SEGURIDAD INDUSTRIAL 6.1 Seguridad en el área de trabajo
6.2 Reglas y normas de seguridad en el TALLER
Several problems were encountered during expression of the putative PNGases from D. radiodurans, S. avermitilis and S. solfataricus in E. coli. Expression of SsoPNGase in E. coli was tried, but was unsuccessful (data not shown). This, however, was not unexpected due to the high probability of
SsoPNGase being N-glycosylated (3.3.4.2). E. coli is generally not able to glycosylate proteins, and this post-translational modification can, however, be important for correct folding of glycoproteins. Following these problems another expression host (insect cells) was trialled for recombinant expression of these three proteins and the putative PNGase from A. niger. The latter protein is highly similar to PNGase At from A. tubingensis, which has been successfully expressed in insect cells using the baculovirus expression system (BVES; (Ftouhi Paquin et al., 1998)). As the putative PNGases from S. avermitilis and
expression of PNGase At using the BVES led to the decision to perform expression experiments using this system. DraPNGase was also included, but here two different truncated versions of this protein were cloned. The positions of these truncations were chosen based on a disorder prediction by the PONDR®-server (www.pondr.com; Appendix 3). At the N-terminus of
DraPNGase, 93 amino acid residues were removed (numbering based on the full-length protein, excluding the predicted signal sequence). Two different truncations points were chosen for the C-terminus, the first after residue 561 and the second after residue 613.
For recombinant protein expression in insect cells, the TOPO® and Gateway® Systems and parts of the Bac-to-Bac® Baculovirus expression system (Invitrogen™) were employed using the methods described in 5.2.5 and 5.2.6. The success of each cloning step was verified by colony PCR. The nucleotide sequences of the fragments inserted into the entry vectors were verified by DNA sequencing.
Table 5.1 summarises the intermediate steps and expression results obtained for recombinant protein production in E. coli and Sf9 cells using Gateway® technology.
Table 5.1: Summary of results obtained for recombinant protein production in E. coli and Sf9 cells using Gateway® technology.
Pr ot ei n E. coli BVES PC R p EN TR p DEST 15 p DEST 17 Ex p re ss ion (s ol ubl e) p DEST 8 p DEST 10 Ba cm id Ph a ge s tock (P 2 t itr e ) Ex p re ss ion DraPNGase- 561
-
DraPNGase- 613
-
SavPNGase
-
-
-
-
-
SsoPNGase
-
AniPNGase
-
-
-
-
First, entry clones were generated using directional TOPO®-cloning (5.2.5.1). Primers were designed to generate PCR products that would be suitable for the fusion of N-terminal purification tags after transfer into appropriate Gateway® destination vectors, i.e. no start codon was included in the primer sequence. Only AniPNGase was cloned for subsequent expression as an untagged protein as previously described for PNGase At (Ftouhi Paquin et al., 1998). The inserts for the different targets were obtained using the following primer combinations (Table 2.6): O1 + O2 for AniPNGase, O6 + O7 for DraPNGase-561, O6 + O8 for
DraPNGase-613, O11 + O12 for SavPNGase, and O13 + O14 for SsoPNGase. All targets were successfully inserted into pENTR vectors. For subsequent protein production in E. coli, inserts were transferred from the entry vectors into the destination vectors pDEST15 (N-terminal GST tag) and pDEST17 (N-terminal His6 tag) as described in 5.2.5.2. This step was performed for all targets except
AniPNGase and was successful in all cases except for SavPNGase (data not shown). It is not clear why this sequence was resilient to transfer from the entry vectors into any of the destination vectors as the sequence was correct. Small scale expression trials were performed using E. coli BL21-AI, but no soluble protein was obtained for either of the two DraPNGase proteins and no recombinant protein could be detected for SsoPNGase (data not shown).
For protein expression using the BVES (Figure 5.2) the inserts were transferred from the entry vectors into either pDEST10 (N-terminal His6 tag) or pDEST8 (no tag). These destination vectors were then transformed into E. coli
DH10Bac cells, which contain the bacmid (baculovirus shuttle vector) and a helper plasmid. Recombinant bacmids are generated by transposing a mini-Tn7
element from a donor plasmid (pDEST™ vectors) to the mini-attTn7
attachment site on the bacmid. The Tn7 transposition functions are provided by the helper plasmid. Following verification of the bacmid, Sf9 cells were transfected and a P1 stock was isolated and amplified (5.2.6.2). The titres of the P2 stocks were determined (5.2.6.3) and the following values were obtained:
(i) DraPNGase-561: 5.7 107 pfu/mL
(ii) DraPNGase-613: 7.0 107 pfu/mL
(iv) AniPNGase: 2.0 108 pfu/mL
The P2-stock titres were well above the value given as a guideline by the manufacturer (> 107 pfu/mL) and were used for infection of Sf9 cells in protein expression trials and expression optimisation experiments. After initial expression trials failed to show any recombinant protein the following variables were optimised (based on manufactures recommendations and (Farrell & Iatrou, 2004)):
(i) Cell density at time of infection (6 105, 1 106, 2 106 cells/mL)
(ii) Multiplicity of infection (MOI; 2.5, 5, 10 pfu/cell)
(iii) Time (samples taken after 2, 3, 4, 5 days post infection)
However, no recombinant protein was obtained for any target (data not shown). It is difficult to explain why no protein was produced as all steps leading up to the infection of the cells were successful, including generation of the recombinant bacmids that were used to infect the Sf9 cells. One possibility could be the insect cell line used. It is possible that the expression level of the recombinant proteins was too low to be detected and that the use of other cell lines such as High Five™ (ovarian cells of the cabbage looper, Trichoplusia ni), which can, according to the manufacturer, in some cases yield higher amounts of recombinant protein, may be required.
However, due to the numerous problems encountered during this project and time constraints, this line of research was abandoned and the focus of this work shifted to the generation, expression and characterisation of recombinant (r)PNGase F and its site-specific mutants (Section II).
Section II
rPNGase F and Its Site-Specific
Mutants – Structural and Functional
Characterisation
Chapter 6: rPNGase F Site-Specific Mutants: Generation, Expression and Purification
Chapter 7: Structural Characterisation of rPNGase F
Chapter 8: Kinetic Characterisation of rPNGase F and its Site-Specific Mutant Proteins
Anybody who has been seriously engaged in scientific work of any kind realizes that over the entrance to the gates of the temple of science are written the words: ‘Ye must have faith.’