The pET system chosen for these studies enables stringent control of gene expression. The host strain used for expression was E. coli (BL21) ADE3 pLysS, a lysogenic strain
that contains the gene for T7 RNA polymerase (T7pol), under the control of a lac\JW5
promoter. T7 lysozyme, an inhibitor of T7pol, is produced in low quantities by the compatible plasmid pLysS to provide additional stability through the inhibition of basal levels of T7pol produced before induction. The target genes are inserted into the expression vector downstream of a T7/ac promoter that combines the strong T7 transcription promoter and the lac operator. The vector also carries the natural promoter and coding sequence for the lac repressor {lacV), enabling the control of expression through a constitutive repression of transcription of both the T7pol gene from the host chromosome and of the target gene by any T7pol that is made, where repression is removed upon the addition of IPTG.
Inoculation and induction of cultures for expression was carried out as described in Section 2.17
4.4.1 Amidase expression
A high level of amidase expression was achieved under standard conditions (Section 2.17), with the specific activity on acetamide reaching a maximum of 37U /m g±2 approximately 3.5 hours after induction. A typical expression profile is shown in Fig. 4.5. The activity level observed compares very favorably with that of the native organism, with a specific activity 40 times higher. Using previous estimate (Pereira, 1998) of 95% purity following a four-step purification of the native enzyme and specific activity of 117 U/mg, it was estimated that the recombinant enzyme comprised approximately 30% of the total soluble protein.
Fig. 4.5: Expression profile of recombinant amidase Specific Activity Biomass IPTG 20 § < 30 60 90 120 150 180 210 240 285 300 330 360 390
Ferm entation tim e (m ins)
Fig. 4.6: SDS-PAGE analysis of the soluble protein fractions (-lOp-g) of amidase (arrowed) expressed in E. coli (BL21) 1DE3 pLysS. Lanes: 1, molecular weight marker; 2, BL21only; 3, pET21a un-induced; 4, pNH223 un-induced; 5, pET21a induced; 6, pNH223 - 1 hour; 7 pNH223 - 2 hours; 8 pNH223 - 3 hours; 9 pNH223 - 4 hours. ' 1 2 3 4 5 6 7 8 9 60kDa 50 40 _ 30 25 20 15 10 125
4.4.2 Nitrile hydratase expression
In preliminary studies of NHase expression from constructs pNH461 (data not shown) and pNH512 (Fig. 4.4), very low NHase activities were observed. Plasmid stability experiments revealed that cultures of both strains were prone to overgrowth with bacteria that had lost the expression plasmid. The problem was overcome by using carbenicillin as the selective antibiotic at a concentration of 100|xg/ml in place of ampicillin; in addition, the growth medium of the seed cultures was replaced twice prior to inoculation of larger cultures for expression in order to remove secreted p-lactamase.
To investigate the effect of the addition of cobalt ions to the growth medium, a series of expression experiments were performed with varying concentrations of C0CI2. However initial experiments revealed that the addition of cobalt inhibited the growth of the E. coli host, even at concentrations as low as 0.1 mM (data not shown). For this reason cobalt was not added until 15-20 minutes prior to induction.
Expression from pNH512 in the absence of Co^^ resulted in the production of a large quantity of soluble protein (Fig. 4.8) but very low activity. Very similar activities were observed when cobalt was added to cultures at concentrations of 0.1 and 0.5mM (within the range used by previous workers), (Table 4.1). A maximum level of specific activity (on acetonitrile) of 49 U/mg ± 4 was achieved by supplementation with 0.1 mM C0CI2
approximately 3 hours after induction. The recombinant protein was estimated to account for at least 30% of the total cell protein by SDS-PAGE analysis (Fig. 4.8). Calculations based on specific activity determined for the native pure protein were not feasible as the reported value was significantly lower than observed for crude extracts of recombinant protein.
Attempts to express the NHase genes from cells containing pNH461 (containing the NHase a and P subunit genes and the attenuated P14K gene) resulted in very low levels of activity, reaching a maximum of -5 U/mg 2 hours after induction. The addition of cobalt to the growth medium made no effect on the level of activity expressed. When crude extracts were analysed by SDS-PAGE, it was not possible to detect bands corresponding to either the NHase subunits or P14K protein.
Table 4.1: Effect of cobalt addition to cell cultures on NHase activity. Cell extracts of cultures containing pNH512 were prepared 3 hours after induction and assayed for NHase activity on acetonitrile for 10 minutes at 50°C.
C o ^ d d ^ K m M ^ 0 0.1 0.5
NHase activity (U/mg) 5 ± 2 49 ± 4 44 ± 4
Fig. 4.7: Expression profile of recombinant NHase
2 1. 8 I Specific Activity Biomass IPTG 60 90 120 150 170 200 230 260 290 320 350 380 410
Fermentation time (m ins)
Fig. 4.8: SDS-PAGE analysis of the soluble protein fractions (-lOpg) of NHase expressed in E. coli (BL21) 1DE3 pLysS. Lanes: 1 & 14, molecular weight marker; 2, BL21 only; 3, pET21a un-induced; 4, pNH512 un-induced; 5, pET21a induced; 6-9, pNH512, no Co^\ 1 -4hours; 10-13, pNH512 + Co^\ 1 -4hours. Alpha and beta subunits are indicated by arrows, by convention the smaller subunit is referred to as the
a subunit and the larger the p subunit.
! I 1 2 3 4 5 6 90kj 80 ' 70 : 60 » - 50 ' 40 V 4%» M 30