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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Optimization of Culture Conditions for Expression of Anti-lipopolysaccharide Single Domain Antibody Clone in Pichia pastoris

A
Anu Malik1
A
Akhil Kumar Gupta1,*
J
Jaideep Kumar1
A
Anshul Lather1
P
Parveen Kumar1
M
Mahavir Singh2
S
Swati Dahiya1
N
Naresh Kumar Kakker1
1Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
2College Central Lab., Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

ABSTRACT

Background: Previously, a single domain antibody clone (dAbCl26) selected from phage display library of LPS-immunized Indian desert camel was expressed in prokaryotic expression system and found to inhibit the LPS-induced inflammatory cytokine both in vitro and in vivo. However, the level of expression in prokaryotic expression system was low and also there was problem of endotoxin contamination. Pichia pastoris has emerged as an alternative host and economical approach for the expression of recombinant protein as it has the ability to properly refold the protein, gives high yield and produce protein without endotoxin contamination. Encouraged by the earlier findings of in vitro and in vivo inhibition by dAbCl26, the expression of dAbCl26 was investigated in yeast expression system P. pastoris.

Methods: The dAbCl26 was cloned into the Pichia expression vector pPICZαA and transformed into P. pastoris strain X33. Mutant phenotypes of selected clones were assessed using agar plate methods. For expression of the dAbCl26 in P. pastoris strain X33, the final growth conditions were optimized in buffered minimal methanol media with induction at OD600 4.0 and methanol induction at 0.5%.

Result: Using the optimized conditions, a yield of about 4 mg/L of dAbCl26 was obtained in shake flask culture which is 2 fold higher than previously reported. The expressed protein was purified by using Ni-NTA chromatography and confirmed by Western blotting. The method developed in this study will be used as base for expression of other single domain antibodies and to scale up the expression on large scale. Based on its already reported functional properties, the Pichia expressed dAbCl26 can be a suitable candidate for further development as a therapeutic agent for endotoxaemia in animals.

INTRODUCTION

Different in vitro selection technologies such as phage display antibody provides a solution for the generation of recombinant antibodies (Chiu and Gilliland, 2016). Phage display technology has been found to be one of the most effective, robust, easy-to-perform methods to develop recombinant antibodies with high affinity and target specificity and has been utilized to generate single-domain antibodies or variable domain of heavy chain of heavy chain antibodies (VHHs), also widely known as nanobodies (Roncolato et al., 2015; Homayouni et al., 2016; Kazemi-Lomedasht et al., 2016).
       
The Department of Veterinary Microbiology, LUVAS, Hisar, Haryana (India) had previously constructed a phage display library of single-domain antibodies of LPS-immunized Indian desert camel for diagnostic and therapeutic use (Singh, 2009; Banerjee et al., 2020). From this library, one of the clone i.e. dAbCl26 was found to inhibit the LPS-induced inflammatory cytokine response in buffalo and murine macrophages in vitro (Gupta, 2014). Also, the dAbCl26 inhibited the LPS-induced TNF-α release in Swiss albino mice model. The dAbCl26 also neutralized the E. coli 0153 LPS in chicken embryo lethality model and was found to cross react with LPS isolated from different Gram negative organisms i.e. P. multocida B:2, E. coli 0153, Salmonella typhimurium, Pseudomonas aeruginosa and Klebsiella spp. (Gupta, 2014; Banerjee, 2015). These findings suggested that dAbCl26 could be a novel candidate to prevent endotoxaemia and sepsis. However, utilizing prokaryotic expression systems for producing antibodies or other therapeutic proteins presents significant challenges. One major issue is the relatively low level of protein expression in these systems. Additionally, the production process is often complicated by endotoxin contamination, as prokaryotic cells, particularly Gram negative bacteria, inherently contain LPS in their outer membranes. This contamination can pose serious risks, especially when producing treatments for conditions like sepsis, where endotoxins are a primary concern (Fux et al., 2024).
       
To address these limitations, the Pichia pastoris expression system offers an efficient and economical approach for producing wide variety of heterologous proteins in a correctly refolded form with disulfide bridges (White et al., 1994; Kurtzman, 2009; Anangi et al., 2012; Swichkow et al., 2022). Therefore, in this study, dAbCl26 was cloned and expressed in P. pastoris. Initially, a zeocin-resistant clone was selected and further optimized for culture conditions, including medium type, induction timing and methanol concentration, to maximize protein yield. After purification, the dAbCl26 was characterized by SDS-PAGE and Western blotting. By leveraging the unique properties of dAbCl26 and the advantages of the P. pastoris expression system, our aim was to create an efficient and scalable production method for this promising therapeutic agent.

MATERIALS AND METHODS

Ethics statement
 
Permission to work on Pichia pastoris was obtained from Institutional Biosafety committee (IBSC) vide reference no. LUVAS/ABT/2020/29 Dated 07.01.2020. The experimental work was carried out at Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana over a period extending from October, 2019 to November, 2020.
 
Strains and media used
 
The EasySelect™ Pichia Expression Kit (Thermo Scientific, K1740-01) was used for the expression of dAbCl26 in P. pastoris. P. pastoris X-33 strain, TOP10FA E. coli, P. pastoris vector (pPICZαA), zeocin, vector specific sequencing primers and media supplied with the kit were used in the present study.
 
Cloning of dAbCl26 in P. pastoris pPICZαA vector
 
The dAbCl26 gene (Acc no. KF990217) was cloned in Pichia vector (pPICZαA) in frame with c-myc and His tag. The dAbCl26 gene was amplified using gene specific primer (Table 1) by gradient PCR with annealing temperature ranging from 59-63oC. The PCR product was purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega, A9281).

Table 1: Primers used for cloning of dAbCl26 into Pichia vector pPICZaA (Resrtriction sites shown in bold).


       
The vector pPICZαA and purified PCR product of dAbCl26 were double digested with EcoRI (Thermo Scientific, FD0274) and XbaI (Thermo Scientific, FD0684) restriction enzymes and the purified RE digested dAbCl26 was cloned into the vector pPICZαA keeping the insert:vector molar ratio to 3:1. The ligated gene product was transformed into chemi-competent Top10FA E. coli cells using E. coli transformation buffer set kit (Zymo Research, T3002). The Top10F′ E. coli cells were then plated with 150 μl volume/plate on 90 mm low salt LB/zeocin (25 μg/ml) agar plates. Finally, plates were incubated overnight at 37oC. Next day, five transformed clones were randomly screened with gene specific primers and vector specific primers.
 
