Expressed protein ligation: a general method for protein engineering

Abstract

A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

Figure 1

Phosphotyrosine tails in Src and Csk. (A) Phosphorylation of the Src tail on tyrosine-527 is catalyzed by Csk. This phosphorylation results in a conformational change involving an intramolecular interaction between the Src tail and the SH2 domain. (B) Csk is highly homologous to Src but lacks a C-terminal tyrosine-containing tail. Proposed ligation of a phosphotyrosine tail might lead to a conformational change like that found in Src. Csk is not a Src family member and in addition to its lack of a C-terminal tail, Csk also lacks two key Src features: an activating tyrosine-containing loop, as well as an N-terminal myristoylation site (16, 17).

Figure 2

The principle of expressed protein ligation. In the first step, the gene or gene fragment is cloned into the commercially available PCYB2-IMPACT vector (New England Biolabs) by using the NdeI and SmaI restriction sites. This cloning strategy results in the addition of a Pro-Gly appended to the native C terminus of the protein of interest. The presence of a C-terminal glycine has been shown to accelerate native chemical ligation (24) and thus reduces the chance of side reactions. After expression and affinity purification of the fusion protein by binding to the chitin resin, the chemical ligation step is initiated by incubating the resin-bound protein with thiophenol and synthetic peptide in buffer. This results in the in situ generation of a highly reactive phenyl ^αthioester derivative of the protein that then rapidly ligates with the synthetic peptide to afford the desired semisynthetic protein.

Figure 3

Characterization of semisynthetic proteins. (A) A Coomassie-stained 10% SDS/PAGE gel of Csk^PEP crude reaction product mixture. Lane 1: molecular mass markers from the top: 97, 66, 45, 31, and 21.5 kDa. Lane 2: Wild-type Csk. Lane 3: Csk^PEP crude ligation product mixture; a combination of N-terminal sequencing and electrospray MS indicated that the bands at 56 kDa and 69 kDa were GroEL and DnaK, respectively. Lane 4: Comixture of wild-type Csk and Csk^PEP crude ligation product mixture. (B) A Coomassie-stained 10% SDS/PAGE gel of the purified semisynthetic Csk proteins. Lane 1: Molecular mass markers as listed in A. Lane 2: Wild-type Csk. Lane 3: Csk^pPEP purified by reverse-phase HPLC. Lane 4: Csk^PEP purified by phosphotyrosine affinity chromatography. (C) Characterization of semisynthetic proteins by electrospray MS. (Top) Full-length wild-type Csk, expected mass = 50,705 Da (average isotope). (Middle) Csk^PEP, expected mass = 52,540 Da (average isotope). (Bottom) Csk^pPEP, expected mass = 52,619 Da (average isotope). Each sample was isolated by reverse-phase HPLC and mass analyzed by using a Perkin–Elmer-Sciex (Thornhill, ON, Canada) API-100 mass spectrometer. Predicted masses were calculated by using the program macbiomass (S. Vemuri and T. Lee, City of Hope, Duarte, CA). Note, the ligated Csk products were engineered to have the sequence Pro-Gly added to their C termini, and Edman sequencing indicated that the N-terminal methionine had been removed from the Csk expressed in the pCYB2 vector. That the ligation products contained only one N terminus (i.e., from Csk) combined with the MS data provides unambiguous characterization of the semisynthetic proteins.

Figure 4

Fluorescence imaging of an SDS/PAGE showing the results of proteolytic digestions of Csk^PEP and Csk^pPEP with subtilisin. Lane 1: Csk^pPEP − subtilisin; lane 2: Csk^pPEP + subtilisin; lane 3: Csk^PEP − subtilisin; lane 4: Csk^PEP + subtilisin. Reactions conditions: Csk^PEP and Csk^pPEP (1 μg) in 20 μl buffer (20 mM Tris⋅acetate, pH 8.0/10% glycerol/2 mM DTT) treated with subtilisin Carlsberg (12.5 ng) for 30 min at 4°C. Fluorescence imaging was done on a Storm instrument (Molecular Dynamics). Proteolysis reactions were performed in parallel under identical conditions with different protein preparations on four different occasions and all gave comparable results to those shown here.

Figure 5

Phosphorylation of Lck catalyzed by semisynthetic Csk proteins. (A) Time course of Csk^PEP and Csk^pPEP-catalyzed phosphorylation of Lck. •, Csk^pPEP; ○, Csk^PEP. Relative product formation is corrected for the amount of Csk^PEP and Csk^pPEP utilized. [Lck] = 1 μM. (B) Kinase activity vs. Csk^PEP and Csk^pPEP concentration with the substrate Lck. •, Csk^pPEP; ○, Csk^PEP. [Lck] = 1 μM. Reaction time = 2 min. (C) Rate of phosphorylation of Lck catalyzed by Csk^PEP and Csk^pPEP vs. Lck concentration. •, Csk^pPEP; ○, Csk^PEP. The data for Csk^pPEP could not be strictly fit to standard Michaelis-Menten kinetics because of apparent substrate inhibition. For Csk^pPEP the K_m (apparent) (Lck) estimated as the substrate concentration necessary for 50% maximal velocity is 0.4 ± 0.1 μM. The data for Csk^PEP was fit to the Michaelis-Menten equation and gave K_m (apparent) (Lck) = 2 ± 0.5 μM. The Lck K_m with wild-type Csk enzyme under these conditions is 3–5 μM (25). All assays were performed as described (25) on a 15 μl scale at 30°C by using catalytically impaired Lck (N-terminal 63 aa deleted, containing a K273R mutation) at fixed and near-saturating ATP concentration [10 μM, ≈3 × K_m (apparent)] and optimal MnCl₂ concentration (5 mM) under initial conditions (<10% turnover of the limiting substrate). All assays were carried out at least two times and duplicate points generally agreed within 10%. Kinase activity was shown to be identical for the phosphotyrosine-affinity purified and unpurified semisynthetic proteins by using Lck as substrate. Kinase specificity for the 505-tyrosine of Lck was confirmed by demonstrating that 505-phosphorylated Lck was not detectably phosphorylated by either semisynthetic protein using conditions described (25).