Enhanced expression of recombinant proteins in insect cells using a baculovirus vector containing a bacterial leader sequence.

Baculovirus/insect cell expression systems have been in use for several years, often resulting in very high levels of expression of recombinant proteins (1). For expression of mature proteins the preferred vectors, such as pVL941 (2) or p36C (3), utilise the strong polyhedrin promoter and leader sequence. We recently reported the finding that the presence of a 24 bp bacterial sequence immediately upstream of the initiation codon unexpectedly supported high expression levels of tetanus toxin fragment C (4). The bacterial leader sequence is derived from the 3' end of the E. coli trpE gene where it functions as part of the ribosome binding site of the adjacent trpD gene (5). This sequence also enabled P69 pertactin from Bordetella pertussis to be expressed at high levels by baculovirus-infected cells (unpublished observations). We have investigated this bacterial sequence further, by comparing its effect on the expression of three different heterologous genes. A 40 kDa fragment (P40) from B.pertussis, which is found in purified extracts of P69 pertactin (6) and which contains the main antigenic determinants of the P69 molecule (7) was expressed in the insect/baculovirus system. Two constructs were made: either with (vAc36CP40-pl) or without (vAc36CP40) the 24 bp bacterial leader sequence (Figure 1), in both cases by ligating the P40 part of the P69 pertactin gene and an appropriate oligonucleotide into the BamHI site of p36C (3). From these transfer vectors were generated recombinant baculovirus as previously described (8). Both recombinant baculovimses were used to infect Spodoptera frugiperda cells. In common with other higher eukaryotic expression systems, variation in expression levels between different batches of insect cells, infected by the same recombinant baculovirus, can be quite large. Consequently, both recombinant baculoviruses were used to infect the same batch of cells at the same time. Both sets of infected cells were harvested after 72 hours and expression levels were analysed by densitometer scanning of stained SDS-polyacrylamide gels. The presence of the bacterial leader sequence increased expression by a factor of 2 (Table 1). This comparison was repeated by infections of a different batch of insect cells and again the presence of the bacterial leader sequence resulted in the higher expression by a similar factor (data not shown). The metalloproteinase enzyme stromelysin (9) was also expressed using two analogous constructs (Figure 1). Expression of stromelysin was much lower than P40 and was quantitated by assaying its protease activity. Again the bacterial leader stimulated expression two-fold (Table 1). Again a repeat pair of infections gave a similar stimulation (data not shown).