High-throughput generation of P. falciparum functional molecules by recombinational cloning

Abstract

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Figure 1

Flowchart of high-throughput recombinational cloning using the Gateway system. The schematic follows a single malaria ORF (CSP, green) as one example for the workflow. (1) The establishment of master clones is generated in a 96-well format including: ORF amplification, PCR purification, BP reaction, E. coli transformation, and recombinant clone screening. On average, we achieved 84% cloning efficiency for the BP reaction. Quality control is monitored and documented using PCR and DNA sequencing followed by archiving the master clones as purified DNA and glycerol stocks. (2) The simultaneous subcloning of ORFs into multiple expression vectors is achieved though the LR reactions. The illustration shows three destination vector examples: DNA vaccine (red), recombinant protein (green), and Y2H (yellow). Following the LR reaction and E. coli transformation (3), recombinant clones are screened by either single-colony (100% efficiency) or bulk-culture (93% efficiency) PCR as represented by the deep-well plate. (4) After screening and DNA purification, expression clones are then processed directly into functional assays.

Figure 2

PCR screening BP and LR reactions. (A) Recombinant clones in the pDONR/Zeo master plasmid were screened by colony PCR using plasmid-specific primers (M13 forward and reverse). The figure shows amplified DNA products from 12 BP reactions representing different genes in groups of four colonies (a, b, c, d) per clone analyzed on a 1% agarose gel. (B) The screening of recombinants clones in the VR1020-DV destination vector was done by PCR on DNA from 4 single colonies (a, b, c, d) as well as bulk cultures (B) from the same transformation. Clones shown were picked at random. (M) 1-kb DNA extension ladder.

Figure 3

Size distribution of cloned and noncloned ORFs. A total of 303 amplified P. falciparum ORFs were screened for cloning into the Gateway entry plasmid by PCR and DNA sequencing. The horizontal lines to the right of the data points are the mean and the upper and lower 95% confidence limits.

Figure 4

Immunogenicity, stage expression, and localization of P. falciparum proteins encoded by ORFs cloned into DNA vaccine plasmids. In all, 95 genes from Panel A were batch cloned into the VR1020-DV DNA vaccine vector, and used to immunize 5 CD-1 mice per gene. The murine sera were pooled and screened by IFAT against the sporozoite, asexual erythrocytic (trophozoites and schizonts), and sexual erythrocytic (gametocyte) stages of P. falciparum. The asexual erythrocytic stage was a mixture of early and late trophozoites and schizonts. Sporozoites were isolated from mosquitos' salivary glands and gametocytes were percoll-purified from in vitro cultures. Of the 95 constructs screened, the number of positive genes were shown. The titer range is the lowest and the highest end-point IFA dilution scored for any positive group. Numbers on the left indicate the accession number for ORFs.