Genetic strategies have become important in the design ofefficient processes for production of recombinant proteins.When constructing a production scheme, inherent properties,purity and quality requirements, as well as the final use ofthe protein, have to be considered. In this thesis, geneticdesign has been applied to differentEscherichia coliproduction processes for human peptidehormones, facilitating the down-stream processing andincreasing the expression yields.
UponE. coliproduction of insulin-like growth factor I(IGF-I), misfolded and aggregated forms of IGF-I have reducedthe yield of correctly folded IGF-I. Extensive in vitrorefolding schemes have thus been considered necessary forobtaining acceptable yields. We investigated whethercoexpression of the IGF binding protein type 1 (IGFBP-1) wouldincrease the yield of correctly folded IGF-I. In this study,the two fusion proteins BB-IGFBP-1 and Z-IGF-I were used,containing the serum albumin binding affinity tag BB and theIgG-binding affinity tag Z, respectively. It was demonstratedthat correctly folded IGF-I could be recovered by twosubsequent affinity chromatography steps, verifying thatZ-IGF-I/BB-IGFBP-1 heterodimers were formed in vivo.Furthermore, the addition of a glutathione redox buffer duringcultivation significantly improved the relative yields ofcorrectly folded IGF-I, suggesting that affinity-assisted invivo folding could be considered as an attractive strategy forrecombinant proteins secreted to theE. coliperiplasm.
Aiming for an efficient process for production of both humaninsulin and proinsulin C-peptide, the possibility to integratethe removal of an affinity handle with the processing ofproinsulin to insulin and C-peptide, was investigated.Expression vectors encoding three different ZZ-proinsulinfusion proteins were constructed. Between the two IgG-binding Zdomains and proinsulin, trypsin-sensitive cleavage sites,consisting of either arginine, lysine-arginine or lysine wereengineered. A study of cleavage kinetics, in which the threefusion proteins were treated with trypsin and carboxypeptidaseB, demonstrated that the construct with a single arginineresidue was most efficiently processed. This fusion protein,which was found to be expressed to high levels in a fed-batchcultivation, accumulated intracellularly as inclusion bodies.After solubilization, refolding was performed by oxidativesulfitolysis. IgG affinity purification was used for singlestep recovery of pure proinsulin fusion protein. Afterenzymatic cleavage of the fusion protein, human insulin andC-peptide were recovered with good yields by preparativereversed-phase chromatography.
To investigate if increased production levels of theC-peptide could be obtained by gene fragment multimerization,DNA constructs encoding one, three or seven copies of theC-peptide gene were genetically fused to BB. Each C-peptidegene was flanked with codons for arginine, enabling enzymaticrelease of native C-peptide by trypsin/carboxypeptidase Btreatment. The three fusion proteins were produced to similarlevels as soluble and proteolytically stable intracellular geneproducts. Analysis of released C-peptide after enzymatictreatment of the fusion proteins, showed a six-fold increasedyield of C-peptide obtained from the heptameric fusion protein,as compared to the one-copy fusion protein. Based on theheptameric fusion protein BB-C7, an integrated process forproduction of proinsulin C-peptide was developed, whichincluded a heat treatmentprocedure for efficient release ofthe soluble fusion protein into the culture medium. The heattreatment also served as a purification step, precipitating themajority of the host cell proteins. In the production processpresented, chromatographic steps suitable for large-scalepurification, were used. The overall yield of native C-peptidewith a purity exceeding 99%, was 400 mg/l culture,corresponding to an overall recovery of 56%.
Taken together, the genetic strategies investigated havedemonstrated to be useful in schemes for facilitated productionof recombinant human peptide hormones inE. coli.
Key words:affinity-assisted in vivo folding, affinityfusion, carboxypeptidase B, C-peptide, enzymatic cleavage,Escherichia coli, gene multimerization, heat treatment,human peptide hormone, IGF-I, IGFBP-1, insulin, proinsulin,staphylococcal protein A, streptococcal protein G, trypsin.
Institutionen för biokemi och biokemisk teknologi , 1999. , 58 p.