secreted eukaryotic HIS-recombinant protein

Wolfgang Schechinger novalidaddress at nurfuerspam.de
Tue Apr 4 04:33:48 EST 2006


Here is the relevant part of the methods section

Essentially no polyhistidine-tagged hßG was recovered
after passage of culture medium through an Ni-NTA column
under different conditions. Batch processing allowed the recovery
of up to 25% of the recombinant hßG, but only
when large amounts of the Ni-NTA resin were employed
(20-50 %, v/v). Extensive dialysis of the culture medium
against the Ni-NTA-binding buffer did not improve product
recovery (results not shown). In contrast, ammonium
sulphate precipitation before Ni-NTA affinity chromatography
increased the recovery of recombinant proteins. As
can be seen from Figure 1(A), near-quantitative recovery of
hßG and sCD13 was achieved by ammonium sulphate precipitation
of the culture medium. After dissolving the
precipitate in Ni-NTA-binding buffer, sCD13 bound well to
Ni-NTA, resulting in a sharp peak after elution with excess
imidazole (Figure 1B). The final yield was 3-5 mg of purified
sCD13 per litre of culture medium with an overall recovery
of approx. 40%. As shown in Figure 2(A), sCD13 could
not be seen after SDS/PAGE of the original culture medium,
whereas the final product consisted of a major band with the
expected molecular mass of approx. 150 kDa (Figure 2A).

For citations, see here:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14725509&query_hl=1&itool=pubmed_docsum

Wolfgang



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