An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris

Accepted Manuscript An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris Quan Chen, Yuhang Zhou, Jianli Yu, Wenshuai Liu, Fei Li, Mo Xian, Rui Nian, Haipeng Song, Dongxiao Feng PII:

S1046-5928(18)30525-4

DOI:

https://doi.org/10.1016/j.pep.2018.11.001

Reference:

YPREP 5351

To appear in:

Protein Expression and Purification

Received Date: 10 October 2018 Accepted Date: 5 November 2018

Please cite this article as: Q. Chen, Y. Zhou, J. Yu, W. Liu, F. Li, M. Xian, R. Nian, H. Song, D. Feng, An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris, Protein Expression and Purification (2018), doi: https://doi.org/10.1016/j.pep.2018.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

An Efficient Constitutive Expression System for Anti-CEACAM5 Nanobody

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Production in the Yeast Pichia pastoris

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Quan Chena,#, Yuhang Zhoub,#, Jianli Yub, Wenshuai Liua, Fei Lib, Mo Xiana, Rui

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Niana,*, Haipeng Songb,*, Dongxiao Fengc,*

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a

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Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road,

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Qingdao 266101, China

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b

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China

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CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and

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Shenzhen Innova Nanobodi Co., Ltd., No. 7018 Caitian Road, Shenzhen 518000,

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c

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Road, Yantai, 264003, China

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#

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*Corresponding authors.

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E-mail addresses: [email protected] (R. Nian); [email protected] (H.

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Song); [email protected] (D. Feng)

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These authors contributed equally to this work.

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School of Pharmaceutical Sciences, Binzhou Medical University, No. 346 Guanhai

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ACCEPTED MANUSCRIPT Abstract:

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Nanobodies offer multiple advantages over conventional antibodies in terms of size,

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stability, solubility, immunogenicity, and production costs, with improved tumor

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uptake and blood clearance. Additionally, the recombinant expression of nanobodies

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is robust in various expression systems, such as Escherichia coli, Saccharomyces

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cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for

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nanobody production, but all or almost all expression vectors developed for this

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system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1)

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that requires methanol for full induction. In this study, a constitutive anti-CEACAM5

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nanobody

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glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) promoter. The effects of

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different carbon sources and pH on nanobody expression were evaluated in shaking

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flask cultures. After 96 h of constitutive expression in shaking flask, a yield of 51.71

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mg/L was obtained. In addition, this constitutive expression system produced

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nanobodies at equivalent yield and affinity to that produced by methanol-induced

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expression. The results of this study indicated that the use of a constitutive expression

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system is a promising alternative for the production of nanobodies applied for cancer

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diagnosis and therapy.

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Keywords: Nanobody, constitutive expression, GAP promoter, Pichia pastoris,

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anti-CEACAM5

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ACCEPTED MANUSCRIPT 1. Introduction

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Monoclonal antibodies (mAbs) are widely used for disease diagnosis and therapy, due

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to their incomparable specificity and sensitivity (Nguyen et al. 2012).Nanobodies

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represent the new generation of antibodies and are usually derived from camelids,

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which naturally possess a unique subset of immunoglobulin that consists of heavy

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chains (HCs) only, and therefore lack light chains (LCs) (Hamers-Casterman et al.,

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1993). Nanobodies have many advantages over conventional antibodies such as size,

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stability, solubility, immunogenicity, and production costs, with improved tumor

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uptake and blood clearance(Muyldermans et al., 2009;Harmsen et al., 2007 ).

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Unlike to conventional antibodies, nanobodies could be easily expressed in various

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microorganism systems, such as Escherichia coli, Saccharomyces cerevisiae and

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Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody

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production, but all or almost all expression vectors developed for this system are

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based on the control of alcohol oxidase 1 promoter (AOX1) (Harmsen et al., 2007;

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Baghban et al., 2016; Rahbarizadeh et al., 2006; Ezzine et al., 2012).However, the

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methanol-induced system may have some drawbacks: methanol is toxic and easily

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causes

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methanol-induced system needs to shift carbon sources, and the time of fermentation

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lasts

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glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter is a good alternative

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promoter to AOX1, and the GAP expression system can be applied to many

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heterologous protein production, with expression levels of recombinant proteins that

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are comparable to the AOX1 system (Liu et al., 2012; Bo et al., 2013). Furthermore,

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the GAP expression system may have greater potential in large-scale production of

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recombinant proteins (Cos et al., 2006; Goodrick et al., 2001; Khasa et al., 2007).

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However, until now, there has been no report of use of the GAP promoter to express

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nanobodies.