Confirmation of recombinant plasmid
 
For the confirmation of recombinant plasmid, recombinant vector plasmids (dAbCl26 + pPICZαA) and empty vector plasmids (pPICZαA) were double digested with EcoRI and XbaI enzymes. Meanwhile, the recombinant vector plasmid (dAbCl26 + pPICZαA) was linearized by single digestion with restriction enzyme SacI (Thermo Scientific, FD1134). This linearized plasmid was later used for transformation in P. pastoris strain X33. These digested products were resolved on 1.5% agarose gel for confirmation.
 
Transformation into P. pastoris strain X33
 
The recombinant linear plasmid (dAbCl26+pPICZαA) was chemically transformed into P. pastoris X-33 cells using Pichia EasyComp Transformation Kit as per manufacturer’s protocol (Invitrogen, K173001). After transformation, the entire mixture was plated on the Yeast extract peptone dextrose (YPD)/zeocin (100 µg/ml) agar plates and incubated for 3 days at 28oC. From the plate, transformed clones were regrown on YPD plates supplemented with progressively increasing concentrations of Zeocin (100, 200 and 400 µg/ml). This step was undertaken to select the clone exhibiting hyper-resistance to Zeocin. The Pichia transformants surviving on 400 µg/ml Zeocin were screened by PCR using gene specific primers and vector specific primers. The PCR products were resolved in a midi gel horizontal electrophoresis apparatus using 1.25% agarose gel with 5 µg ml-1 ethidium bromide in 1X TAE buffer at 5V/cm gel length and visualized in UV-transilluminator (Spectroline™).
 
Determining the Mut+/MutS phenotype of selected clones
 
The PCR positive clones were screened for determining their Mut phenotype. To determine the Mut phenotype of the transformed clones, zeocin resistant Pichia transformants were selected. One half portion of the selected colony was picked and streaked on minimal dextrose (MD) agar plate and the remaining half was streaked on minimal methanol (MM) agar plate. The plates were incubated at 28oC for 2 days and the growth on MD and MM agar plates was compared. After confirming the Mut phenotype of the selected clones, Mut+ or MutS protocol was followed for inducing expression in P. pastoris strain X-33.
 
Optimization of culture conditions for expression of recombinant dAbCl26 in Pichia pastoris
 
Effect of different media on recombinant protein expression
  
A single recombinant Pichia colony was inoculated in 100 ml of Buffered glycerol-complex (BMGY) medium in 500 ml baffled flask and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 4.0. The cells were harvested by centrifugation at 6000 rpm for 7 min and re-suspended in 25 ml of different media: Minimal methanol (MM), Buffered minimal methanol (BMM), Buffered methanol-complex medium (BMMY) and BMMY supplemented with 2% Casamino acid. The culture was induced with methanol to final concentration of 0.5% (added after every 24 h). The culture supernatant was collected every 24h interval till 120 h. The cells were precipitated with 10% Trichloroacetic acid (TCA) and checked on SDS-PAGE for protein expression.
 
Effect of time of induction on protein expression
 
A single recombinant Pichia colony was inoculated in 100 ml of BMGY medium in 500 ml baffled flask in triplicate and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 2.0, 4.0 and 6.0 for each flask. The cells of each flask were harvested by centrifugation at 6000 rpm for 7 min and the pellet was re-suspended in BMM media and induced with 0.5% methanol (added every 24 h till 120 h). The culture supernatant was collected every 24 h interval till 120 h, precipitated with 10% TCA and checked on SDS-PAGE for protein expression.
 
Effect of methanol on induction of protein expression
 
A single recombinant Pichia colony was inoculated in 100 ml of BMGY medium in 500 ml baffled flask in triplicate and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 4.0 for each flask. The cells of each flask were harvested by centrifugation at 6000 rpm for 7 min and the pellet was re-suspended in BMM media and the flask was induced with 0.5%, 1% and 2% methanol, respectively. The methanol was added in the respective flasks after every 24 h till 120 h. The culture supernatant was collected every 24 h interval till 120 h, precipitated with 10% TCA and checked on SDS-PAGE for protein expression.
 
Purification of recombinant protein
 
The recombinant protein was purified using Ni-NTA purification system. The supernatant collected 96 h after induction was cleared by centrifugation at 7000 rpm for 10 min and was dialyzed against binding buffer (50mM NaH2PO4.H2O, 300mM NaCl, pH 8.0) for 3 days with buffer changed after every 12 h. One ml of Ni-NTA agarose beads were settled down in column and washed with 5 volume of binding buffer. These equilibrated Ni-NTA beads were incubated with dialyzed culture supernatant overnight at 4oC. Next morning, the culture supernatant packed in column was washed with 5 volume of washing buffer (binding buffer with 0.1% triton X). The 6xHis tagged protein was eluted with elution buffer (binding buffer with 300 mM imidazole). The eluates were collected in small fractions of 1 ml each and checked by SDS-PAGE.
 
Western blot analysis of Pichia expressed dAbCl26
 
For Western blotting, Pichia expressed dAbCl26 protein was resolved on 15% SDS-PAGE to obtain polypeptide profiles and the same were electro-blotted onto PVDF membrane (0.22 µm) in a semi-dry trans-blot apparatus (Atto® corporation, Japan) at 1.0 mA/cm2 of the gel for 60 min at room temperature. The PVDF membrane was blocked in 3% BSA-PBST (0.05% Tween-20) blocking buffer for 1 hr in shaking incubator at room temperature. After washing with PBST (0.05% Tween-20), the membrane was probed with anti-His HRP conjugated antibody (1:5000 dilution) and developed in 3,3-diaminobenzidine tetrahydrochloride hydrate (Sigma, D5637) and hydrogen peroxide (DAB/H2O2) substrate solution.

RESULTS AND DISCUSSION

Cloning of dAbCl26 in Pichia expression vector pPICZα​A
 
The recombinant plasmid (dAbCl26+pPICZαA) was transformed in TOP10FA E. coli competent cells for the purpose of storage and propagation of recombinant plasmid to get high copy number for the successful transformation in P. pastoris strain X-33. It had been well documented that for the optimization of protein expression, high copy number appeared to have dominant positive influence and to achieve the same P. pastoris plasmids were propagated in E. coli prior to transformation (Vogl et al., 2018). Gradient PCR was conducted for the optimization of annealing temperature for the designed primers with the temperature range 59-63oC. It was observed that better amplification was obtained at the annealing temperature of 60oC. The vector pPICZαA and purified dAbCl26 PCR product were double digested, ligated and transformed in the competent TOP10FA E. coli cells and plated on the low salt LB/zeocin agar plates. After incubation at 37oC for 18 h, five randomly selected clones were found positive by PCR using both gene specific primers and vector specific primers, respectively. The gene specific primers showed a band of 400 bp while vector specific primers a band of 929 bp was observed.
 
