Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

Abstract

Background: Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies.

Results: We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells.

Conclusion: A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.

Figure 1

Evaluation of 70% percoll for self-forming gradients. t = 0 hour: before centrifuge; t = 1, 2, 3 hour: 1, 2 or 3 hours after centrifugation at 27,000 g. A set of colored density marker beads from American density materials (Staunton, VA, USA) was used as density standards to determine the range of the buoyant density formed in the tubes. The density values of the beads are shown on the right side of the tubes.

Figure 2

Light microscopic pictures of cells used in the study. Uninduced QC1101 (A) and induced QC1101 cells (B) expressing HC124 peptide; uninduced QC1525 (C) and induced QC1525 cells (D) expressing HC415 peptide. Note cells expressing HC415 inclusion bodies in panel D are distinctly larger than cells expressing HC124 inclusion bodies shown in panel B. The 5 µm scale bar is indicated.

Figure 3

Density gradient analysis of QC1525 cells growing under different conditions. Top panel of the figure shows the banding patterns of cells on 70% percoll gradient. U, uninduced; I, induced. Lower panel shows SDS-PAGE analysis of the extracted bands as indicated by the arrows in the top panel. Numbers on the lanes on SDS-PAGE correspond to the numbers of the bands on density gradient. The protein band corresponding to ~28kD, where the arrow points, is the peptide of interest, which was only observed in lane 2, 5 and 8.

Figure 4

Density gradient analysis of mixed cultures of induced QC1101 and QC1525. Top panel of the figure shows the banding patterns of individual cultures and the mixed cultures on 70% percoll density gradient. I, induced. Arrows indicate the bands that were extracted for SDS-PAGE analysis. Lower panel shows the SDS-PAGE analysis of the extracted bands. Numbers on the lanes on SDS-PAGE correspond to the numbers of bands on density gradient. Arrowheads show HC124 peptide, while arrows show HC415 peptide in the SDS-PAGE gel.

Figure 5

Density gradient analysis of isolates of interest. U, uninduced; I, induced. Notice increase in buoyant density and population of induced cells obtained from colony 181 (top panel). The induced cells from colony 181 also show higher percentage of POI production (bottom panel, lane 8) compared to the QC1101 induced cell control (lane 3). The numbers on the lanes on SDS-PAGE correspond to the numbers of the bands on density gradient.

Figure 6

Microdensity gradient centrifugation of selected isolates from density gradient sorting of the QC3800 library. Top panel is the whole cells (WC) and bottom panel is the inclusion bodies (IB). The control BW25113 containing the expression plasmid is labeled BW. The density positions of the controls are indicated by the straight lines. The density positions of isolates F1 and A12 are indicated by the arrows.

Figure 7

Confirmation of increased buoyant density in ΔgltA. Density gradient analysis of the whole cells producing the inclusion bodies (top panel WC) and the crude inclusion bodies (bottom panel IB) from the parent strain BW25113 and the JW0710 strain with ΔgltA. Density standard beads of various buoyant densities were separated under the same conditions