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TBV25H Expressed in Yeast and Purified Using Nickel-NTA Agarose

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TBV25H Expressed in Yeast and Purified Using Nickel-NTA Agarose © 1994 Nature Publishing Group http://www.nature.com/naturebiotechnology /RESEARCH• Production, Purification and lmmunogenicity of a Malaria Transmission-Blocking Vaccine Candidate: TBV25H Expressed in Yeast and Purified Using Nickel-NTA Agarose David C. Kaslow* and Joseph Shlloach 1 Molecular Vaccine Section, Laboratory of Malaria Research, National Institute of Allergy and Infectious Diseases. 'Biotechnology Unit, Laboratory of Cellular and Developmental Biology, National Insitute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892. *Corresponding author (e-mail: [email protected]). We have constructed a second generation malaria transmission-blocking vaccine candidate based on Pfs25, the predominate surface protein of Plasmodium falciparum zygotes, to overcome potential produc­ tion problems with the original construct. Four modifications were made: (1) addition of the last cysteine residue of the fourth epidermal growth factor like-domain of Pfs25; (2) mutagenesis of asparagine-linked glycosylation sites with glutamine rather than alanine; (3) addition of a six histidine tag at the carboxy­ terminus for highly efficient purification ofrecombinant protein on nickel-NTA agarose; and (4) fermenta­ tion that combines continuous glucose fed-batch methodology with pH-controlled glucose addition and a terminal ethanol feed. The resulting product, TBV25H (Transmission-Blocking Vaccine based on Pfs25 with a Histidine tag), appears to be a more potent antigen and immunogen than the original construct, and the fermentation and post-fermentation processing methodology easily lend themselves to technology transfer to the ultimate users, newly industrialized countries. Received 8 March 1994; accepted 28 March 1994. alaria, a mosquito-transmitted disease afflicting sequence fused to the four domains of Pfs25. To eliminate two of hundreds of millions of residents living in more four potential N-linked glycosylation sites, the coding sequence than 100 tropical countries, is an immense public of Pfs25 was truncated before the last cysteine residue of the health problem. Despite initial successes of vec­ fourth EGF-like domain. The remaining two sites were muta­ tor control and chemotherapy in controlling the genized by substituting Ala for Asn. Nevertheless, Pfs25-B is spread of malaria parasites, recent progress has immunogenic in laboratory animals, and despite not recreating been severely hampered by the spread of insecti­ all of the tertiary structure of native Pfs25, Pfs25-B elicited cide-resistant mosquitoes and drug-resistant parasites (for a transmission-blocking antibodies in mice and monkeys5•6 • Prob­ recent review see ref. 1). One long term solution actively being lems arose, however, when attempts were made to scale-up sought to stem the resurgence of malaria is a malaria vaccine production. In particular, there was serious loss of product due aimed at disrupting the parasites life cycle at one or more of to degradation of the protein. To circumvent the problems we three stages (sporozoite stage or pre-erythrocytic, asexual para­ have constructed a second generation vaccine, TBV25H. In site blood stage or erythrocytic, and sexual stage or sporogonic). addition to adding back the last cysteine residue of the fourth The primary goal of an anti-sexual stage vaccine is to block EGF-like domain, the protein and purification scheme were transmission of malaria parasites by interrupting gamete devel­ altered by addition of a "His6-tag" on the carboxy-terminus and opment in the mosquito midgut [for recent review see ref. 2]. by using nickel-NTA agarose to affinity purify the secreted Antibodies against proteins expressed on the surface of sexual protein with an intact carboxy-terminus, thus minimizing co­ stage parasites are ingested along with the blood meal and have purification of degradation products. The purification method been shown to interfere with normal development of the parasite and fermentation protocol described here can be easily trans­ in the mosquito2• Although any malaria vaccine that completely ferred to newly industrialized malaria endemic countries for protects an individual from developing malaria parasites in the in situ production. bloodstream will also block transmission, those directed against the sexual stages are referred to as transmission-blocking vac­ Results cines because anti-sexual stage vaccines do not directly protect Plasmid construction. Using overlapping synthetic oli­ the individual vaccinated. Rather, these anti-sexual stage vac­ gonucleotides, the Pfs25 coding region (not including the secre­ cines prevent infected individuals from transmitting the parasite, tory signal sequence