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Carcinoembryonic antigen (CEA) was first found in tissue extracts of human colon

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cancer in 1965 (Gold et al., 1965) and is used as an important clinical tumor marker

fire

long

in

large-scale

(Delroisse

et

fermentations;

al.,

2005;

fermentation

Oledzka

et

al.,

process

of

2003).The

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ACCEPTED MANUSCRIPT for gastrointestinal cancers (Duffy et al., 2001).Early detection and accurate

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measurements of CEA level in serum can be used to determine the appropriate

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treatment strategy for patients, and this measurement is currently achieved by various

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immunoassay methods that rely on highly specific anti-CEA antibodies (Qu et al.

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2013).In this study, an anti-CEACAM5 nanobody 11C12 gene was synthesized and

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expressed efficiently under the control of the constitutive GAP promoter in P. pastoris.

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To our knowledge, this is the first report of nanobody expression under a constitutive

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promoter in P. pastoris.

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2. Materials and methods

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2.1 Screening of anti-CEACAM5 nanobody

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To obtain the anti-CEACAM5 nanobodies, we constructed a phage display nanobody

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library from the peripheral blood lymphocytes of llamas immunized with CEACAM5

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antigen. And then, we panned the phage nanobody library, screened and

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functional/biophysical charactered

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previously (Pardon et al., 2014; Rossi et al., 2014). After 3 rounds of screening, 21

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clones were selected, and clone 11C12 having higher affinity was chosen for further

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study.

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2.2 Strains and growth conditions

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E. coli strain DH5α was used as a host for DNA manipulation. P. pastoris strain

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GS115 was used for the expression of recombinant proteins. E. coli DH5α was

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cultured at 37oC in LB medium with zeocin (25 µg/mL). P. pastoris GS115 was

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grown at 30oC in YPG medium (1% yeast extract (w/v), 2% peptone (w/v), and 2%

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glycerol (v/v)) .

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2.3 Transformation and selection of recombinant P. pastoris

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Competent cells of P. pastoris GS115 were freshly prepared and used the same day.

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For transformation, 5-20 µg of pPICZαA-11C12 linearized by Sac Ⅰ or

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pGAPZαA-11C12 linearized by AvrⅡ was separately transformed into P. pastoris

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GS115 using a GenePulser XcellTM Electroporation system (Bio-Rad). The cells were

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spread onto YPD selection plates containing different zeocin concentration (100, 300，

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ACCEPTED MANUSCRIPT 500, 1000 and 1500 mg/L), and incubated at 30oC for 2-3 days until individual

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colonies appeared. The genome of colonies was extracted, followed by PCR and

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sequencing to select the positive transformants.

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2.4 Expression of recombinant nanobody in P. pastoris

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The selected colones containing pGAPZαA-11C12 were grown in a 500-mL flask

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containing 50 mL BMGY medium after a preculture step in a 10 mL YPD medium at

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30oC over night. The culture temperature and shaking rate were 28oC and 220 r/min,

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respectively. The effects of different carbon sources (glucose, glycerol, sucrose,

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methanol, and sorbitol) and pH values (5.0-7.0) on nanobody production were

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determined respectively.For the colones containing pPICZαA-11C12, the cells were

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centrifuged at 1500 g for 5 min and the cell pellet was re-suspended in BMMY

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medium (the same composition as BMGY but the 1%（v/v） glycerol was replaced

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with 1% （v/v） methanol) to induce expression after growing for about 24 h.

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Methanol was added to a final concentration of 1%（v/v） every 24 h to maintain

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induction.All data are expressed as the means ± SD of triplicate determinations.

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2.5 SDS-PAGE and western blotting analysis

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was

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performed with a 12% polyacrylamide gel at 120 V for about 1.5 h on a Mini-protein

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Tetra apparatus (Bio-Rad, USA). Protein molecular weight marker was Invitrogen™

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PageRuler.Proteins were stained with Coomassie Brilliant Blue R-250. The PVDF

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membrane was blocked with 5% skim milk for overnight at 4oC. Goat anti-His-HRP

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antibody was used at 1:5000 dilution to detect the specificity of nanobody.

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Anti-His-HRP solution was added to the membrane and incubated for 1-2 h at room

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temperature. The membranes were washed with phosphate buffer saline containing

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0.01% Tween 20 (TBST) three times and each for 10 min . The signal was detected

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using the HRP-DAB TMB kit (TIANGEN). Yeast extract of GS115-pPICZαA or

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GS115-pGAPZαA was used as a negative control.