Confirmation of the recombinant plasmid (pPICZα​A + dAbCl26)
 
The recombinant plasmid and empty vector were double digested with EcoRI and XbaI enzymes. Double digested recombinant plasmid showed two bands, one band of 3.5 kbp and other of 400 bp, as expected of the insert. The empty vector pPICZαA also showed a single band of 3.5 kbp. The single RE digested recombinant plasmid showed a single band of 3.9 kbp indicating that gene of interest was cloned in pPICZαA vector (Fig 1).

Fig 1: Agarose gel electrophoresis of RE digested products.


 
Transformation and screening of recombinant clones transformed in P. pastoris X-33 strain
 
For transformation, the recombinant plasmid linearized with enzyme SacI was transformed into P. pastoris X-33 by chemical method. After 3 days incubation at 28oC, more than 50 colonies were observed on YPD agar plates containing 100 µg/ml of Zeocin. These colonies were regrown on YPD plates supplemented with progressively increasing concentrations of Zeocin (100, 200 and 400 µg/ml). This step was aimed at selecting clones that exhibit hyper-resistance to Zeocin, ensuring the selection of the most robust and genetically stable clones for subsequent protein production. This approach aligns with findings by Nordén et al. (2011), who reported an intimate correlation between hyper-resistance against Zeocin and enhanced expression of foreign proteins in P. pastoris. Therefore, adopting this strategy was crucial for selecting hyper-resistant P. pastoris strains, which are likely to exhibit improved expression of the desired recombinant proteins. From YPD/zeocin (400 µg/ml) agar plates, twenty-four clones were randomly selected and screened by PCR using both gene specific and vector specific primers, respectively. On agarose gel electrophoresis, out of twenty-four clones, 23 were found positive (400 bp) with gene specific primers (Fig 2) and 22 clones were found positive (~929 bp) with vector specific primers (Fig 2). This confirmed the integration of dAbCl26- pPICZαA plasmid in P. pastoris genome.

​

Fig 2: Agarose gel electrophoresis of PCR amplification of transformed Pichia clones using (a) Gene specific primers (EcoRI and XbaI); (Lane L: 100 bp DNA ladder; Lane 1-24: randomly selected clones with the band size of 400 bp); (b) Vector specific primers (5¢ AOX and 3¢ AOX); (Lane L: 100 bp plus DNA ladder; Lanes 1-24: Randomly selected clones with the band size of ~929 bp).


       
Out of 22 positive clones (~929 bp), it was found that 21 clones showed two bands where one band corresponded to the size of AOX gene of ~2.2 kbp size and other band corresponded to dAbCl26 gene amplified with vector specific primers of size ~929 bp. This type of band pattern was found in Mut+ phenotype. Remaining one clone had only one band of ~929 bp which specified MutS phenotype. Based on PCR results, out of 22 positive clones, 21 clones were of Mut+ phenotype and only one clone was of MutS phenotype.
 
Confirmation of mut phenotype of the selected clones
 
The Mut phenotype of the positive clone was further confirmed by growing these zeocin resistant Pichia transformants on MD agar plate and MH agar plate. The Mut+ is the methanol utilization wild phenotype and has equal growth on the MM and MD agar plate while, MutS is the methanol utilization slow phenotype that has slow growth on MM plates than MD plates. Based on growth, it was found that MutS phenotype clone by PCR was also of MutS phenotype, since more growth was observed on MD agar plate as compared to growth on MH agar. Also, Mut+ phenotype clone by PCR was confirmed to be Mut+ phenotype as the growth was almost similar on both agar plates. Based on growth, it was found that out of 22 PCR positive clones 21 were of Mut+ phenotype and one was of MutS phenotype (Fig 3).

​

Fig 3: Confirmation of Mut phenotype of transformed clones.



Optimization of culture conditions for expression of recombinant dAbCl26 in P.  pastoris X-33
 
The expression level of heterologous proteins in P. pastoris can be significantly enhanced by adopting various strategies (Macauley-Patrick et al., 2005; Yu et al., 2010). In the present study, different conditions like type of media, biomass production and methanol induction, were optimized to obtain better expression results. 
       
For optimization of effect of different media on protein expression, dAbCl26 clone was grown in different culture media like MM, BMM, BMMY and BMMY + 2% Casamino acid. Following induction with 0.5% methanol every 24 h over a 120 h period, the supernatant samples were analyzed by SDS-PAGE. A band of about 22-24 kDa was detected and it was found that BMM media showed better expression of protein in comparison to other media used.
       
For optimization the effect of absorbance on protein expression, dAbCl26 in BMM media was induced with 0.5% methanol every 24 h till 120 h at OD600  2.0, 4.0 and 6.0 for each flask. The culture supernatants collected after every 24 h interval till 120 h were analyzed by SDS-PAGE. It was observed that protein expression was considerably higher when induced at absorbance of OD600 4.0 for 96 h than for other cultures when induced at absorbance of OD600 2.0 or 6.0.
       
For optimizing the effect of methanol induction on protein expression, dAbCl26 in BMM media was induced with 0.5%, 1% and 2% methanol, respectively every 24 h till 120 h at OD600 4.0. The culture supernatants collected after every 24 h interval till 120 h were analyzed by SDS-PAGE. It was observed that the expression of dAbCl26 was better after inducing with 0.5% methanol than other two samples induced with 1% and 2% methanol. Previous studies have indicated that suitable methanol concentrations for induction typically range between 0.1% and 3% (v/v) (Mu et al., 2008; Zhang et al., 2009; Minjie and Zhongping, 2013). These findings highlighted the importance of optimizing culture conditions for each specific protein of interest in P. pastoris to achieve maximum expression levels.
       
The final optimized conditions for expression of dAbCl26 in Pichia strain X-33 were BMM growth media, induction at OD600 4.0 and methanol 0.5%. The SDS-PAGE profile of expressed recombinant protein dAb26 in Pichia strain X-33 using the final optimized conditions has been shown in Fig 4. The size of protein expressed in P. pastoris was found slightly greater (~22-24 kDa) than that of the earlier expressed dAbCl26 protein in E. coli (~17 kDa). The increased size was probably due to the unique phenomenon of glycosylation by P. pastoris. Teh et al., (2011) expressed recombinant human erythropoietin (rhEPO) gene in P. pastoris where the estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa and the variation in size was attributed to the presence of glycosylation analogs.

