Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin

摘要

class="kwd-title">Abbreviations: CNBr, cyanogen bromide; DTT, dithiothreitol; GSH/GSSG, reduced glutathione/oxidized glutathione; PS/DVB, polystyrene/divinylbenzene; RPC, reversed-phase chromatography; RP-HPLC, reversed-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid class="kwd-title">Method name: Improved purification of recombinant proinsulin class="kwd-title">Keywords: Proinsulin, Prohormone, Protein expression, Inclusion bodies, Protein purification, HPLC protein analysis class="head no_bottom_margin" id="idm139752070027728title">AbstractThe goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:. class="first-line-outdent">

• Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.

• Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.

• Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.