for DNA cloning.¹²

The PCR is a three-step reaction: first the double-stranded DNA is denatured resulting in two single strands (95 - 98°C). In the second step, the temperature is decreased (≈50 - 60°C) allowing the primer to anneal to the DNA. In the third step, typically performed at 72°C, the applied thermo-stable polymerase finally creates the new DNA strand complementary to the DNA template strand. These three steps were repeated (about 25-35 cycles) resulting in the exponential amplification of the DNA.¹²

Within this thesis, the PCR technique was used for the site-directed mutagenesis of the HTLV-1 PR 1-116 (L40I, Y114N) construct: for the identification of the following optimized PCR conditions, different ratios of the components of the PCR solution as well as different hybridization temperatures were tested.

Finally, the following composition of the PCR solution and parameters of the PCR program were used:

10 µl 5x HiFi-buffer (BioCat)

1 µl PRECISOR high-fidelity DNA Polymerase, 250 U (BioCat) 5 µl plasmid (encoding HTLV-1 PR 1-116, L40I, Y114N) 1 µl primer 1 (20 nM)

1 µl primer 2 (20 nM)

1 µl dNTP-Mix (each 25 mM) 31 µl water (sterile)

The PCR was performed in a Thermocycler PCR Mini Cycler (MJ Research). Steps 2-4 were repeated 30 times (30 PCR cycles).

step time temperature

1 60s 98°C

2 30s 98°C denaturation

3 30s 63°C hybridisation

4 7min 72°C extension

5 ∞ 4°C

Before transforming the new plasmid into E. coli cells, first a DpnI digestion must be performed to remove the starting plasmid and the PCR product has to be purified. The DpnI digestion was performed by incubating 30 µL of the PCR product solution and 3 µL DpnI (10 U/µL, Fermentas) for 2 h at 37°C. For purification of the PCR product a purification kit (peQLab) was used.

After the plasmid minipreparation, the new plasmid (L40I) was sequenced (Eurofins, Germany) to confirm the mutation.

2.In-House Establishment of a HTLV-1 Protease Technology Platform 37

2.2.2 Expression System

The full-length protease (HTLV-1 PR 1-125) as well as the truncated protease (HTLV-1 PR 1-116) were expressed in the E. coli strain Rosetta 2 (DE3).

First, the respective plasmids, both bearing the L40I mutation, were transformed via heat shock into the Rosetta 2 (DE3) competent cells according to a standard protocol and a glycerol stock was prepared and stored at -80°C.

2.2.3 Inclusion Bodies

According to the literature, the HTLV-1 protease is expressed as inclusion bodies in E.

coli. Inclusion bodies often result from overexpression of recombinant proteins through aggregation of the expressed and un- or partially folded protein. For the recovery of active protein from inclusion bodies, they firstly need to be extracted from the E. coli cells, followed by the solubilization of the aggregated protein, typically using strong denaturants, such as for example highly concentrated urea or guanidine hydrochloride, thiocyanate salts or detergents causing denaturation. Finally, the protein usually has to be refolded, for example by dilution or by chromatography. In this step the denaturing agent is removed to enable the refolding of the protein. Finding suitable refolding conditions is often quite challenging, due to aggregation and misfolding of the protein.

Therefore it seems in general to be easier to work with soluble proteins instead of insoluble inclusion bodies. Process parameters that might influence the production of soluble protein are e.g. the media composition, the expression temperature, the production rate, and the availability of chaperones. To address the problem of protein insolubility, also special E. coli cell lines are available, providing a further approach to increase the yield of soluble protein, like e.g. the ArcticExpress cells.¹³ However, working with inclusion bodies might also have some advantages: soluble impurities can easily be separated by washing steps and centrifugation. Usually the expression level is higher than in soluble systems, the protein product is protected from proteolytic degradation, and in case of target proteins with toxic properties towards the used expression organism, the cells are usually protected against their toxicity.^14,15

2.2.4 Protein Expression

The protein expression protocol is based on the literature procedure from Li et al.¹ The pre-culture was prepared in 10 ml LB medium containing the antibiotics ampicillin (100 mg/l) and chloramphenicol (20 mg/l) at 37°C and 220 rpm shaking overnight. For the main culture two pre-cultures (20 ml) were transferred into 1.6 l LB medium containing the same antibiotics as in the pre-culture and shaken at 37°C until an OD600

