Figure 2
From: High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers
Analysis of the functional activity and protein expression level of recombinant LHyal.
(a) Determination of HAase activity using a typical plate assay. Cylindrical holes were injected with the fermentation of LHyal-expressing (left) or control P. pastoris GS115 (right). The clear circle corresponds to the distance diffused by LHyal, while the control (undigested HA) precipitated in the gel upon the addition of cetylpyridinium chloride. (b) Effects of various length N-terminal His-tags on LHyal expression. The gene copy numbers of all recombinant strains were screened with G418 (3 mg ml−1) on YPD plates. Typical time course of recombinant LHyal (C) and H6LHyal (d) expression by P. pastoris in a 3-L fermenter during the methanol induction phase. LHyal production (red 
, U ml−1) and cell growth density (green 
, OD600 value) were measured at regular intervals. SDS-PAGE analysis of LHyal (e) and H6LHyal (f) expression in the 3-L fermenter at regular intervals during the methanol induction phase. The protein production of LHyal and H6LHyal is consistent with the enzymatic activity shown in Fig 3c and 3d, respectively. Black arrows indicate the target protein.
