Image Credit:

Engineering the pPICZαA Vector for Optimized Recombinant Protein Expression in Pichia pastoris: Substitution of Antibiotic Resistance and Auxotrophic Selection Markers

Author(s):
Salimeh HassaniSalimeh Hassani1, Elham BehvandiElham Behvandi1, Ghasem BagherpourGhasem Bagherpour2,*
1Student Research Committee, Zanjan University of Medical Sciences, Zanjan, Iran
2Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran

Koomesh:Vol. 28, issue 1; e165404
Published online:Jan 31, 2026
Article type:Research Article
How to Cite:Hassani S, Behvandi E, Bagherpour G. Engineering the pPICZαA Vector for Optimized Recombinant Protein Expression in Pichia pastoris: Substitution of Antibiotic Resistance and Auxotrophic Selection Markers. koomesh. 2026;28(1):e165404. doi: https://doi.org/10.69107/koomesh-165404

Abstract

References

  • 1.
    Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 1987;15(9):3859–76. [PubMed ID:3108861]. [PubMed Central ID:PMC340787]. https://doi.org/10.1093/nar/15.9.3859.
  • 2.
    Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000;24(1):45–66. [PubMed ID:10640598]. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x.
  • 3.
    Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol. 1989;9(3):1316–23. [PubMed ID:2657390]. [PubMed Central ID:PMC362724]. https://doi.org/10.1128/mcb.9.3.1316-1323.1989.
  • 4.
    Baron M, Reynes JP, Stassi D, Tiraby G. A selectable bifunctional beta-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells. Gene. 1992;114(2):239–43. [PubMed ID:1601306]. https://doi.org/10.1016/0378-1119(92)90581-9.
  • 5.
    Drocourt D, Calmels T, Reynes JP, Baron M, Tiraby G. Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance. Nucleic Acids Res. 1990;18(13):4009. [PubMed ID:1695734]. [PubMed Central ID:PMC331125]. https://doi.org/10.1093/nar/18.13.4009.
  • 6.
    Behvandi E, Bagherpour G, Nedaei K, Kaboli S, Johari B. [Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris]. Koomesh. 2024;26(2):e149316. Persian. https://doi.org/10.69107/koomesh-149316.
  • 7.
    Mohammadgholizad F, Hashemi A. Construction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM. Koomesh. 2019;21(4):743–50. Persian.
  • 8.
    Chen J, Stubbe J. Bleomycins: towards better therapeutics. Nat Rev Cancer. 2005;5(2):102–12. [PubMed ID:15685195]. https://doi.org/10.1038/nrc1547.
  • 9.
    Lanza AM, Kim DS, Alper HS. Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells. Biotechnol J. 2013;8(7):811–21. [PubMed ID:23450727]. https://doi.org/10.1002/biot.201200364.
  • 10.
    Oliva-Trastoy M, Defais M, Larminat F. Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells. Mutagenesis. 2005;20(2):111–4. [PubMed ID:15755800]. https://doi.org/10.1093/mutage/gei016.
  • 11.
    Hagen L, Sharma A, Aas PA, Slupphaug G. Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection. Biochimica et Biophysica Acta (BBA) - Proteins Proteomics. 2015;1854(1):84–90. https://doi.org/10.1016/j.bbapap.2014.10.016.
  • 12.
    Li Cc, Hu R, Hua Xm, Ni Yx, Ge L, Zhang L, et al. Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10. Microbiol Spectr. 2023;11(6):e0120623. [PubMed ID:37905802]. [PubMed Central ID:PMC10714783]. https://doi.org/10.1128/spectrum.01206-23.
  • 13.
    Abdemami B, Shokrgozar MA, Shahreza HK, Ghavami M. Design and construction of two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Avicenna J Med Biotechnol. 2011;3(1):11.
  • 14.
    Fani R, Tamburini E, Mori E, Lazcano A, Lio P, Barberio C, et al. Paralogous histidine biosynthetic genes: evolutionary analysis of the Saccharomyces cerevisiae HIS6 and HIS7 genes. Gene. 1997;197(1-2):9–17. [PubMed ID:9332345]. https://doi.org/10.1016/s0378-1119(97)00146-7.
  • 15.
    Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988;74(2):527–34. [PubMed ID:3073106]. https://doi.org/10.1016/0378-1119(88)90185-0.
  • 16.
    Imadeartika. The Use of HIS6 Gene as a Selectable Marker for Yeast Vector. HAYATI J Biosciences. 2009;16(1):40–2. https://doi.org/10.4308/hjb.16.1.40.
  • 17.
    Bar-Nun S, Shneyour Y, Beckmann JS. G-418, an elongation inhibitor of 80 S ribosomes. Biochimica et Biophysica Acta (BBA) - Gene Structure Expression. 1983;741(1):123–7. https://doi.org/10.1016/0167-4781(83)90018-0.
  • 18.
    Lin-Cereghino J, Hashimoto MD, Moy A, Castelo J, Orazem CC, Kuo P, et al. Direct selection of Pichia pastoris expression strains using new G418 resistance vectors. Yeast. 2008;25(4):293–9. [PubMed ID:18327886]. [PubMed Central ID:PMC2504081]. https://doi.org/10.1002/yea.1587.
  • 19.
    Mignon C, Sodoyer R, Werle B. Antibiotic-free selection in biotherapeutics: now and forever. Pathogens. 2015;4(2):157–81. [PubMed ID:25854922]. [PubMed Central ID:PMC4493468]. https://doi.org/10.3390/pathogens4020157.
  • 20.
    Raiford DW, Heizer EM, Jr., Miller RV, Doom TE, Raymer ML, Krane DE. Metabolic and translational efficiency in microbial organisms. J Mol Evol. 2012;74(3-4):206–16. [PubMed ID:22538926]. https://doi.org/10.1007/s00239-012-9500-9.

Crossmark
Crossmark
Checking
Share on
Cited by
Metrics

Purchasing Reprints

  • Copyright Clearance Center (CCC) handles bulk orders for article reprints for Brieflands. To place an order for reprints, please click here (   https://www.copyright.com/landing/reprintsinquiryform/ ). Clicking this link will bring you to a CCC request form where you can provide the details of your order. Once complete, please click the ‘Submit Request’ button and CCC’s Reprints Services team will generate a quote for your review.
Search Relations

Author(s):

Related Articles