​

Fig 4: SDS-PAGE profile of recombinant protein expressed in Pichia pastoris strain X-33.


 
Purification of recombinant protein
 
The culture supernatant collected 96 h after induction was purified using Ni-NTA purification system. The purified protein was seen as a band of approximately 22-24 kDa (Fig 5). Also, a total yield of 4 mg/L of the culture was obtained which is two-fold higher as compared to previous reports (Gupta, 2014).

​

Fig 5: SDS-PAGE profile of recombinant protein purified by Ni-NTA column.


 
Western blot analysis of Pichia expressed dAbCl26
 
The expression of dAbCl26 was further confirmed by immunoblotting using anti-His HRP conjugated antibody (Invitrogen, R93125). A band of ~22-24 kDa was observed, thus confirming the results of SDS-PAGE analysis (Fig 6).

​

Fig 6: Western blotting of Pichia expressed dAbCl26.

CONCLUSION

In conclusion, the work described here provided a feasible method to clone, express and purify the recombinant single domain antibody clone dAbCl26 in P. pastoris. Furthermore, the method developed in this study will serve as a foundation for expressing other single-domain antibodies and for scaling up expression to a large scale. Given its already reported functional properties, the P. pastoris expressed dAbCl26 presents itself as a suitable candidate for further development as a therapeutic agent for endotoxemia in animals.

ACKNOWLEDGEMENT

This work is a part of the M.V.Sc thesis submitted to Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, India by the first author. The funding received from Education Division of Indian Council of Agricultural Research (ICAR) is duly acknowledged for carrying out the present research work under the Niche Area of Excellence project entitled “Phage display techniques for Production of Veterinary Immunobiologicals without sacrificing animals”.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

REFERENCES


  1. Anangi, R., Koshy, S., Huq, R., Beeton, C., Chuang, W.J., King G.F. (2012). Recombinant expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An efficient method for production of KV1.3 channel blockers. PLoS One. 7: e52965. 

  2. Banerjee, S. (2015) Functional characterization of a lipopoly- saccharide binding single domain antibody clone selected from phage display library. [Masters dissertation, Veterinary Immunology, Department of Veterinary Microbiology, LUVAS, Hisar].

  3. Banerjee, S., Singh, A., Rawat, J., Bansal, N., Maan, S. (2020). Dataset of next-generation sequence reads of nanobody clones in phage display library derived from Indian desert camel (Camelus dromedarius L.). Data Brief. 34:106663. doi: 10.1016/j.dib.2020.106663.

  4. Chiu, M.L. and Gilliland, G.L. (2016). Engineering antibody therapeutics. Current Opinion in Structural Biology. 38: 163-173. doi: 10.1016/j.sbi.2016.07.012.

  5. Fux, A.C., Casonato Melo, C., Schlahsa, L., Burzan, N.B., Felsberger, A., Gessner, I., Fauerbach, J.A., Horejs-Hoeck, J., Droste, M., Siewert, C. (2024). Generation of endotoxin-specific monoclonal antibodies by phage and yeast display for capturing endotoxin. International Journal of Molecular Sciences. 25: 2297.

  6. Gupta, A.K. (2014). Studies on endotoxin-neutralizing effects of three camelid single-domain antibody clones. [Doctoral dissertation, Veterinary Immunology, Department of Veterinary Microbiology, LUVAS, Hisar]. 

  7. Homayouni, V., Ganjalikhani-Hakemi, M., Rezaei, A., Khanahmad, H., Behdani, M., Lomedasht, F.K. (2016). Preparation and characterization of a novel nanobody against T-cell immuno- globulin and mucin-3 (TIM-3). Iranian Journal of Basic Medical Sciences. 19(11): 1201.

  8. Kazemi-Lomedasht, F., Behdani, M., Habibi-Anbouhi, M., Shahbazzadeh, D. (2016). Production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 35(3): 167-171.

  9. Kurtzman, C.P. (2009). Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. Journal of Industrial Microbiology and Biotechnology. 36(11): 1435.

  10. Macauley-Patrick, S., Fazenda, M.L., Mcneil, B., Harvey, L.M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast. 22: 249-270. 

  11. Minjie, G. and Zhongping, S. (2013). Process control and optimization for heterologous protein production by methylotrophic Pichia pastoris. Chinese Journal of Chemical Engineering. 21: 216-226.

  12. Mu, X., Kong, N., Chen, W., Zhang, T., Shen, M., Yan, W. (2008). High- level expression, purification and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris. Protein Expression and Purification. 59: 282-288.

  13. Nordén, K., Agemark, M., Danielson, J.Å., Alexandersson, E., Kjellbom, P., Johanson, U. (2011). Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris. BMC Biotechnology. 11: 47.

  14. Roncolato, E.C., Campos, L.B., Pessenda, G., e Silva, L.C., Furtado, G.P., Barbosa, J.E. (2015). Phage display as a novel promising antivenom therapy: A review. Toxicon. 93: 79-84.

  15. Singh, A. (2009). DBT research project on production of phage display library of Indian desert camel. Final report submitted to the Department of Biotechnology, Government of India, New Delhi. 

  16. Swichkow, C.R., Bloom, J.S., Kruglyak, L. (2022). Exploring the genetic architecture of protein secretion in Komagataella phaffii (Pichia pastoris) for biotechnology applications. PLOS Biology. 20(12): e3001911.

  17. Teh, S.H., Fong, M.Y., Mohamed, Z. (2011). Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris. Genetics and Molecular Biology. 34: 464-470.

  18. Vogl, T., Gebbie, L., Palfreyman, R.W., Speight, R. (2018). Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Applied and Environmental Microbiology. 84(6): e02712-17.

  19. White, C.E., Kempi N.M., Komives, E.A. (1994). Expression of highly disulfide-bonded proteins in Pichia pastoris. Structure. 2: 1003-1005.

  20. Yu, M., Wen, S., Tan, T. (2010). Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris. Engineering in Life Sciences. 10: 458-464. 

  21. Zhang, T., Gong, F., Peng, Y., Chi, Z. (2009). Optimization for high- level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase. Process Biochemistry. 44: 1335- 1339.
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    Full Research Article

    Optimization of Culture Conditions for Expression of Anti-lipopolysaccharide Single Domain Antibody Clone in Pichia pastoris

    A
    Anu Malik1
    A
    Akhil Kumar Gupta1,*
    J
    Jaideep Kumar1
    A
    Anshul Lather1
    P
    Parveen Kumar1
    M
    Mahavir Singh2
    S
    Swati Dahiya1
    N
    Naresh Kumar Kakker1
    1Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.
    2College Central Lab., Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India.

