High level expression and facile purification of recombinant silk-elastin-like polymers in auto induction shake flask cultures

Abstract

Silk-elastin-like polymers (SELPs) are protein-based polymers composed of repetitive amino acid sequence motifs found in silk fibroin (GAGAGS) and mammalian elastin (VPGVG). These polymers are of much interest, both from a fundamental and applied point of view, finding potential application in biomedicine, nanotechnology and as materials. The successful employment of such polymers in such diverse fields, however, requires the ready availability of a variety of different forms with novel enhanced properties and which can be simply prepared in large quantities on an industrial scale. In an attempt to create new polymer designs with improved properties and applicability, we have developed four novel SELPs wherein the elastomer forming sequence poly(VPGVG) is replaced with a plastic-like forming sequence, poly(VPAVG), and combined in varying proportions with the silk motif. Furthermore, we optimised a simplified production procedure for these, making use of an autoinduction medium to reduce process intervention and with the production level obtained being 6-fold higher than previously reported for other SELPs, with volumetric productivities above 150 mg/L. Finally, we took advantage of the known enhanced stability of these polymers in developing an abridged, non-chromatographic downstream processing and purification protocol. A simple acid treatment allowed for cell disruption and the obtention of relative pure SELP in one-step, with ammonium sulphate precipitation being subsequently used to enable improved purity. These simplified production and purification procedures improve process efficiency and reduce costs in the preparation of these novel polymers and enhances their potential for application.

Figure 1

Schematic representation of the block units for the four SELP copolymers, where Sx represents the number of silk-like blocks and Ey the number of elastin-like blocks. Each hybrid block (SxEy) was self-ligated through a series of ligation reactions to achieve a number of tandem repeats corresponding to a molecular weight of approximately 55 kDa.

Figure 2

Electrophoretic patterns of cell crude extracts during growth of auto-induced pCM13(SELP-1020-A)-BL21(DE3) cultures in A) LB+lac at 37°C, B) TB+lac at 37°C and C) TB+lac at 30°C. To facilitate direct comparison of protein expression, all samples were normalised for the same cell density before loading on gel. The sampling time is given above each lane whereas the OD600 at that time point is indicated below. Target recombinant protein is indicated by arrows and the molecular weight marker is in the left of each gel. The abnormal gel mobility of the recombinant protein was previously observed by other authors (Teng et al. ; Lyons et al. ; McPherson et al. 1992).

Figure 3

Cell growth and SELP-1020-A expression using auto-induction medium with different flask volume ratios after 22 hours of fermentation. All samples were normalised for the same cell density prior to loading on gel. This figure was assembled from pictures of different gels. No modifications were made to the images other than cutting, pasting and resizing. Cell density is indicated below each gel with target recombinant protein indicated by arrows. Molecular weight marker is represented on the right and left lanes.

Figure 4

Expression levels with respect to the concentration of inducer and time course of induction. Cultures of E. coli harboring pCM13(SELP-1020-A) in LB were induced with 0.5 mM or 1 mM of IPTG when the OD₆₀₀ reached 1. Samplings were taken at the indicated intervals. Cell density is indicated below each lane with target recombinant protein indicated by arrows.

Figure 5

Purification of SELP-1020-A by A) affinity chromatography and B) molecular weight determination by mass spectrometry. A) SDS-PAGE of SELP-1020-A: lane 1 – soluble lysate before column loading; lane 2 – flow through; lane 3 – fraction eluted with 20 mM imidazole; lane 4 – fraction eluted with 40 mM imidazole; lane 5 – fraction eluted with 80 mM imidazole. Fully purified polymer fraction was obtained by elution with buffer containing 80 mM of imidazole with high recovery rate. B) Image represents the MALDI-TOF mass spectrum of SELP-1020-A. The molecular ion peak at 54125 confirms the theoretical calculated value (54117 Da).

Figure 6

Effect of the acidic treatment of the soluble lysate. The clear supernatant of the lysate from SELP-1020-A (lane 4) was adjusted to pH 4 (lane 1), 3.5 (lane 2) and 3 (lane 3). After pH adjustment, the precipitated pellet was removed and the clear supernatant was analysed by SDS-PAGE. Nearly pure SELP-1020-A was obtained after the acidic treatment at pH 3.

Figure 7

Cell crude extract obtained from bacterial cell cultures producing the different copolymers. This figure was assembled from pictures of different gels. No modifications were made to the images other than cutting, pasting and resizing.

Figure 8

Purification of the recombinant copolymers by ammonium sulphate precipitation. The soluble acid-treated lysates of SELP-1020-A (A), SELP-520-A (B) and SELP-59-A (C) were saturated with increasing concentrations of ammonium sulphate (indicated above each gel). Depending on the concentration used, the copolymers either precipitated or stayed in the supernatant. (D) ( Increased purity was obtained by resuspending the precipitated copolymer (with 20% ammonium sulphate) in water and letting at 4°C with agitation followed by centrifugation or filtration (lane 1 – SELP-1020-A, lane 2 – SELP-520-A, lane 3 – SELP-59-A). The abnormal gel mobility of the recombinant protein was previously observed by other authors (Teng et al. ; Lyons et al. ; McPherson et al. 1992) and attributed to the hydrophobic nature of the proteins.

Figure 9

Evaluation of cell death and protein release in bacterial cultures of SELP-59-A. (A) – number of CFUs in untreated cell crude extracts (U) and in cell extracts submitted to sonication (S), acid-based cell lysis (Ac) and acid-based cell lysis followed by sonication (AcS). Cell dilutions are represented in parenthesis. (B) – Graphic representation of the percentage of cell survival for the different treatments with results expressed as percentage of survival as compared to the untreated sample. (C) – Evaluation of protein release in samples at different concentrations of wet cell weight after acid-based cell lysis (Ac) and acid-based cell lysis followed by sonication (AcS). The same volume was applied in each lane. Samples legend: U – untreated cell crude extract; Ac – acid-based cell lysis; AcS – acid-based cell lysis followed by sonication. (D) – FTIR spectra displaying the amide I (1600 – 1700 cm-1) and amide II (1500 – 1600 cm-1) band regions of pure lyophilized SELP-59-A samples without (a) and with (b) the acidic treatment.