Advances on the large-scale bacterial production and stable phosphorylation of Hsc70

Kley N, Hecht-Bucher M, Itzen A
Protein Expression and Purification :106923 (2026 Mar)

doi: 10.1016/j.pep.2026.106923

Submitted to SASBDB: 2026 Jan 12 Published in SASBDB: 2026 Mar 26

SASDYK4 – Heat shock cognate 71 kDa protein (Hsc70) T204A mutant with ATP (2 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.1 nm Dmax 17.5 nm VolumePorod 143 nm3

SASDYL4 – Heat shock cognate 71 kDa protein (Hsc70) T204A mutant with ATP (3 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.1 nm Dmax 18.3 nm VolumePorod 143 nm3

SASDYM4 – Heat shock cognate 71 kDa protein (Hsc70) T204A mutant with ATP (4 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.3 nm Dmax 18.4 nm VolumePorod 160 nm3

SASDYN4 – Heat shock cognate 71 kDa protein (Hsc70) T204A mutant with ATP (5 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.4 nm Dmax 19.1 nm VolumePorod 167 nm3

SASDYV4 – Heat shock cognate 71 kDa protein (Hsc70) T204A mutant with ADP (1 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.2 nm Dmax 22.0 nm VolumePorod 252 nm3

SASDYW4 – Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (2 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.5 nm Dmax 22.9 nm VolumePorod 291 nm3

SASDYX4 – Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (3 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.7 nm Dmax 23.8 nm VolumePorod 340 nm3

SASDYY4 – Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (4 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.7 nm Dmax 23.5 nm VolumePorod 317 nm3

SASDYZ4 – Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (7 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.9 nm Dmax 25.5 nm VolumePorod 334 nm3

SASDY25 – Heat shock cognate 71 kDa protein (Hsc70) T204A APO (0.5 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.6 nm Dmax 24.0 nm VolumePorod 324 nm3

SASDY35 – Heat shock cognate 71 kDa protein (Hsc70) T204A apo (2 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.8 nm Dmax 25.0 nm VolumePorod 351 nm3

SASDY45 – Heat shock cognate 71 kDa protein (Hsc70) T204A apo (3 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.6 nm Dmax 25.0 nm VolumePorod 348 nm3

SASDY55 – Heat shock cognate 71 kDa protein (Hsc70) T204A apo (4 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.7 nm Dmax 24.5 nm VolumePorod 351 nm3

SASDY65 – Heat shock cognate 71 kDa protein (Hsc70) T204A apo (5 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.7 nm Dmax 24.7 nm VolumePorod 352 nm3

SASDY75 – Heat shock cognate 71 kDa protein (Hsc70) T204A apo (7 mg/ml) Sample: Heat shock cognate 71 kDa protein, 71 kDa protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.6 nm Dmax 25.0 nm VolumePorod 360 nm3

SASDY85 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ATP (1 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 3.6 nm Dmax 15.3 nm VolumePorod 122 nm3

SASDY95 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ATP (2 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 3.7 nm Dmax 16.4 nm VolumePorod 131 nm3

SASDYA5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ATP (3 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 3.9 nm Dmax 16.7 nm VolumePorod 141 nm3

SASDYB5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ATP (4 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.3 nm Dmax 16.7 nm VolumePorod 149 nm3

SASDYD5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ATP (7 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1 mM TCEP, 1 mM ATP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.2 nm Dmax 18.1 nm VolumePorod 162 nm3

SASDYE5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (1 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.4 nm Dmax 20.3 nm VolumePorod 169 nm3

SASDYF5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (2 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.8 nm Dmax 22.3 nm VolumePorod 203 nm3

SASDYG5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (3 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.9 nm Dmax 22.0 nm VolumePorod 218 nm3

SASDYH5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (4 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.0 nm Dmax 22.8 nm VolumePorod 226 nm3

SASDYJ5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (7 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.5 nm Dmax 24.4 nm VolumePorod 250 nm3

SASDYK5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (1 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 4.8 nm Dmax 21.0 nm VolumePorod 195 nm3

SASDYL5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (2 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.0 nm Dmax 22.0 nm VolumePorod 222 nm3

SASDYM5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (3 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.3 nm Dmax 23.0 nm VolumePorod 235 nm3

SASDYN5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (4 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.2 nm Dmax 24.0 nm VolumePorod 245 nm3

SASDYP5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (5 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.4 nm Dmax 23.5 nm VolumePorod 254 nm3

SASDYQ5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A apo (7 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM M gCl2, 1mM TCEP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.5 nm Dmax 25.0 nm VolumePorod 267 nm3

SASDYR5 – Phosphorylated (T495) Heat shock cognate 71 kDa protein (Hsc70) T204A with ADP (5 mg/ml) Sample: Phosphorylated Heat shock cognate 71 kDa protein, 71 kDa Homo sapiens protein Buffer: 20 mM HEPES, 150 mM KCl, 10 mM MgCl2, 1mM TCEP, 1mM ADP, pH: 7.5 Experiment: SAXS data collected at EMBL P12, PETRA III on 2025 Mar 6 RgGuinier 5.3 nm Dmax 22.8 nm VolumePorod 236 nm3