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. Author manuscript; available in PMC: 2019 Jan 7.
Published in final edited form as: Biochemistry. 2017 Nov 28;56(49):6423–6433. doi: 10.1021/acs.biochem.7b00700

Figure 3.

Figure 3.

Analysis of two-step downstream purification of rhTIMP-2– 6XHis following bioprocess scale expression. Samples from the bioprocess purification were analyzed as shown in the (A) PageBlue Protein-stained SDS−PAGE gel of the fractions obtained from the IMAC (HisTrap column) purification step. Lane M contained the molecular weight standards (SeeBlue Plus 2 Prestained Standards, Invitrogen, catalog no. LC 5925). Lane CM contained the starting condition medium sample obtained from the HEK-293-F suspension culture. Lane FT contained the flow-through (unbound) fraction obtained during sample loading. Lane NSE contained the nonspecific elution obtained during step gradient elution with 20 mM imidazole. Lane SE contained the specific elution fraction obtained following 250 mM imidazole step elution. (B) PageBlue stained SDS−PAGE gel showing that the rhTIMP-2-6XHis-containing fractions from the IMAC (HisTrap) purification contain a predominant 22 kDa band with minor higher-molecular weight contaminants. The reverse phase HPLC purification effectively removed these higher-molecular weight contaminants, resulting in a single 22 kDa band with >95% purity as estimated by densitometry using a Bio-Rad ChemiDoc XRS+ instrument with Image Lab software. (C) Western Blot analysis of the IMAC fractions using anti-TIMP-2 (top) anti-6XHis tag (bottom) antibodies. The lanes are labeled the same as in panel A.