Abstract
BackgroundMembrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein.Methods and FindingsWe describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold.ConclusionsThe results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.
Highlights
Membrane proteins comprise up to 30% of the proteome of any organism [1] and, in humans, are the direct targets of 60% of all pharmaceuticals [2]
The bacterium Escherichia coli is the standard prokaryotic protein production host but, since membrane proteins encompass molecules that greatly differ with respect to structure, sugar decoration, lipid requirements and folding-factors needed, a broad set of hosts may have to be screened to find one best suited for production of a certain protein [5]
Based on a characterization of the E. coli BL21(DE3)-derived C41(DE3) and C43(DE3) strains, which are known for their increased ability to produce membrane proteins [6], Wagner et al [7] designed E. coli Lemo21(DE3), a strain with a wider applicability due to the tunable activity of the T7 RNA polymerase driving the production of recombinant proteins
Summary
Membrane proteins comprise up to 30% of the proteome of any organism [1] and, in humans, are the direct targets of 60% of all pharmaceuticals [2]. The biological and medical relevance of membrane proteins is quite clear, they have been largely neglected due to a number of technical constraints, such as the production and purification of appropriate quantities of these proteins in their native form. This notion is driving many initiatives and international consortia [3] and has led to the development of novel ways of producing membrane proteins, such as cell-free expression systems [4]. Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. The complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein
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