Purification, proteolytic digestion and solubilization profile of human growth hormone and asparaginase inclusion bodies.

<p>(a) Purification of inclusion bodies by sucrose density gradient ultracentrifugation, tube one : asparaginase IB, tube two: hGH inclusion bodies (b) SDS-PAGE analysis of purified hGH (21 kDa, lane 1) and asparaginase (37 kDa, lane 3) inclusion bodies. Lane 2 and 4, LMW marker: 97, 66, 45, 30, 20.1 and 14.4 kDa. (<b>c</b>) Kinetics of proteolytic digestion of inclusion body aggregates by Proteinase K. SDS-PAGE of solubilized inclusion body supernatants. (<b>d</b>) hGH IBs. Lane 1–9, supernatants of 0 to 8 molar urea. (<b>e</b>) Asparaginase IBs. Lane 1–9, supernatants of 0 to 8 molar urea; lane M, LMW marker (97, 66, 45, 30, 20.1 and 14.4 kDa).</p

Size distribution patterns of IBs isolated from <i>E. coli</i> cells harvested at different time points after induction.

<p>(<b>a</b>) Asparaginase IBs. (<b>b</b>) hGH IBs. Colored bars indicate the harvesting time.</p

Amino acid sequence of hGH (Swiss-Prot, P01241).

<p>Bold and italics regions indicate presence of hydrophobic residues involves in making hydrophobic and amphipathic helices.</p

Proteinase K degradation profiles of hGH and asparaginase inclusion bodies isolated after 4 hours of induction.

<p>(<b>a</b>) Rate of degradation of IBs with time. (<b>b</b>) Rate of change in degradation rate of IBs with decrease in turbidities.</p

Transmission electron micrograph of purified asparaginase inclusion bodies.

<p>(<b>a</b>), (<b>b</b>), (<b>c</b>) and (<b>d</b>) asparaginase IBs isolated after 1, 2, 3 and 4 hours of IPTG induction respectively. Bar represents 2 µm.</p

Second derivative FTIR spectra of hGH and asparaginase IBs.

<p>(<b>a</b>) Second derivative spectrum of hGH IBs. (<b>b</b>) Second derivative spectrum of asparaginase IBs in amide band region. Broader peak in 1620–1640 cm-1 and 1680–1685 cm-1 range indicates large beta sheet content.</p

Transmission electron micrograph of purified hGH inclusion bodies.

<p>(<b>a</b>), (<b>b</b>), (<b>c</b>) and (<b>d</b>) are hGH IBs isolated after 1, 2, 3 and 4 hours of IPTG induction respectively. Bar represents 2 µm.</p

Solubilization profiles of inclusion bodies isolated from cell harvested at different time points after induction.

<p>(<b>a</b>) hGH IBs (<b>b</b>) Asparaginase IBs. Colored bars indicate the harvesting time.</p

Spectral characteristics of Th-T binding with IBs.

<p>(<b>a</b>) Asparaginase and (<b>b</b>) hGH inclusion bodies isolated at different time points after induction. Concentrations of Th-T and IBs used for assay were 75 µM and 50 µg/ml respectively.</p

Spectral characteristics of Congo-Red (CR) and difference spectra of IBs.

<p>(<b>a</b>) Asparaginase and (<b>b</b>) hGH inclusion bodies isolated at different time points after induction. Concentrations of CR and IBs used for assay were 10 µM and 25 µg/ml respectively.</p