Optimisation of surface expression using the AIDA autotransporter

Abstract

Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

Figure 1

Surface expression vector. Components are shown to approximate and relative sizes. The construct contains the native signal sequence for AIDA (SP), the protein Z passenger (Z), a linker region (L) and the C-terminal β-barrel domain (AIDA^c) of AIDA.

Figure 2

Strain dependent expression of protein Z. Comparison of surface expression of protein Z in the wild type and in the OmpT-negative E. coli strain 0:17. (A): Western blot of the outer membrane protein fraction, developed using antiserum against AIDA^c. Lane 1: Marker, Lane 2: 0:17, Lane 3: 0:17ΔOmpT, Lane 4: 0:17 with empty vector, Lane 5: 0:17ΔOmpT with empty vector, Lane 6: 0:17 with vector containing protein Z, Lane 7: 0:17ΔOmpT with vector containing protein Z. (B): Cells analysed by flow cytometry using human IgG linked to AlexaFluor488. Red: 0:17 with empty expression vector, Blue: 0:17 with vector containing protein Z, Green: 0:17ΔOmpT with vector containing protein Z.

Figure 3

Effects of carbon starvation on surface expression. Shake flask cultivations of 0:17ΔOmpT showing growth and surface expression of the Z protein in relation to the entry into stationary phase. Three cultivations are shown: one with 10 g l^-1 initial glucose (squares) and two with 3 g l^-1 initial glucose (circles and diamonds). Open symbols show cell growth measured by optical density and filled symbols show surface expression measured by flow cytometry. Arrow: a second addition of glucose was made to one of the two flasks with low starting glucose (circles). All curves are normalized to enter stationary phase simultaneously, and the entry into the stationary phase is marked with a vertical line.

Figure 4

Effects of growth medium. (A) Comparison of surface expression of protein Z in minimal medium with glucose (grey), LB medium (white) and LB medium with glucose (black). Samples were taken at OD₆₀₀ = 0.5 and 3. (B) Consumption of amino acids and production of NH₃ in LB medium (filled symbols) and LB medium with glucose (open symbols) as a function of cultivation time. Legend: Serine (circles), aspartate (squares), threonine (upwards pointing triangle), proline (downwards pointing triangle) and NH₃ (diamonds). Arrow: depletion of the first amino acid (serine) in the LB cultures.

Figure 5

Fed-batch cultivation. Progress of cell growth and surface expression before and after feed start (marked with a vertical line). Filled symbols show cell growth and open symbols show surface expression. Legend: μ = 0.1 h^-1 (circles), μ = 0.2 h^-1 (squares) and μ = 0.4 h^-1 (triangles).

Figure 6

Batch cultivation with repeated glucose addition. (A) Cell growth measured as optical density (filled squares) and dry weight (open squares), and surface expression measured by flow cytometry (circles) as a function of cultivation time. Growth curves are fitted to exponential functions to approximate the growth rates (optical density and dry weight). (B) Growth rate (diamonds), acetic acid concentration in the growth medium (triangles) and dissolved oxygen tension (DOT, line) as a function of time. Downward pointing arrows indicate time points for the five additions of glucose. The leftmost upwards pointing arrow indicates the starting point for manual DOT regulation by increasing of the stirring or the air-flow, and the rightmost upwards pointing arrow indicates the point at which the maximum stirrer speed was reached.

Figure 7

Typical fluorescence peaks characterising batch and fed-batch expression. Comparison of fluorescence peak width between a selected but typical fed-batch experiment here from a cultivation at a feed corresponding to a growth rate of 0.4 h^-1 (green) and the batch experiment with repeated glucose additions (purple).