Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin

Abstract

The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin-like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from E. coli extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex E. coli extracts by repeated precipitation/re-dissolution steps at near physiological temperature.•The method is based on the inverse thermal transition process.•The "green" method is affordable for basic laboratories and can be easily transferred to new users.

Keywords: HUG; Human elastin-like fusion proteins; Inverse thermal transition; Lab-scale production of the recombinant fusion protein HUG; Protein purification method.

Graphical abstract

Fig. 1

Protein expression profile of the induced bacterial culture. Aliquots of the bacterial cultures obtained from 4 clones were taken before (0.4 mL) and after (0.2 mL) induction with IPTG. Lane 1: molecular weight markers; lane 2: the HELP elastin-like protein purified by ITC; lanes 3–5–7–9: bacterial extract before induction; lanes 4–6–8–10: bacterial extract after IPTG induction.

Scheme 1

Sequence for the recovery of pelleted E. coli cells expressing HUG.

Fig. 2

Bacterial pellets as appeared after the centrifugation.

Fig. 3

(A). Homogenization of bacterial pellets in extraction buffer by the OV5 homogenizer. (B) Appearance of mixture after disruption of bacterial cells.

Fig. 4

Example of bacterial lysate clarification. (A) Separation of HUG from bacterial debris after centrifugation. (B) Collected supernatant ready to be stored at −20 °C.

Scheme 2

Overview of crude extracts preparation.

Fig. 5

HUG precipitation starting from a 2 × 240 mL cell extract. (A) Supernatant from extraction before precipitation. (B) Supernatant after adding 2 × 160 mL 5 M NaCl and precipitate appearance.

Fig. 6

Pellets obtained after the first coacervation step.

Fig. 7

Insoluble materials removed by centrifugation.

Fig. 8

Supernatant at 42 °C. (A) Before the addition of NaCl; (B) After the addition of NaCl; (C) After 15 min at 42 °C in the presence of NaCl, leading to complete coacervation.

Fig. 9

Pellet after final centrifugation.

Fig. 10

Freeze-dried HUG protein.

Scheme 3

Sequential steps for purification of HUG.

Scheme 4

Outline of quality controls of purified HUG.

Fig. 11

UV–vis spectra of serially diluted HUG solutions in water. The absorbance values at λ =280 nm were plotted against the HUG concentration. The angular coefficient of the curve represents the experimental extinction coefficient of HUG in water (ε = 18,747 ± 1262; R2 = 0.9728).

Fig. 12

SDS-PAGE analysis of three different purified HUG preparations.

Fig. 13

Fluorescence values as a function of HUG (µg) in the presence of bilirubin excess.