Figure 6

Cloning strategy for expression of LASV proteins GP1, GP2, and NP in E. coli using pMAL vectors. To generate MBP-LASV gene fusions for E. coli expression, PCR-amplified LASV gene sequences were restricted and cloned in-frame at the 3' end of the mal E gene, beyond the cleavage site for Factor Xa (IQGR). The LASV GP1 gene sequence comprised a.a. 59–259 in the native GPC, spanning the first a.a. beyond the known SPase cleavage site at position 58 to the junction between GP1 and GP2 domains, which is cleaved by the SKI-1/S1P protease at a.a. 259. The LASV GP2 gene sequence comprised a.a. 260–427, spanning the first a.a. of mature GP2 to the last a.a. before the predicted TM domain. The LASV NP gene sequence comprised the complete ORF of the gene, with the exception of the N-terminal Met. The 3' oligonucleotides used for amplification of each gene sequence were engineered to contain two terminator codons separated by a single nucleotide. All genes were cloned into vectors pMAL-p2x and pMAL-c2x for periplasmic and cytoplasmic expression of fusion proteins, respectively, in E. coli Rosetta 2(DE3) or gami 2 strains. The a.a. position of each LASV gene domain is noted, as are REN sites. Abbreviations include: MBP gene (mal E), MBP promoter (P_tac), philamentous phage origin of replication (M13 ori), bacterial origin of replication (pBR322 ori), beta-lactamase gene (bla), E. coli terminator (rrnB), the LacZ alpha-complementation domain (LacZα), and the lacI repressor gene (lacI^q). The periplasmic secretory domain in pMAL-p2x is indicated by a black box on the 5' end of the mal E gene sequence.