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2.6 Nanobody purification and quantitative determination

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The yeast cells were pelleted by centrifugation at 10,000 g for 15 min at 4oC. Solid

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ACCEPTED MANUSCRIPT (NH4)2SO4 was added to the supernatant gradually, until solution gradually reached

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saturation at 4oC. The mixture was then separated by centrifugation at 10,000 g for 10

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min. The precipitate was dissolved in a 50 mM Tris, 300 mM NaCl buffer (pH 8.0),

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dialyzed overnight in the same buffer, and then collected as a crude nanobody solution

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and filtered through a 0.22 µm filter.

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The crude nanobody solution was purified using 1 mL of Ni-NTA affinity column

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(Bio-Rad). After washing, the 6His-tagged proteins were eluted with phosphate buffer

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pH 7.5 containing 100 mM imidazole. After dialysis, the protein was further separated

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via a HiTrapTM SP HP column, ultimately obtaining the purified nanobody. The purity

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of the protein was checked by SDS-PAGE. The nanobody concentration was

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evaluated with the BCA Easy II Protein Quantitative Kit (TransGen).

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2.7 Affinity analysis

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All experiments were performed using a Biacore T100 instrument with a CM5 series

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S sensor chip from GE Healthcare. Peptide (CEACAM5) immobilization was

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measured according to a previously described method (Rossi, 2014 ). All solutions

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and buffers were prepared with Milli-Q water, and the running buffer HBS-EP+ 10×

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(0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA, and 0.05% (v/v) surfactant P20) were

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purchased from GE Healthcare.The 11C12 nanobody (at different initial

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concentrations tested by the BCA Easy II Protein Quantitative Kit) was diluted in

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running buffer to final concentrations of 80, 40, 20, 10 and 5 µg/mL.Each

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concentration was tested in duplicate.Diluted samples were injected for 60 - 120 s at a

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flow rate of 30 µL/min,and then flushed for 200 s at a flow rate of 30 µL/min. Finally

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the chip surfaces were regenerated with a glycine solution (10 mM, pH 1.5) for 30 s

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and a NaOH solution (0.1 M) for 60 s at a flow rate of 30 µL/min.

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3. Results

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3.1 Construction of the recombinant vector pGAPZαA-11C12

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The full-length anti-CEACAM5 nanobody (named 11C12) genes were synthesized

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and cloned into the yeast expression vector, pGAPZαA, under the control of the GAP

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promoter, to derive the expression vector, pGAPZαA-11C12 (Fig. 1).

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ACCEPTED MANUSCRIPT 3.2 Influence of codon optimization on 11C12 expression in P. pastoris

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The 305 codons of the 11C12 gene were optimized according to the codon bias of P.

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pastoris. To compare the protein expression level of the native and codon-optimized

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11C12 genes in P. pastoris, we chose P. pastoris transformants with single copy

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number native and codon-optimized 11C12 genes, and cultured these transformants in

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shaking flasks for 96 h. SDS-PAGE and western blotting analysis of cell culture

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supernatant were performed and are shown in Fig. 2a and 2b. Significantly higher

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protein expression was obtained in yeast cells for the codon-optimized variants, as

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compared to the native ones. After constitutive expression for 96 h, the

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codon-optimized 11C12 protein was 42.2 mg/L in the fermentation broth compared to

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24.6 mg/L of the native 11C12 (Fig. 2c). Thus, codon optimization significantly

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enhanced nanobody expression level in P. pastoris.

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3.3 Effects of carbon source and initial pH on 11C12 production in shaking flasks

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To investigate the possible effects of various carbon sources on the codon-optimized

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11C12 protein expression level during shake-flask culture, the strain with the highest

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expression level of 11C12 protein was cultured in BMY medium supplemented with

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different carbon sources (glucose, glycerol, sucrose, methanol and sorbitol). The

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results (Fig. 3a) showed that glycerol was the best carbon source for expression, with

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a 11C12 expression level of about 47.3 mg/L after 96 h of culture.As a result, glycerol

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was chosen as the optimal carbon source for 11C12 production in P. pastoris.

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In order to evaluate the effects of the initial pH of the medium on cell growth and

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11C12 protein production, cells were grown in buffered YPG medium at pH 5.0, 5.5,

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6.0, 6.5, and 7.0. As shown by the results presented in Fig. 3b, the initial pH of the

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culture medium had a slight effect on cell growth and 11C12 expression. 11C12

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expression level increased slightly with increasing pH values below 6.5. At pH 6.5,

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cell dry weight (CDW) was 11.23 g/L and 11C12 yield was 46.68 mg/L.In this study,

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the optimal pH value for high level 11C12 expression was set as 6.5.

