Expression in Pichia pastoris and characterization by circular dichroism and NMR of rhodostomin

Abstract

Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is a disintegrin that inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alpha(IIb)beta3 of platelets. Rho produced in Escherichia coli inhibited platelet aggregation with a K(I) value of 263 nM. Although functional, Rho produced in E. coli is misfolded based on our 2D and 3D NMR studies. In order to correct the folding problem, Rho was expressed in Pichia pastoris. The recombinant Rho expressed in P. pastoris inhibited platelet aggregation with a resulting K(I) value of 70 nM. This is the same potency as that of native Rho. CD analysis showed that the secondary structures of Rho are pH-independent and contain 3.5-7.9% alpha-helix, 48.2-50.5% beta-structures, and 42.3-47% coil. The sequential assignment and structure analysis of Rho were obtained using 2D and 3D 15N-edited NMR spectra. These results provide the first direct evidence that highly disulfide-bonded disintegrin can be expressed in P. pastoris with the correct fold. This evidence may serve as the basis for exploring the structure and function relationships as well as the dynamics of disintegrin and its variants.

Structural Analysis of a Glycoside Hydrolase Family 11 Xylanase from Neocallimastix patriciarum

“…Similar phenomenon was observed in our previous study that analyzed the structure of rhodostomin, which contains six pairs of disulfide bonds. NMR examinations indicated that the E. coli-produced rhodostomin was misfolded despite being capable of inhibiting platelet aggregation (46). In contrast, P. pastoris produced correctly folded rhodostomin with all of six intramolecular disulfide pairings formed, which also exhibited higher activity than that from E. coli.…”

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

“…16,44 Den and Rho expression in P. pastoris was achieved using yeast transfer vector (pPICZa A; Invitrogen) and an expression kit (EasySelect version A; Invitrogen) with minor modifications to the manufacturer's instructions. The structural gene of Den was amplified using a polymerase chain reaction (PCR) with the sense primer:…”

“…15 It consists of 68 amino acids, which includes 12 residues of cysteine and a PRGDMP sequence at positions [48][49][50][51][52][53]. We previously showed 16 that Rho expressed in Pichia pastoris has the same function and structure as native protein. Rho binds to unstimulated and ADP-stimulated platelets in a saturable manner with the dissociation constants of 76 and 74 nM, respectively.…”

“…Expression of three-fingered toxins with the correct fold has been obtained; however, the yield is less than 1 mg/L, or refolding of the protein is required. [18][19][20][21][22] Many reports 2,16,23 have shown that highly disulfide-bonded proteins can be expressed in P. pastoris with high yields. In the present study, we expressed a three-fingered toxin, Den, in P. pastoris with high yield and compared its backbone dynamics with those of Rho.…”

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

“…The Pichia pastoris yeast-based expression system is appropriate for the expression of eukaryotic genes and has been used with great success over the last decade (Cregg et al, 2000;Guo et al, 2001;Fang et al, 2004). As a eukaryote, P. pastoris has many advantages: improved expression of higher eukaryotic proteins due to correct processing, folding, and post-translational modification of the recombinant protein akin to that of the natural protein; retention of antigenic determinants yielding robust immune responses; and the potential for the protein to be secreted directly into liquid medium, which facilitates purification.…”