Efficient solubilization of inclusion bodies

Abstract

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.

Techno‐economic evaluation of an inclusion body solubilization and recombinant protein refolding process

“…The efficiency of this method, judged on the basis of the total amount of protein solubilized and the fraction of soluble aggregates formed, has been shown to be dependent on the combined effect of the pH, the concentration of urea and the concentration of DTT. 26 As distinct from the pH_indSR system, this solubilization method can be applied to solubilize practically any IBs, making it generally applicable. This solubilization method works by targeting the forces (noncovalent and covalent) holding the protein together in the form of an IB, urea (chaotrope) disrupts the interactions based on noncovalent forces (e.g., hydrophobic and hydrogen bonding), whereas DTT (RA) disrupts the interactions based on covalent forces (i.e., intrachain and interchain disulfide bonds).…”

“…The pH_IndSR system offers advantages including (a) IBs solubilization is achieved without a chaotrope or using a relatively low chaotrope concentration ( 2 M); (b) no RA is used; and (c) IBs solubilization and protein refolding are attained in one step. The limitations of the pH_IndSR include (a) inefficient solubilization of the IBs, represented by the inability to prevent or minimize the formation of soluble aggregates 26 ; (b) proteins exposed to relatively high alkaline pH, for relatively long periods of time, may undergo irreversible chemical modifications 7 ; and (c) the success of the pH_IndSR system is highly protein dependent, as not all IBs can be solubilized by pH alone. Figure 1A presents a block flow diagram (BFD) depicting the pH_IndSR system, showing the blocks solubilization, and refolding and DSP.…”

“…24 The fact that this system, from now on referred to as pHinduced solubilization and refolding (pH_IndSR), does require minimum or no chaotrope agent and no RA, is most likely the most attractive feature of pH_IndSR, because it makes it a relatively cheap process. The main limitations of the pH_IndSR systems include (1) inefficient solubilization of the IBs, because pH alone may not be able to minimize the formation of soluble aggregates 26 and (2) a highly protein-dependent system, because not all IBs can be efficiently solubilized by pH alone and most proteins, once exposed for long periods to a pH [ 12, will most likely undergo irreversible chemical modifications. 7 In summary, up to this point four distinct process configurations have been introduced and each of them is well suitable to perform IBs solubilization and protein refolding.…”

Pemurnian Pretrombin-2 Manusia Rekombinan di Escherichia coli untuk Produksi Trombin sebagai Komponen Lem Fibrin

“…Perbedaan persentase kandungan GC antara gen target dengan inang ekspresi menyebabkan adanya bias kodon, dan selanjutnya bias kodon yang tinggi akan menurunkan tingkat ekspresi karena diperkirakan akan dapat menyebabkan kesalahan translasi (Merkl, 2003;Gustafsson et al, 2014), sehingga menghasilkan protein agregat kompleks tidak aktif yang lazim dikenal sebagai badan inklusi (Freydell et al, 2007). Nukleotida PT2 sintetik, selain banyak memiliki kodon jarang juga kandungan GC nya tinggi, sehingga diperlukan optimasi kodon gen sesuai preferensi kodon E. coli melalui memanfaatkan teknologi gen sintetik.…”

“…Beberapa peneliti melaporkan bahwa nilai CAI dapat digunakan untuk memprediksi tingkat ekspresi protein heterolog (Carbone et al, 2003;Xia, 2007;Fox & Erill, 2010). Optimasi kodon gen PT2 dapat meningkatkan nilai CAI PT2 sintetik menjadi 1,00 dan secara teori nilai CAI tersebut dianggap bagus untuk ekspresi (Carbone et al, 2003;Xia, 2007 Pembentukan badan inklusi selama produksi protein dalam inang prokariot juga dapat dicegah dengan mengurangi suhu selama ekspresi protein, karena suhu rendah biasanya meningkatkan kelarutan protein (Freydell et al, 2007). Namun, penurunan suhu dapat memperlambat pertumbuhan sel, sehingga tingkat sintesis protein lebih rendah.…”