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3.4 Expression comparison of P. pastoris under methanol-induced and constitutive

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expression

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promoter in P. pastoris by growing cultures in shaking flasks. The time-courses for

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cell growth and expression of 11C12 under both conditions are shown in Fig. 4. The

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expression level of 11C12 increased with cell growth in both induced conditions.

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Compared to methanol-induced expression, the strain under constitutive induction

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grew faster, and achieved the highest concentration of 12.89 mg/L at 60 h. After 96 h

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of constitutive expression, the 11C12 yield reached 51.71 mg/L, higher than the 42.38

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mg/L obtained under methanol induction.

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3.5 Purification and affinity analysis of 11C12 produced in P. pastoris

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The ammonium sulfate precipitated 11C12 with a C terminus containing 6His-tag was

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dissolved, dialyzed, and loaded onto a Nickel-affinity chromatography column. And

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then, it was eluted with elution buffer of different concentrations of imidazole (50,

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100, 150 and 300 mM). Lane 2 of Fig. 5a shows that the purity of the 11C12 reached

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approximately 85% in 100 mM imidazole eluate. After dialysis, the 11C12 was

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further purified using a HiTrapTM SP HP column, ultimately obtaining the purified

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11C12 (Lane 3 of Fig. 5b).

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To analyze the nanobody affinity constant KD (kd/ka), we evaluated the interactions

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between each purified 11C12 sample and the immobilized CEACAM5 peptide with a

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1:1 binding model. As results shown in Fig. 6, the 11C12 produced by

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methanol-induction

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constant values (0.92 × 10−9 M and 2.33 × 10−9 M, respectively).

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4. Discussion

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To date, almost all studies on nanobody expression in P. pastoris used AOX1 as their

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vector promoter for expression. However, AOX1 may not be the best promoter for the

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expression of nanobodies, and GAP or another promoter might allow higher

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expression level of nanobodies with similar affinity. In this study, we constructed a

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constitutive expression vector pGAPZαA-11C12 in P. pastoris for anti-CEACAM5

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nanobody production. The recombinant vector pGAPZαA-11C12 was integrated into

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the genome of P. pastoris GS115. The effect of various carbon sources on 11C12

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constitutive

expression

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similar

affinity

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ACCEPTED MANUSCRIPT nanobody expression was evaluated in shaking flask cultures. We found that

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recombinant P. pastoris grew well and more efficiently secreted 11C12 into the

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medium when using glycerol as carbon source. To test parameters of the process that

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affect production, the influence of initial pH was also evaluated. Compared with

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inducible expression, the strain under constitutive expression grew faster and steadily

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secreted nanobody 11C12. The 11C12 yield produced with constitutive expression

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reached 51.71 mg/L, slightly higher than that of 42.38 mg/L obtained by inducible

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expression. In addition, the affinity of 11C12 produced in both induced conditions

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remained 10−9 M. A simple fermentation process and higher nanobody yield made the

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constitutive expression process better than that described for AOX1-derived

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expression system. Moreover, more carbon sources are available for nanobody

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production by use of the GAP promoter-driven expression system. Therefore, the

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constitutive expression system is expected to be more efficient and desirable for

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large-scale production of nanobodies.

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Acknowledgments

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This work was supported by the National Natural Science Foundation of China (Grant

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no.

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20163702021237) and the Primary Research Development Plan of Shandong

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Province (Grant no. 2016GSF121006)

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the

Nanobodi-QIBEBT

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Collaboration

Project

(Grant

no.

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Fig. 1 Constitutive expression vector pGAPZαA-11C12

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Fig. 2 Comparison of native and codon-optimized 11C12 expression in P. pastoris under constitutive expression by a) SDS-PAGE, b) Western blotting and c) 11C12 yield. Red boxes indicate the position of the ～16 KDa 11C12 nanobody. M: protein marker; 1: negative control; 2: native 11C12; 3: codon-optimized 11C12

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CDW of GS115/pPICZαA-11C12 CDW of GS115/pGAPZαA-11C12 Nanobody yield of GS115/pPICZαA-11C12 Nanobody yield of GS115/pGAPZαA-11C12

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AC C

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TE D

Time (h)

16

80

100

0 -10

ACCEPTED MANUSCRIPT

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Fig. 5 SDS-PAGE showing the purification of 11C12 expressed in P. pastoris. M: protein marker; 1: flow through via a Ni-NTA affinity column; 2: eluate via a Ni-NTA affinity column; 3: purified 11C12 via a HiTrapTM SP HP column

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ACCEPTED MANUSCRIPT Fig. 6 Affinity of 11C12 produced under the control of a) pGAP or the b) pAOX1

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promoter

18