High-yield expression of isotopically labeled peptides for use in NMR studies

Abstract

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.

Figure 1.

(A) GB-1 fusion protein construction, with location of methionine residues denoted by M. (B) Peptide name and amino acid sequence of expressed peptides.

Figure 2.

An 8%–22% gradient SDS-PAGE gel of expressed fusion proteins. MW markers (lane 1), uninduced cells (lane 2), induced cells (lane 3), French press lysate supernatant (lane 4), and purified GB-1_cIp-RR-20_His (lane 5). Lanes 6–15 contain induced cells and the French press lysate supernatant of each indicated fusion protein construct, with regions of fusion protein constructs denoted by an asterisk.

Figure 3.

CNBr cleavage of GB-1_cIp-RR-20_His fusion protein as monitored by ¹H-NMR. Reduction of peak intensity of methionine ɛ-CH₃ side chains as a function of time indicates efficient cleavage of the peptide amide bond.

Figure 4.

(A) {¹H, ¹⁵N-HSQC} and (B) {¹H, ¹³C-HSQC} NMR spectra of ¹⁵N- and ¹⁵N/¹³C-cIp-RR-20. Backbone amide assignments for {¹H, ¹⁵N-HSQC} are as indicated.