    ABSTRACT

    Background: Previously, a single domain antibody clone (dAbCl26) selected from phage display library of LPS-immunized Indian desert camel was expressed in prokaryotic expression system and found to inhibit the LPS-induced inflammatory cytokine both in vitro and in vivo. However, the level of expression in prokaryotic expression system was low and also there was problem of endotoxin contamination. Pichia pastoris has emerged as an alternative host and economical approach for the expression of recombinant protein as it has the ability to properly refold the protein, gives high yield and produce protein without endotoxin contamination. Encouraged by the earlier findings of in vitro and in vivo inhibition by dAbCl26, the expression of dAbCl26 was investigated in yeast expression system P. pastoris.

    Methods: The dAbCl26 was cloned into the Pichia expression vector pPICZαA and transformed into P. pastoris strain X33. Mutant phenotypes of selected clones were assessed using agar plate methods. For expression of the dAbCl26 in P. pastoris strain X33, the final growth conditions were optimized in buffered minimal methanol media with induction at OD600 4.0 and methanol induction at 0.5%.

    Result: Using the optimized conditions, a yield of about 4 mg/L of dAbCl26 was obtained in shake flask culture which is 2 fold higher than previously reported. The expressed protein was purified by using Ni-NTA chromatography and confirmed by Western blotting. The method developed in this study will be used as base for expression of other single domain antibodies and to scale up the expression on large scale. Based on its already reported functional properties, the Pichia expressed dAbCl26 can be a suitable candidate for further development as a therapeutic agent for endotoxaemia in animals.

    INTRODUCTION

    Different in vitro selection technologies such as phage display antibody provides a solution for the generation of recombinant antibodies (Chiu and Gilliland, 2016). Phage display technology has been found to be one of the most effective, robust, easy-to-perform methods to develop recombinant antibodies with high affinity and target specificity and has been utilized to generate single-domain antibodies or variable domain of heavy chain of heavy chain antibodies (VHHs), also widely known as nanobodies (Roncolato et al., 2015; Homayouni et al., 2016; Kazemi-Lomedasht et al., 2016).
           
    The Department of Veterinary Microbiology, LUVAS, Hisar, Haryana (India) had previously constructed a phage display library of single-domain antibodies of LPS-immunized Indian desert camel for diagnostic and therapeutic use (Singh, 2009; Banerjee et al., 2020). From this library, one of the clone i.e. dAbCl26 was found to inhibit the LPS-induced inflammatory cytokine response in buffalo and murine macrophages in vitro (Gupta, 2014). Also, the dAbCl26 inhibited the LPS-induced TNF-α release in Swiss albino mice model. The dAbCl26 also neutralized the E. coli 0153 LPS in chicken embryo lethality model and was found to cross react with LPS isolated from different Gram negative organisms i.e. P. multocida B:2, E. coli 0153, Salmonella typhimurium, Pseudomonas aeruginosa and Klebsiella spp. (Gupta, 2014; Banerjee, 2015). These findings suggested that dAbCl26 could be a novel candidate to prevent endotoxaemia and sepsis. However, utilizing prokaryotic expression systems for producing antibodies or other therapeutic proteins presents significant challenges. One major issue is the relatively low level of protein expression in these systems. Additionally, the production process is often complicated by endotoxin contamination, as prokaryotic cells, particularly Gram negative bacteria, inherently contain LPS in their outer membranes. This contamination can pose serious risks, especially when producing treatments for conditions like sepsis, where endotoxins are a primary concern (Fux et al., 2024).
           
    To address these limitations, the Pichia pastoris expression system offers an efficient and economical approach for producing wide variety of heterologous proteins in a correctly refolded form with disulfide bridges (White et al., 1994; Kurtzman, 2009; Anangi et al., 2012; Swichkow et al., 2022). Therefore, in this study, dAbCl26 was cloned and expressed in P. pastoris. Initially, a zeocin-resistant clone was selected and further optimized for culture conditions, including medium type, induction timing and methanol concentration, to maximize protein yield. After purification, the dAbCl26 was characterized by SDS-PAGE and Western blotting. By leveraging the unique properties of dAbCl26 and the advantages of the P. pastoris expression system, our aim was to create an efficient and scalable production method for this promising therapeutic agent.

    MATERIALS AND METHODS

    Ethics statement
     
    Permission to work on Pichia pastoris was obtained from Institutional Biosafety committee (IBSC) vide reference no. LUVAS/ABT/2020/29 Dated 07.01.2020. The experimental work was carried out at Department of Veterinary Microbiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana over a period extending from October, 2019 to November, 2020.
     
    Strains and media used
     
    The EasySelect™ Pichia Expression Kit (Thermo Scientific, K1740-01) was used for the expression of dAbCl26 in P. pastoris. P. pastoris X-33 strain, TOP10FA E. coli, P. pastoris vector (pPICZαA), zeocin, vector specific sequencing primers and media supplied with the kit were used in the present study.
     
    Cloning of dAbCl26 in P. pastoris pPICZαA vector
     
    The dAbCl26 gene (Acc no. KF990217) was cloned in Pichia vector (pPICZαA) in frame with c-myc and His tag. The dAbCl26 gene was amplified using gene specific primer (Table 1) by gradient PCR with annealing temperature ranging from 59-63oC. The PCR product was purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega, A9281).

    Table 1: Primers used for cloning of dAbCl26 into Pichia vector pPICZaA (Resrtriction sites shown in bold).


           
    The vector pPICZαA and purified PCR product of dAbCl26 were double digested with EcoRI (Thermo Scientific, FD0274) and XbaI (Thermo Scientific, FD0684) restriction enzymes and the purified RE digested dAbCl26 was cloned into the vector pPICZαA keeping the insert:vector molar ratio to 3:1. The ligated gene product was transformed into chemi-competent Top10FA E. coli cells using E. coli transformation buffer set kit (Zymo Research, T3002). The Top10F′ E. coli cells were then plated with 150 μl volume/plate on 90 mm low salt LB/zeocin (25 μg/ml) agar plates. Finally, plates were incubated overnight at 37oC. Next day, five transformed clones were randomly screened with gene specific primers and vector specific primers.
     
