Efficient expression systems for cysteine proteases of malaria parasites

Figures & data

Figure 1. Multiple alignment of amino acid sequences (confirmed or predicted mature form) of several plasmodial cysteine hemoglobinases and some closely-related isoforms. Numbers of amino acids are marked at the right side of each of the alignments. Hyphens (-) represent gaps introduced to maximize alignment and asterisks (*) indicate conserved active-site residues. Clear boxes indicate the position of conserved cysteine residues implicated in disulfide bonds (based on the Falcipain-2 structure).⁷ Shaded boxes indicate the position of a short α-helix (α1) within the N-terminal extension playing a critical role in folding of the mature protein and the C-terminal motif involved in hemoglobin binding. Amino acid sequences of chabaupain-1 (AAP43629); vinckepain-1 (AAL48319); berghepain-1 (XP_677643); yoelipain-1 (XP_729023); vivapain-1 (XP_001615807); falcipain-1 (AAA29578); falcipain-2 (AF282975); falcipain-2´ (AAX77225); falcipain-3 (AAN35746); vivapain-2 (XP_001615274); vivapain-3 (XP_001615273); vivapain-4 (XP_001615272); chabaupain-2 (AAP43630); vinckepain-2 (AAL48319); berghepain-2 (AAL48318); yoelipain-2 (XP_726900) were obtained from GeneBank database (www.ncbi.nlm.nih.gov/genbank/). Papain sequence (AAB02650) was included for comparison.

Table 1. Heterologous expression systems of Papain-like cysteine proteases from human and rodent malaria parasites

Enzyme	Specie	Host	Plasmid	Induction cond.	Expression yield	Solubility and Activity	Refolding	Final yield	Reference
Falcipain-1	P. falciparum	E. coli BL21 (DE3)	pET-9d, pRSET	IPTG 0.4 mM	Abundant	I. bodies	Not achieved	-	14
Falcipain-1	P. falciparum	Insect cells Trichoplusia ni	pBlueBac2	-	ND	Soluble and active	-	low	14
Falcipain-1	P. falciparum	E. coli BL21 (DE3)	pGEX-6P-1, pMAL-c	IPTG 1 mM 25˚C	Low-to-moderate	Soluble and active	-	ND	21
Falcipain-1	P. falciparum	Several E. coli strains	pET22b, pET32b, pQE30	IPTG 1 mM 37˚C	~ 5 mg/L	I. bodies	Rapid dilution, optimized composition	~ 1 mg/L	15
Falcipain-2	P. falciparum	E. coli M15(pREP4)	pQE30	IPTG 0.25 mM 37˚C	Abundant	I. bodies	Rapid dilution, optimized composition	ND	5
Falcipain-2	P. falciparum	Several E. coli strains	MAL-c, pET-30a(+), pQE30	IPTG 0.25 mM 37˚C	~ 48 mg/L (10% of total proteins)	I. bodies	Rapid dilution, optimized composition	2.4 mh/L	20
Falcipain-2	P. falciparum	E. coli BL21(DE3),TB1	pET24d, pET24a, pGEX-6P-1, pThioHisA, pMAL-c	IPTG 0.1 mM 25˚C	Abundant	Soluble and active	-	low	22
Falcipain-2	P. falciparum	Pichia pastoris X33, KM71H	pPICZA	MeOH 0.5%	Not achieved	-	-	-	22
Falcipain-2	P. falciparum	E. coli M15(pREP4), RosettaBlue (DE3)	pQE-30	IPTG 0.5 mM 37˚C	ND	I. bodies	Rapid dilution, optimized composition	Low-to-moderate	7
Falcipain-2	P. falciparum	Several E. coli strains	pBAD24, pTrcHis2A	Lactose 0.2% 37˚C	~460 mg/L (42% of total proteins)	I. bodies	Rapid dilution, optimized composition	55 mg/L	19
Falcipain-2	P. falciparum	E. coli BL21 (DE3)	pMAL-c	IPTG 1 mM 25˚C	Moderate-to-abundant	Soluble and active	-	low	24
Falcipain-2	P. falciparum	E. coli Bl21 (DE3)/pLysS	pRSET-B	IPTG 0.25 mM 37˚C	ND	I. bodies	Rapid dilution, optimized composition	ND	8
Falcipain-2	P. falciparum	E. coli M15(pREP4)	pQE-30, pMAL-c2X	IPTG 0.25 mM 37˚C	Abundant	Soluble and active	-	ND	23
Falcipain-3	P. falciparum	E. coli M15(pREP4)	pQE-30	IPTG	Low-to-moderate	I. bodies	Rapid dilution, optimized composition	High	6
Vivipain-2	P. vivax	E. coli M15(pREP4)	pQE-30	IPTG 1 mM	Low-to-moderate	I. bodies	Rapid dilution, optimized composition	ND	9
Vivipain-3	P. vivax	E. coli M15(pREP4)	pQE-30	IPTG 1 mM	Low-to-moderate	I. bodies	Rapid dilution, optimized composition	ND	9
Vivipain-4	P. vivax	E. coli M15(pREP4)	pQE-30	IPTG 1 mM 37˚C	Abundant	I. bodies	Rapid dilution, optimized composition	ND	10
Vinckepain-2	P. vinckei	E. coli M15(pREP4)	pQE-30	IPTG	Low-to-moderate	I. bodies	Rapid dilution, optimized composition	ND	11
Berghepain-2	P. berghei	E. coli Bl21 (DE3)/pLysS	pRSET-A	IPTG 0.5 mM 37˚C	Low-to-moderate	I. bodies	Rapid dilution, optimized composition	ND	12
Chabaupain-1	P. chabaudi	E. coli XL1 Blue	pGEX-6P-1, pQE30	IPTG 0.5 mM 17˚C	Moderate-to-abundant	Partially soluble	-	ND	13
Chabaupain-2	P. chabaudi	E. coli XL1 Blue	pQE30	IPTG 0.5 mM 37˚C	ND	I. bodies	Rapid dilution, optimized composition	ND	13

