Expression, Purification, and Mass Spectrometric Analysis of 15N, 13C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

Abstract

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed 15N, 13C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with 15N and 13C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone 15N, 13C and 1H resonance assignments of the r-RGD-Hirudin. The 15N-1H HSQC spectrum of uniformly 15N, 13C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the 15N-lH correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization

“…The 15 N-HSQC spectrum displays one peak for each bound 15 N-1 H pair of atoms (i.e., one pick per amino acid in the polypeptide backbone except proline and also other two peaks for side chain of glutamine, asparagine and one for tryptophan) [34]. The adequate dispersion and the correct number of 15 N-1 H cross-peaks observed in the 15 N rCmPI-II HSQC spectrum indicated adequate and homogeneous 15 N isotope incorporation, and that protein is pure and well folded.…”

“…On the other hand, several strategies have been used in the expression of isotopically-labeled proteins in P. pastoris in shaker flask, as well as fermenters [27,34]. For the homogeneous labeling with 15 N, the most frequently used nitrogen sources are ( 15 NH 4 ) 2 SO 4 [27,38] and 15 NH 4 Cl [39] due to the prohibitive price of 15 NH 4 OH (traditional source of nitrogen in expression of non-labeled protein in P. pastoris) [41].…”

“…For example, Wood and co-workers [39] obtained 90 mg of TMEGF45/L of culture using 46 g of labeled ammonium salts, whilst Huang and coworkers (2012) [34] reported the expression of 166 mg/L of culture of RDG-hirudin using 120 g of labeled ammonium salts. The analysis of the rCmPI-II expression yield in terms of mg of protein/g of labeled ammonium salts used (1.8 mg of protein/g of labeled ammonium salt), is similar to the yields reported in the literature above (1.3-2.4 mg of protein/g of labeled ammonium salt).…”

“…The selection of STREAMLINE Direct HST matrix as a first step of purification allowed applying the culture supernatant directly without previous downstream steps, as dialysis or gel filtration [27,34,40,42]. In addition, due to the very high binding capacity of this matrix [43], it was possible to process a large amount of sample at the beginning of the purification procedure.…”

Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity

“…The novel recombinant RGD-hirudin, which contains an Arg-Gly-Asp (RGD) adhesion site recognition sequence, is a bi-functional molecule based on the structure of wild-type hirudin variant 2 [ 14 ]. In the recombinant version, several amino acid residues in the C-terminus have been replaced by negatively charged residues (Asp 62 and Asp 65 ).…”