    Confirmation of recombinant plasmid
     
    For the confirmation of recombinant plasmid, recombinant vector plasmids (dAbCl26 + pPICZαA) and empty vector plasmids (pPICZαA) were double digested with EcoRI and XbaI enzymes. Meanwhile, the recombinant vector plasmid (dAbCl26 + pPICZαA) was linearized by single digestion with restriction enzyme SacI (Thermo Scientific, FD1134). This linearized plasmid was later used for transformation in P. pastoris strain X33. These digested products were resolved on 1.5% agarose gel for confirmation.
     
    Transformation into P. pastoris strain X33
     
    The recombinant linear plasmid (dAbCl26+pPICZαA) was chemically transformed into P. pastoris X-33 cells using Pichia EasyComp Transformation Kit as per manufacturer’s protocol (Invitrogen, K173001). After transformation, the entire mixture was plated on the Yeast extract peptone dextrose (YPD)/zeocin (100 µg/ml) agar plates and incubated for 3 days at 28oC. From the plate, transformed clones were regrown on YPD plates supplemented with progressively increasing concentrations of Zeocin (100, 200 and 400 µg/ml). This step was undertaken to select the clone exhibiting hyper-resistance to Zeocin. The Pichia transformants surviving on 400 µg/ml Zeocin were screened by PCR using gene specific primers and vector specific primers. The PCR products were resolved in a midi gel horizontal electrophoresis apparatus using 1.25% agarose gel with 5 µg ml-1 ethidium bromide in 1X TAE buffer at 5V/cm gel length and visualized in UV-transilluminator (Spectroline™).
     
    Determining the Mut+/MutS phenotype of selected clones
     
    The PCR positive clones were screened for determining their Mut phenotype. To determine the Mut phenotype of the transformed clones, zeocin resistant Pichia transformants were selected. One half portion of the selected colony was picked and streaked on minimal dextrose (MD) agar plate and the remaining half was streaked on minimal methanol (MM) agar plate. The plates were incubated at 28oC for 2 days and the growth on MD and MM agar plates was compared. After confirming the Mut phenotype of the selected clones, Mut+ or MutS protocol was followed for inducing expression in P. pastoris strain X-33.
     
    Optimization of culture conditions for expression of recombinant dAbCl26 in Pichia pastoris
     
    Effect of different media on recombinant protein expression
      
    A single recombinant Pichia colony was inoculated in 100 ml of Buffered glycerol-complex (BMGY) medium in 500 ml baffled flask and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 4.0. The cells were harvested by centrifugation at 6000 rpm for 7 min and re-suspended in 25 ml of different media: Minimal methanol (MM), Buffered minimal methanol (BMM), Buffered methanol-complex medium (BMMY) and BMMY supplemented with 2% Casamino acid. The culture was induced with methanol to final concentration of 0.5% (added after every 24 h). The culture supernatant was collected every 24h interval till 120 h. The cells were precipitated with 10% Trichloroacetic acid (TCA) and checked on SDS-PAGE for protein expression.
     
    Effect of time of induction on protein expression
     
    A single recombinant Pichia colony was inoculated in 100 ml of BMGY medium in 500 ml baffled flask in triplicate and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 2.0, 4.0 and 6.0 for each flask. The cells of each flask were harvested by centrifugation at 6000 rpm for 7 min and the pellet was re-suspended in BMM media and induced with 0.5% methanol (added every 24 h till 120 h). The culture supernatant was collected every 24 h interval till 120 h, precipitated with 10% TCA and checked on SDS-PAGE for protein expression.
     
    Effect of methanol on induction of protein expression
     
    A single recombinant Pichia colony was inoculated in 100 ml of BMGY medium in 500 ml baffled flask in triplicate and incubated at 28oC in shaking incubator at 270 rpm till OD600 reached 4.0 for each flask. The cells of each flask were harvested by centrifugation at 6000 rpm for 7 min and the pellet was re-suspended in BMM media and the flask was induced with 0.5%, 1% and 2% methanol, respectively. The methanol was added in the respective flasks after every 24 h till 120 h. The culture supernatant was collected every 24 h interval till 120 h, precipitated with 10% TCA and checked on SDS-PAGE for protein expression.
     
    Purification of recombinant protein
     
    The recombinant protein was purified using Ni-NTA purification system. The supernatant collected 96 h after induction was cleared by centrifugation at 7000 rpm for 10 min and was dialyzed against binding buffer (50mM NaH2PO4.H2O, 300mM NaCl, pH 8.0) for 3 days with buffer changed after every 12 h. One ml of Ni-NTA agarose beads were settled down in column and washed with 5 volume of binding buffer. These equilibrated Ni-NTA beads were incubated with dialyzed culture supernatant overnight at 4oC. Next morning, the culture supernatant packed in column was washed with 5 volume of washing buffer (binding buffer with 0.1% triton X). The 6xHis tagged protein was eluted with elution buffer (binding buffer with 300 mM imidazole). The eluates were collected in small fractions of 1 ml each and checked by SDS-PAGE.
     
    Western blot analysis of Pichia expressed dAbCl26
     
    For Western blotting, Pichia expressed dAbCl26 protein was resolved on 15% SDS-PAGE to obtain polypeptide profiles and the same were electro-blotted onto PVDF membrane (0.22 µm) in a semi-dry trans-blot apparatus (Atto® corporation, Japan) at 1.0 mA/cm2 of the gel for 60 min at room temperature. The PVDF membrane was blocked in 3% BSA-PBST (0.05% Tween-20) blocking buffer for 1 hr in shaking incubator at room temperature. After washing with PBST (0.05% Tween-20), the membrane was probed with anti-His HRP conjugated antibody (1:5000 dilution) and developed in 3,3-diaminobenzidine tetrahydrochloride hydrate (Sigma, D5637) and hydrogen peroxide (DAB/H2O2) substrate solution.

    RESULTS AND DISCUSSION

    Cloning of dAbCl26 in Pichia expression vector pPICZα​A
     
    The recombinant plasmid (dAbCl26+pPICZαA) was transformed in TOP10FA E. coli competent cells for the purpose of storage and propagation of recombinant plasmid to get high copy number for the successful transformation in P. pastoris strain X-33. It had been well documented that for the optimization of protein expression, high copy number appeared to have dominant positive influence and to achieve the same P. pastoris plasmids were propagated in E. coli prior to transformation (Vogl et al., 2018). Gradient PCR was conducted for the optimization of annealing temperature for the designed primers with the temperature range 59-63oC. It was observed that better amplification was obtained at the annealing temperature of 60oC. The vector pPICZαA and purified dAbCl26 PCR product were double digested, ligated and transformed in the competent TOP10FA E. coli cells and plated on the low salt LB/zeocin agar plates. After incubation at 37oC for 18 h, five randomly selected clones were found positive by PCR using both gene specific primers and vector specific primers, respectively. The gene specific primers showed a band of 400 bp while vector specific primers a band of 929 bp was observed.
     
