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M.ENG. THESIS

Process Optimization for the Productionof Recombinant 30Kc19α-Runx2 Protein

30Kc19α-Runx2재조합단백질의생산공정최적화

BY

ERI KWON

February 2020

DEPARTMENT OF ENGINEERING PRACTICEGRADUATE SCHOOL OF ENGINEERING PRACTICE

SEOUL NATIONAL UNIVERSITY

M.ENG. THESIS

Process Optimization for the Productionof Recombinant 30Kc19α-Runx2 Protein

30Kc19α-Runx2재조합단백질의생산공정최적화

BY

ERI KWON

February 2020

DEPARTMENT OF ENGINEERING PRACTICEGRADUATE SCHOOL OF ENGINEERING PRACTICE

SEOUL NATIONAL UNIVERSITY

Abstract

Process Optimization for the Production of Recombinant 30Kc19α-Runx2

Protein

Eri Kwon

Department of Engineering Practice

Graduate School of Engineering Practice

Seoul National University

Due to the expedite growth in protein therapeutics identified in biopharmaceuti-

cal market, an effective manufacturing of therapeutic drugs is in great demand. In this

study, the recombinant 30Kc19α-Runx2 protein was analyzed by SDS-PAGE assay

to suggest optimized process for expression of soluble protein. Twenty (20) exper-

imental groups were analyzed based on cultivation protocol. The constructed pET-

23α/30Kc19α-Runx2 plasmid was transformed in E. coli and cultivated at 37 ◦C. Four

cultivation temperature (20, 25, 30, and 37 ◦C) and five cultivation time (1, 2, 4, and 6

hours, and overnight) after IPTG induction to express soluble form in E. coli were in-

vestigated. The results were analyzed using SDS-PAGE and coomassie blue staining.

The amount of soluble expression of 30Kc19α-Runx2 was increased with cultivation

time through 4 hours at induced culture temperature of 20, 25, and 30 ◦C. The signifi-

cant increase in soluble fraction was observed at 20 ◦C with cultivation time of 4 hours

after IPTG induction. The production at cultivation time of 4 hours at induced culture

temperature of 20 ◦C showed the largest amount of soluble protein expression. The

results suggested that the process optimized cultivation time for 4 hours after temper-

ature shift to 20 ◦C increased the amount of soluble expression of 30Kc19α-Runx2.

i

Keywords: therapeutic drugs, biopharmaceutical, 30Kc19α-Runx2 recombinant

protein, soluble expression, process optimization

Student number: 2018-27514

ii

Contents

Abstract i

Contents ii

List of Tables iv

List of Figures v

1 Introduction 1

2 Literature Review 3

2.1 Biopharmaceutical market trends . . . . . . . . . . . . . . . . . . . . 3

2.2 Production of soluble recombinant protein in E. coli . . . . . . . . . . 6

2.3 Recombinant 30Kc19α-Runx2 protein . . . . . . . . . . . . . . . . . 9

3 Materials and Methods 10

3.1 30Kc19α-Runx2 protein expression . . . . . . . . . . . . . . . . . . 10

3.2 SDS-PAGE Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.3 Western blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.4 Assessment of soluble fraction by SDS-PAGE . . . . . . . . . . . . . 16

3.5 Assessment of protein expression by SDS-PAGE . . . . . . . . . . . 16

3.6 Protein purification and quantification . . . . . . . . . . . . . . . . . 16

ii

4 Results 18

4.1 Effect of temperature shift on growth profile of E. coli . . . . . . . . . 18

4.2 Process optimization for soluble expression of recombinant 30Kc19α-

Runx2 protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.3 SDS-PAGE analysis of soluble fraction of recombinant 30Kc19α-Runx2

protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4.4 SDS-PAGE analysis of total amount of soluble recombinant 30Kc19α-

Runx2 protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4.5 Expression and purification of 30Kc19α-Runx2 protein . . . . . . . . 33