Where more than one expression vector was used, that finally used for large-scale expression is indicated in bold letter. ND, not described.

Figure 2. Comparison of plasmodial genes for cysteine proteases (●) with highly expressed Escherichia coli genes (٭) corresponding to glycolytic and tricarboxylic acid cycle enzymes. The cluster corresponding to vivipain genes is indicated with dashed lines. The optimized falcipain-2 gene is also indicated with a star. CAI values and global A+T content were calculated using the CAIcal server (http://genomes.urv.es/CAIcal/).²⁷

Figure 3. Comparison of several eukaryotic systems (hosts) for the expression of plasmodial genes for cysteine proteases (P. pastoris: Pichia pastoris; S. cerevisiae: Saccharomyces cerevisiae; T. ni: Trichoplusia ni; S. frugiperda: Spodoptera frugiperda; C. griseus: Cricetulus griseus; H. sapiens: Homo sapiens). The mean for each group is indicated by a horizontal line. The cluster corresponding to vivipain genes is indicated with dashed lines.

Figure 4. Comparative profile of relative codon frequencies in plasmodial cysteine protease, E. coli (A) and P. pastoris (B) genes. Differences in frequency (per thousand) of codon usage (FCU_PLASMODIUM – FCU_HOST) were calculated for each codon, using codon composition from plasmodial cysteine protease genes and information contained in codon usage tables for E. coli and P. pastoris, obtained from Codon Usage Database.³⁰ Those codons showing modular differences ≥ 20% are indicated.

Table 2. Nucleotide composition of plasmodial genes in comparison to E. coli and P. pastoris genes

Host/parameter	A+TGlobal (%)	A+T(1) (%)	A+T(2) (%)	A+T(3) (%)
Plasmodial cysteine protease genes	68.2	60.2	71.4	72.9
E. coli genes	47.7	39.2	59. 4	44.4
P. pastoris genes	57.3	51.3	62.7	57.8

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