Abstract
Objectives
To develop a host-vector system for heterologous protein co-expression and purification in the Trichoderma reesei (teleomorph Hypocrea jecorina) industrial strain RUT-C30.
Results
The co-expression and purification system is based on: (i) an efficient and reliable selectable marker using bar (phosphinothricin acetyltransferase gene); (ii) a compact hygromycin B resistance marker; and (iii) a versatile integration plasmid for gene expression and knockout. Fluorescent protein genes were introduced into T. reesei and the corresponding proteins were purified from fermentation broth. The host-vector system was used in a proof-of-principle approach to achieve the co-expression of an alkaline endoglucanase and an alkaline cellobiohydrolase.
Conclusions
This protocol can be used at an industrial scale to produce large amounts of proteins in T. reesei.
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Acknowledgments
This research was supported by the National Natural Science Foundation of China (No. C010302-31500066), the National High Technology Research and Development Program of China (863, Program No. 2012AA101806 and 2013AA102109), and National Basic Research Program of China (973, Program No. 2012CB721103).
Supporting information
Supplementary Table 1—Transformants of T. reesei RUT-C30 constructed in this study.
Supplementary Table 2—Sequences of oligos.
Additional results:
Phosphinothricin purification
Transformation of T. reesei
Immobilized metal-ion affinity chromatography
RNA preparation and quantitative real-time reverse transcription PCR
Plasmid construction
Supplementary Fig. 1—Restriction enzyme map of pPK2, pPK1s and two based vectors pSB902 and pSB903.
Supplementary Fig. 2—The transformation efficiency and relative hpt mRNA expression level using five short promoters.
Supplementary Fig. 3—Alkaline endoglucanase activities in s-pSB902-V3 transformants and RUT-C30 with no additional plasmids.
Supplementary Fig. 4—Copy number of the deletion cassette in two Δcbh1 transformants (CBH1-rgf.1 and CBH1-rgf.7).
Additional result: DNA sequences of plasmids
Additional references.
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Zhang, L., Zhang, S., Jiang, X. et al. A novel host-vector system for heterologous protein co-expression and purification in the Trichoderma reesei industrial strain RUT-C30. Biotechnol Lett 38, 89–96 (2016). https://doi.org/10.1007/s10529-015-1948-4
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DOI: https://doi.org/10.1007/s10529-015-1948-4