Physiological Adaptation of the Bacterium Lactococcus lactis in Response to the Production of Human CFTR

Abstract

Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

“…In both LIC and VBEx procedures, rare restriction sites ( Swa I and Sfi I) were used. This strategy allowed for the successful expression of MPs from prokaryotic and eukaryotic origins [ 44 , 45 , 46 ].…”

“…As for yeast mitochondrial carriers, human ADP/ATP translocators (AAC1, AAC2, and AAC3) were also expressed in L. lactis . Other human MPs from diverse families and topologies (1–12 TM helices) have been expressed, with levels from almost undetectable (<0.1%) to 1% (Bcl-Xl) ( Table 4 ), including the ABC transporter, CFTR with a very high number of TM helices (12 helices), and size (168 kDa) expressed at very low levels (below 0.1% of TMP; [ 46 ]).…”

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].…”

Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells

“…The instability of pSTVA1 could therefore result from an increase in the metabolic energy necessary for plasmid maintenance and function or from the general deleterious effect on the bacterial growth rate (32,33). There are many reports that show that overexpressed heterologous membrane proteins can affect the bacterial growth rate by imposing a metabolic burden, an overload of the membrane biogenesis machinery, a membrane stress, or local membrane disruptions in bacteria (33)(34)(35). Thus, one possible explanation for the lack of VmpA detection in the absence of tetracycline is that the expression of this protein decreases S. citri fitness and produces counter selection, leading to the loss of VmpA expression.…”