    Confirmation of the recombinant plasmid (pPICZα​A + dAbCl26)
     
    The recombinant plasmid and empty vector were double digested with EcoRI and XbaI enzymes. Double digested recombinant plasmid showed two bands, one band of 3.5 kbp and other of 400 bp, as expected of the insert. The empty vector pPICZαA also showed a single band of 3.5 kbp. The single RE digested recombinant plasmid showed a single band of 3.9 kbp indicating that gene of interest was cloned in pPICZαA vector (Fig 1).

    Fig 1: Agarose gel electrophoresis of RE digested products.


     
    Transformation and screening of recombinant clones transformed in P. pastoris X-33 strain
     
    For transformation, the recombinant plasmid linearized with enzyme SacI was transformed into P. pastoris X-33 by chemical method. After 3 days incubation at 28oC, more than 50 colonies were observed on YPD agar plates containing 100 µg/ml of Zeocin. These colonies were regrown on YPD plates supplemented with progressively increasing concentrations of Zeocin (100, 200 and 400 µg/ml). This step was aimed at selecting clones that exhibit hyper-resistance to Zeocin, ensuring the selection of the most robust and genetically stable clones for subsequent protein production. This approach aligns with findings by Nordén et al. (2011), who reported an intimate correlation between hyper-resistance against Zeocin and enhanced expression of foreign proteins in P. pastoris. Therefore, adopting this strategy was crucial for selecting hyper-resistant P. pastoris strains, which are likely to exhibit improved expression of the desired recombinant proteins. From YPD/zeocin (400 µg/ml) agar plates, twenty-four clones were randomly selected and screened by PCR using both gene specific and vector specific primers, respectively. On agarose gel electrophoresis, out of twenty-four clones, 23 were found positive (400 bp) with gene specific primers (Fig 2) and 22 clones were found positive (~929 bp) with vector specific primers (Fig 2). This confirmed the integration of dAbCl26- pPICZαA plasmid in P. pastoris genome.

    ​

    Fig 2: Agarose gel electrophoresis of PCR amplification of transformed Pichia clones using (a) Gene specific primers (EcoRI and XbaI); (Lane L: 100 bp DNA ladder; Lane 1-24: randomly selected clones with the band size of 400 bp); (b) Vector specific primers (5¢ AOX and 3¢ AOX); (Lane L: 100 bp plus DNA ladder; Lanes 1-24: Randomly selected clones with the band size of ~929 bp).


           
    Out of 22 positive clones (~929 bp), it was found that 21 clones showed two bands where one band corresponded to the size of AOX gene of ~2.2 kbp size and other band corresponded to dAbCl26 gene amplified with vector specific primers of size ~929 bp. This type of band pattern was found in Mut+ phenotype. Remaining one clone had only one band of ~929 bp which specified MutS phenotype. Based on PCR results, out of 22 positive clones, 21 clones were of Mut+ phenotype and only one clone was of MutS phenotype.
     
    Confirmation of mut phenotype of the selected clones
     
    The Mut phenotype of the positive clone was further confirmed by growing these zeocin resistant Pichia transformants on MD agar plate and MH agar plate. The Mut+ is the methanol utilization wild phenotype and has equal growth on the MM and MD agar plate while, MutS is the methanol utilization slow phenotype that has slow growth on MM plates than MD plates. Based on growth, it was found that MutS phenotype clone by PCR was also of MutS phenotype, since more growth was observed on MD agar plate as compared to growth on MH agar. Also, Mut+ phenotype clone by PCR was confirmed to be Mut+ phenotype as the growth was almost similar on both agar plates. Based on growth, it was found that out of 22 PCR positive clones 21 were of Mut+ phenotype and one was of MutS phenotype (Fig 3).

    ​

    Fig 3: Confirmation of Mut phenotype of transformed clones.



    Optimization of culture conditions for expression of recombinant dAbCl26 in P.  pastoris X-33
     
    The expression level of heterologous proteins in P. pastoris can be significantly enhanced by adopting various strategies (Macauley-Patrick et al., 2005; Yu et al., 2010). In the present study, different conditions like type of media, biomass production and methanol induction, were optimized to obtain better expression results. 
           
    For optimization of effect of different media on protein expression, dAbCl26 clone was grown in different culture media like MM, BMM, BMMY and BMMY + 2% Casamino acid. Following induction with 0.5% methanol every 24 h over a 120 h period, the supernatant samples were analyzed by SDS-PAGE. A band of about 22-24 kDa was detected and it was found that BMM media showed better expression of protein in comparison to other media used.
           
    For optimization the effect of absorbance on protein expression, dAbCl26 in BMM media was induced with 0.5% methanol every 24 h till 120 h at OD600  2.0, 4.0 and 6.0 for each flask. The culture supernatants collected after every 24 h interval till 120 h were analyzed by SDS-PAGE. It was observed that protein expression was considerably higher when induced at absorbance of OD600 4.0 for 96 h than for other cultures when induced at absorbance of OD600 2.0 or 6.0.
           
    For optimizing the effect of methanol induction on protein expression, dAbCl26 in BMM media was induced with 0.5%, 1% and 2% methanol, respectively every 24 h till 120 h at OD600 4.0. The culture supernatants collected after every 24 h interval till 120 h were analyzed by SDS-PAGE. It was observed that the expression of dAbCl26 was better after inducing with 0.5% methanol than other two samples induced with 1% and 2% methanol. Previous studies have indicated that suitable methanol concentrations for induction typically range between 0.1% and 3% (v/v) (Mu et al., 2008; Zhang et al., 2009; Minjie and Zhongping, 2013). These findings highlighted the importance of optimizing culture conditions for each specific protein of interest in P. pastoris to achieve maximum expression levels.
           
    The final optimized conditions for expression of dAbCl26 in Pichia strain X-33 were BMM growth media, induction at OD600 4.0 and methanol 0.5%. The SDS-PAGE profile of expressed recombinant protein dAb26 in Pichia strain X-33 using the final optimized conditions has been shown in Fig 4. The size of protein expressed in P. pastoris was found slightly greater (~22-24 kDa) than that of the earlier expressed dAbCl26 protein in E. coli (~17 kDa). The increased size was probably due to the unique phenomenon of glycosylation by P. pastoris. Teh et al., (2011) expressed recombinant human erythropoietin (rhEPO) gene in P. pastoris where the estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa and the variation in size was attributed to the presence of glycosylation analogs.

