Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography

1999 ◽  
Vol 855 (1) ◽  
pp. 203-213 ◽  
Author(s):  
Carsten Müller ◽  
Ursula Rinas
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Size Exclusion Chromatography ◽  
Inclusion Bodies ◽  
Recombinant Escherichia Coli ◽  
Size Exclusion ◽  
Platelet Derived Growth Factor ◽  
Exclusion Chromatography

scholarly journals Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli

1993 ◽  
Vol 290 (3) ◽  
pp. 797-800 ◽  
Author(s):  
L Polgár ◽  
F Erdélyi ◽  
E Hajnal ◽  
M Löw ◽  
L Gráf ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Initiation Site ◽  
Full Length ◽  
Size Exclusion ◽  
Truncated Form ◽  
Buffer Solution ◽  
Diluted Solution ◽  
Exclusion Chromatography ◽  
Internal Initiation

Poliovirus protease 3C is a cysteine enzyme that is essential for the processing of the viral precursor polyprotein containing structural proteins and enzymes, including the protease itself. We have constructed the plasmid pSD/PV3C which produced protease 3C as inclusion bodies when expressed in Escherichia coli. In addition to the full-length protease, a truncated form was also generated, starting from an internal initiation site (Met-27). The enzyme was renatured by dilution of a 6 M guanidinium chloride solution of the inclusion bodies, and the proteins were precipitated from the diluted solution with ammonium sulphate. By extracting the precipitate with a buffer solution, the full-length enzyme could be completely separated from its N-terminally truncated form. Size-exclusion chromatography of the extracted protease 3C resulted in an active enzyme which appeared homogeneous by SDS/PAGE. For measuring the activity of the protease, a spectrofluorimetric method was devised to monitor the hydrolysis continuously, which is simpler and more precise than the h.p.l.c. technique used previously.

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