Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing. Part 2. Protein recovery.

The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.

Interactions of phage P22 tailspike protein with GroE molecular chaperones during refolding in vitro.

Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated. At elevated temperature (> 30 degrees C), in the absence of ATP, GroEL effectively trapped refolding tailspike protein and prevented reconstitution. Tailspike protein was released from GroEL by addition of ATP around 35 degrees C or without added ATP upon cooling to 25 degrees C, and native tailspike trimers were formed. In accordance with the cold release, tailspike reconstitution at < or = 25 degrees C was unaffected by GroE. No formation of native tailspike trimers was observed, when refolding was initiated at 42 degrees C in the presence of the GroE proteins and ATP or when tailspike protein was dissociated from a preformed complex with the chaperone by addition of ATP at 42 degrees C. In contrast to other GroE ligands, the tailspike polypeptide was bound by and released from GroE in similar states of folding, and the presence of GroES in addition to GroEL had no effect on reconstitution yields at any temperature. Thus, the GroE proteins may exhibit widely differing interactions even with proteins showing similarly temperature-sensitive yields of folding.