    ​

    Fig 4: SDS-PAGE profile of recombinant protein expressed in Pichia pastoris strain X-33.


     
    Purification of recombinant protein
     
    The culture supernatant collected 96 h after induction was purified using Ni-NTA purification system. The purified protein was seen as a band of approximately 22-24 kDa (Fig 5). Also, a total yield of 4 mg/L of the culture was obtained which is two-fold higher as compared to previous reports (Gupta, 2014).

    ​

    Fig 5: SDS-PAGE profile of recombinant protein purified by Ni-NTA column.


     
    Western blot analysis of Pichia expressed dAbCl26
     
    The expression of dAbCl26 was further confirmed by immunoblotting using anti-His HRP conjugated antibody (Invitrogen, R93125). A band of ~22-24 kDa was observed, thus confirming the results of SDS-PAGE analysis (Fig 6).

    ​

    Fig 6: Western blotting of Pichia expressed dAbCl26.

    CONCLUSION

    In conclusion, the work described here provided a feasible method to clone, express and purify the recombinant single domain antibody clone dAbCl26 in P. pastoris. Furthermore, the method developed in this study will serve as a foundation for expressing other single-domain antibodies and for scaling up expression to a large scale. Given its already reported functional properties, the P. pastoris expressed dAbCl26 presents itself as a suitable candidate for further development as a therapeutic agent for endotoxemia in animals.

    ACKNOWLEDGEMENT

    This work is a part of the M.V.Sc thesis submitted to Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, India by the first author. The funding received from Education Division of Indian Council of Agricultural Research (ICAR) is duly acknowledged for carrying out the present research work under the Niche Area of Excellence project entitled “Phage display techniques for Production of Veterinary Immunobiologicals without sacrificing animals”.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors have no conflict of interest to declare.

    REFERENCES


    1. Anangi, R., Koshy, S., Huq, R., Beeton, C., Chuang, W.J., King G.F. (2012). Recombinant expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An efficient method for production of KV1.3 channel blockers. PLoS One. 7: e52965. 

    2. Banerjee, S. (2015) Functional characterization of a lipopoly- saccharide binding single domain antibody clone selected from phage display library. [Masters dissertation, Veterinary Immunology, Department of Veterinary Microbiology, LUVAS, Hisar].

    3. Banerjee, S., Singh, A., Rawat, J., Bansal, N., Maan, S. (2020). Dataset of next-generation sequence reads of nanobody clones in phage display library derived from Indian desert camel (Camelus dromedarius L.). Data Brief. 34:106663. doi: 10.1016/j.dib.2020.106663.

    4. Chiu, M.L. and Gilliland, G.L. (2016). Engineering antibody therapeutics. Current Opinion in Structural Biology. 38: 163-173. doi: 10.1016/j.sbi.2016.07.012.

    5. Fux, A.C., Casonato Melo, C., Schlahsa, L., Burzan, N.B., Felsberger, A., Gessner, I., Fauerbach, J.A., Horejs-Hoeck, J., Droste, M., Siewert, C. (2024). Generation of endotoxin-specific monoclonal antibodies by phage and yeast display for capturing endotoxin. International Journal of Molecular Sciences. 25: 2297.

    6. Gupta, A.K. (2014). Studies on endotoxin-neutralizing effects of three camelid single-domain antibody clones. [Doctoral dissertation, Veterinary Immunology, Department of Veterinary Microbiology, LUVAS, Hisar]. 

    7. Homayouni, V., Ganjalikhani-Hakemi, M., Rezaei, A., Khanahmad, H., Behdani, M., Lomedasht, F.K. (2016). Preparation and characterization of a novel nanobody against T-cell immuno- globulin and mucin-3 (TIM-3). Iranian Journal of Basic Medical Sciences. 19(11): 1201.

    8. Kazemi-Lomedasht, F., Behdani, M., Habibi-Anbouhi, M., Shahbazzadeh, D. (2016). Production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 35(3): 167-171.

    9. Kurtzman, C.P. (2009). Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis. Journal of Industrial Microbiology and Biotechnology. 36(11): 1435.

    10. Macauley-Patrick, S., Fazenda, M.L., Mcneil, B., Harvey, L.M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast. 22: 249-270. 

    11. Minjie, G. and Zhongping, S. (2013). Process control and optimization for heterologous protein production by methylotrophic Pichia pastoris. Chinese Journal of Chemical Engineering. 21: 216-226.

    12. Mu, X., Kong, N., Chen, W., Zhang, T., Shen, M., Yan, W. (2008). High- level expression, purification and characterization of recombinant human basic fibroblast growth factor in Pichia pastoris. Protein Expression and Purification. 59: 282-288.

    13. Nordén, K., Agemark, M., Danielson, J.Å., Alexandersson, E., Kjellbom, P., Johanson, U. (2011). Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris. BMC Biotechnology. 11: 47.

    14. Roncolato, E.C., Campos, L.B., Pessenda, G., e Silva, L.C., Furtado, G.P., Barbosa, J.E. (2015). Phage display as a novel promising antivenom therapy: A review. Toxicon. 93: 79-84.

    15. Singh, A. (2009). DBT research project on production of phage display library of Indian desert camel. Final report submitted to the Department of Biotechnology, Government of India, New Delhi. 

    16. Swichkow, C.R., Bloom, J.S., Kruglyak, L. (2022). Exploring the genetic architecture of protein secretion in Komagataella phaffii (Pichia pastoris) for biotechnology applications. PLOS Biology. 20(12): e3001911.

    17. Teh, S.H., Fong, M.Y., Mohamed, Z. (2011). Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris. Genetics and Molecular Biology. 34: 464-470.

    18. Vogl, T., Gebbie, L., Palfreyman, R.W., Speight, R. (2018). Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Applied and Environmental Microbiology. 84(6): e02712-17.

    19. White, C.E., Kempi N.M., Komives, E.A. (1994). Expression of highly disulfide-bonded proteins in Pichia pastoris. Structure. 2: 1003-1005.

    20. Yu, M., Wen, S., Tan, T. (2010). Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris. Engineering in Life Sciences. 10: 458-464. 

    21. Zhang, T., Gong, F., Peng, Y., Chi, Z. (2009). Optimization for high- level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase. Process Biochemistry. 44: 1335- 1339.
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