or the hydrophobic carboxy-terminus) was and hence the disease, to others. constructed with yeast-preferred codons. Three of the four Pfs25, a 25-kD cysteine-rich protein consisting of four epi­ potential N-linked glycosylation sites of Pfs25 were mutated by dermal growth factor (EGF)-like domains anchored to the substituting glutamine for asparagine residues. The fourth surface of the late sexual stages of malaria parasites by a glyco­ potential glycosylation site present in the hydrophobic carboxy­ sylphosphatidylinositol moiety, is one of several Plasmodium terminus was deleted. Three new restriction sites were intro­ falciparum (the causative agent of the lethal form of human duced for cloning purposes (Fig. 1). The resulting synthetic malaria) transmission-blocking vaccine candidates3 • gene was used as a template in PCR to construct a series of Pfs25 has been expressed previously in vaccinia virus• and truncated Pfs25 genes. The various genes were subcloned into in Saccha.romyces cerevisiae5. The yeast product, referred to the Kpnl and Spel sites of plasmid pIXY154, a derivative of as Pfs25-B, consists of the a-factor pre-prosecretory signal plXY120 (ref. 7). pTBV5, a construct that terminates at the 494 BIO/TECHNOLOGY VOL. 12 MAY 1994 © 1994 Nature Publishing Group http://www.nature.com/naturebiotechnology Ala22 Glu188 carboxy-terminus so that the product could be purified using pTBV5 Ni-NTA agarose. Kpnl Sal I •Clal Xbal Spel Growth of yeast and production of rPfs25. The amount of Ala 22 Ttv 193 • HisX6 full-length Pfs25 (from Ala-22 to Asn-202), encoded by pTBV25H pTBV25FH, secreted from yeast is considerably lower than Kpnl Sall Clal Xbal Spel TBVS (Fig. 2A, lanes Asn-202 and Glu-188, respectively). Ala 22 Asn 202 - HisX6 Yeast transformed with the construct referred to as pTBV25H, pTBV25FH that encodes Pfs25 from Ala-22 to Thr-193 (i.e., including Cys- Kpnl Sall Clal Xbal 192) and terminates with six histidine residues, secreted about the same amount of Pfs25 protein as control pTBV5 (Fig. 2A, ------Kpnl Pfs25 lanes Thr-183 and Glu-188, respectively). Monoclonal antibody (mAb) 487, which recognizes a linear, reduction-insensitive B­ cell epitope in the third EGF-like domain of Pfs25, bound to TBV25H and TBV5 equally well (Fig. 28). Mab 102, which recognizes a reduction-sensitive 8-cell epitope in the third EGF­ 1 ori like domain of Pfs25, binds TBV25H better than TBV-5 (Fig. 2C), suggesting that the last cysteine of the fourth EGF-like \ domain of Pfs25 is somehow involved in recreating the proper \ . conformation of the third EGF-like domain. Because production levels of rPfs25 were more favorable using pTBV25H than full length Pfs25, pTBV25H was used in all of the experiments FIGURE 1. Expression vectors. Only relevant segments of described below. the plasmid DNA are shown and are not drawn to scale. The Because the trpl gene of the S. cerevisiae strain, XV2181 arrows Indicate the orientation of promoters and open reading (ref. 7), has a point mutation, spontaneous reversion can occur. frames. The amino acid residue numbers refer to those used Strain 2905/6, which has a deletion of part of the trpl gene, does in reference 3. not revert. Although approximately equivalent quantities of rPfs25 were produced by these two strains; a Coomassie-stain­ .· .· ... :,' able band migrating just below rPfs25 was observed occasion­ ally on SOS-PAGE (Fig. 2) in XV2181 culture supematants but B C not in 2905/6 supernatants (Fig. 3, lane 4). Therefore, 2905/6 was used in the fermentation studies described below. j 11 I A starting concentration of 2 to 3 % glucose was found to .- . ! 1 I 1 optimize the yield of TBV25H in shaker flask and in batch ,~ fermentation (data not shown). Above 3% glucose, the yield of .• TBV25H dropped below 0.5mg/L (data not shown). By using a »J··F modification of the fed-batch protocol of Tctttrup and Carlsen9 Coomasale lmml.KlOblol lmml.KlOblol ( consisting of continuous nutrient addition at a rate of I. 5 gm mAb487 mAb 102 glucose/L/hr for 40 hours) in combination with a protocol simi­ lar to that developed by Porro et al. 10 (to compensate for the 16% SDS-PAG and Western FIGURE 2. Coomassie-stained increasing blots of TCA-precipitated yeast culture supernatants. Culture increasing glucose consumption with progressively supernatants from transformed XV2181 cells were precipitated biomass), after which the nutrient feed was switched to ethanol with deoxycholate-trichloracetic acid and the acetone-washed at a rate of l .0gm/L/hr, protein production increased almost pellet size-fractionated by 16% SOS-PAGE. (A) Coomassle­ forty-fold. A typical production pattern of extracellular stalned gel. Control, XV2181 cells transformed with plXY154. TBV25H is shown in Figure 4. Glu 188, XV2181 cells transformed with plXY154 encoding Purification of TBV25H. The purification scheme
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