Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum

Abstract

BackgroundThe production of recombinant proteins with proper conformation, appropriate post-translational modifications in an easily scalable and cost-effective system is challenging. Lactococcus lactis has recently been identified as an efficient Gram positive cell factory for the production of recombinant protein. We and others have used this expression host for the production of selected malaria vaccine candidates. The safety of this production system has been confirmed in multiple clinical trials. Here we have explored L. lactis cell factories for the production of 31 representative Plasmodium falciparum antigens with varying sizes (ranging from 9 to 90 kDa) and varying degree of predicted structural complexities including eleven antigens with multiple predicted structural disulfide bonds, those which are considered difficult-to-produce proteins.ResultsOf the 31 recombinant constructs attempted in the L. lactis expression system, the initial expression efficiency was 55% with 17 out of 31 recombinant gene constructs producing high levels of secreted recombinant protein. The majority of the constructs which failed to produce a recombinant protein were found to consist of multiple intra-molecular disulfide-bonds. We found that these disulfide-rich constructs could be produced in high yields when genetically fused to an intrinsically disorder protein domain (GLURP-R0). By exploiting the distinct biophysical and structural properties of the intrinsically disordered protein region we developed a simple heat-based strategy for fast purification of the disulfide-rich protein domains in yields ranging from 1 to 40 mg/l.ConclusionsA novel procedure for the production and purification of disulfide-rich recombinant proteins in L. lactis is described.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0902-2) contains supplementary material, which is available to authorized users.

Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody

“…This is consistent with previous reports documenting the aberrant migration of Pf MSP2 30 , attributed to its lack of hydrophobic residues. Anomalous migration during SDS-PAGE has been observed with several plasmodial proteins with disordered regions, highly charged repeat domains and/or deficits in hydrophobic residues 46,47 . Likewise, the chimeric r Pf MSP2/8 migrated as a corresponding ~100 kDa band, well above its predicted molecular weight of 67,659 daltons.…”

Pfs230 and Pfs48/45 Fusion Proteins Elicit Strong Transmission-Blocking Antibody Responses Against Plasmodium falciparum

“…However, a purification process based on conventional chromatographic procedures is currently being developed. Although yields and purity remain to be determined, it is likely that high product yields can be obtained through upscaling the fermentation process which is straightforward since there is no requirement for oxygen and vigorous stirring during fermentation (39). Additionally, yields of properly folded protein species may also be increased through protein refolding processes as those developed for R0.6C (20).…”

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…In particular, an L. lactis NZ9000 strain that incorporates nisK and nisR has been developed [58] and along with its derivatives, is in active use for recombinant protein expression [94]. This system, along with others, has been used to produce various prokaryotic as well as eukaryotic proteins of animal and plant origin [95][96][97], with disulfide bonds intact [98]. Of particular note, L. lactis expression system has been proven to be highly efficient in membrane protein expression, where the expressed membrane proteins are localized to the cytoplasmic membrane and the use of mild detergents can readily solubilize these membrane proteins [94].…”