38 2.In-House Establishment of a HTLV-1 Protease Technology Platform

of about 0.7 was observed (≈ 3.5 h). The protein expression was induced by addition of isopropyl-β-D-thiogalactopyranosid (IPTG) (1 mM). The temperature was decreased from 37°C to 14°C to enable a slower protein production, after ca. 15 hours the cells were harvested by centrifugation (5500 rpm, 15 min, 4°C). The cells were resuspended in 70 ml buffer A (Table 2.1) and for cell disruption this solution was first incubated with lysozyme (1 h) followed by sonication on ice (10 times for 90 seconds). For removal of nucleic acids, the solution was incubated for 1 h at 4°C with Benzonase^® Nuclease (1.5 µl). After that a washing step of the inclusion bodies was performed: first, urea was added to the inclusion body solution to finally obtain an urea concentration of 0.5 M, while the solution was again stirred for 1 h at 4°C. Second, the solution was centrifuged (20000 rpm, 30 min, 4°C) and the obtained inclusion bodies were washed three times with buffer B. For solubilization of the inclusion bodies, the pellet was resuspended in buffer C, containing 8 M urea as strong denatured, and stirred at 4°C for 15 h.

2.2.5 Protein Purification

The purification was performed according to the procedure from Li et al.¹ and comprises two purification steps by fast protein liquid chromatography (FPLC). First, the inclusion body solution (supernatant) was passed through a Q Sepharose (anion exchange column) equilibrated with buffer D. Before loading the eluate onto an SP Sepharose (cation exchange columns) equilibrated with buffer E, the pH of the eluate was adjusted to 3.0. The bound protein was eluted with buffer E containing 0.3 M NaCl.

2.2.6 Refolding

The refolding of both protease constructs was performed via dialysis; however, a different refolding protocol was used for each protein.

The refolding of the HTLV-1 PR 1-116 was performed according to the publication from Li et al.¹ using the sodium acetate buffer F. The protein was stored at -80°C until use.

The refolding of the full-length protease (HTLV-1 PR 1-125) was performed as published⁹ and comprised three different refolding steps: first, the protein was dialyzed against buffer G for 15-24 h, followed by buffer H for 12-24 h, and, finally, against buffer I for 24 h. The HTLV-1 PR 1-125 was stored at -20°C until use.

2.In-House Establishment of a HTLV-1 Protease Technology Platform 39

Table 2.1. Used buffers for HTLV-1 PR expression, purification and refolding.

buffer buffer composition buffer buffer composition A 10 mM Tris, pH 7.5

5 mM EDTA F 15 mM sodium acetate, pH 3.0

5 % PEG 300 5 mM DTT B 0.5 M urea

10 mM Tris, pH 7.5 5 mM EDTA

G 25 mM formic acid, pH 2.8

C 8 M urea

10 mM Tris, pH 7.5 5 mM EDTA 10 mM DTT

H 50 mM sodium acetate, pH 5.0 1 mM EDTA

1 mM DTT

D 6 M urea

10 mM Tris, pH 7.5 5 mM EDTA 5 mM DTT

I 20 mM PIPES, pH 7.0 150 mM sodium chloride 10 % glycerol

1 mM EDTA 2 mM DTT

0.5 % Nonidet P-40 E 6 M urea

20 mM sodium acetate, pH 3.0 5 mM EDTA

5 mM DTT

2.2.7 Conclusion and Outlook

Within this thesis a robust protocol for the expression, purification, and refolding of HTLV-1 PR was successfully established in our laboratory which provides continuous access to both HTLV-1 protease constructs. As the SDS gels (Figure 2.1 and 2.2) indicate the expression of both proteins worked well. Via the inclusion body washing steps most of the protein impurities were removed; only two weak protein bands between 35 and 40 kDa were present before the purification via Q and SP Sepharose.

After the purification by the Q and SP Sepharose hardly any impurities were detectable (Figure 2.1 and 2.2).

40 2.In-House Establishment of a HTLV-1 Protease Technology Platform

Figure 2.1. SDS gels of the HTLV-1 PR 1-116.

Figure 2.2. SDS gel of the HTLV-1 PR 1-125.

2.In-House Establishment of a HTLV-1 Protease Technology Platform 41