5 Discussion 35

6 Conclusion 39

Abstract (In Korean) 44

iii

List of Tables

2.1 Therapeutic recombinant protein produced from E. coli [1] . . . . . . 5

3.1 Twenty experimental groups for the optimal condition . . . . . . . . . 14

4.1 Total quantity and soluble fraction of recombinant 30Kc19α-Runx2 . 32

iv

List of Figures

2.1 SWOT analysis of recombinant protein production in E. coli . . . . . 7

3.1 Schematic diagram of the experimental protocol . . . . . . . . . . . . 12

3.2 Experimental condition : (a) temperature shift and (b) growth profile . 13

4.1 A representative SDS-PAGE image of soluble and insoluble expres-

sion of 30Kc19α-Runx2 recombinant protein . . . . . . . . . . . . . 20

4.2 Soluble fraction of recombinant 30Kc19α-Runx2 produced at 20 ◦C . 23

4.3 Soluble fraction of recombinant 30Kc19α-Runx2 produced at 25 ◦C . 24

4.4 Soluble fraction of recombinant 30Kc19α-Runx2 produced at 30 ◦C . 25

4.5 Soluble fraction of recombinant 30Kc19α-Runx2 produced at 37 ◦C . 26

4.6 Summary of soluble fraction in recombinant 30Kc19α-Runx2 . . . . 27

4.7 SDS-PAGE images for soluble recombinant 30Kc19α-Runx2 . . . . . 30

4.8 Total amount of soluble recombinant 30Kc19α-Runx2 . . . . . . . . 31

4.9 Expression and purification of 30Kc19α-Runx2 . . . . . . . . . . . . 34

v

Chapter 1

Introduction

The protein therapeutics, which include recombinant protein, represent one of the

fastest growing and predominant market in therapeutic biopharmaceuticals. The ther-

apeutic protein markets are estimated to account for $315.90 billion by end of 2025,

anticipating at Compound Announce Growth Rate (CAGR) of 8.6 % from 2016 to

2025, rising from US $140,109 million in 2016 [1, 2]. Compare to low molecular or-

ganic drug, due to its nature of specificity for targeting molecules and activity with

less side effect, the therapeutic protein possesses superior benefits for curing incurable

disease, such as cancer and autoimmune disease [3]. As the growing interest in curing

disease with aging society, the growing demand of the protein drug market is expected

to drive the continuous increase of research and development in biopharmaceuticals,

as well as efficient manufacturing of drug.

A highly complex process is required for manufacturing and production of thera-

peutic proteins in biopharmaceuticals. These protein therapeutics have to be manufac-

tured in mammalian or non-mammalian organisms as they cannot be synthesized only

by the chemical processes; therefore, the characteristics of final products are affected

by the production condition, selection of the media, and originated species [4].

The choice of expression system is mainly dependent on the target recombinant

protein. Escherichia coli (E. coli) is one of the favorable expression system as the

1

production from E. coli is simple and inexpensive with characteristic of fast growth

rate that enables accumulation of product at higher level [5]. However, the E. coli

poses significant drawback in relations to protein folding in correct manner and lack of

post-translational modifications [6]. The over-expressed protein in E. coli often leads

to increased inclusion bodies (IBs) formation where the proteins are incorrectly folded

or unfolded. These proteins are biologically inactive [6]. Additional solubilization,

denaturation, and refolding steps can be accomplished to induce biologically active

product from formed IBs [5]. However, the additional steps for refolding from IBs are

considered as undesirable due to the increase in production cost with poor recovery

yield. Therefore, the optimization of production condition for expression of soluble

recombinant protein becomes favorable to the refolding from the IBs [6].

Several studies have identified an effective method to mediate soluble expression

where the formation of IBs is avoided. Quing et. al., used a cold inducible expression

system to prevent forming IBs [7]. Prenchevicius et. al., reduced the IPTG concentra-

tion for induction and lowered the growth temperature of induced cultures to optimize

expression of ArtinM lectin recombinant protein [8].

In previous studies, 30Kc19 protein from silkworm hemolymph was reported to

inhibit apoptosis in various cells and deliver protein cargoes into cells by dimerization

mechanisms with enhanced enzyme stability [9, 10]. The protein is structurally con-

sist of α-helix in N-terminal domain and β-sheet in C-terminal domain. 30Kc19 with α

structure, or 30Kc19α, is known as cell penetrating protein reported to simultaneously

enhance the protein expression in soluble form with high stability and the transcrip-

tional activity [11]. In addition, recombinant 30Kc19α-Runx2 protein was identified

to express in a soluble form with enhanced osteogenic differentiation [12].

In this study, we examined the optimized growth temperature of induced cultures

and cultivation time after induction in production of recombinant 30Kc19α-Runx2

protein in lab-scale experiment.

2

Chapter 2

Literature Review

2.1 Biopharmaceutical market trends

The protein therapeutics account majority of markets with recombinant DNA tech-

nology in various expression system. Of 71 newly accepted biopharmaceuticals in year

2018, the 62 active ingredients are the recombinant protein manufactured in E. coli,

yeast, and mammalian [1]. These recombinant proteins are used for medical applica-

tion, such as vaccines or treatment of disease, or screening of new drugs where the

recombinant proteins are used as a target protein [13].

The continual rise in the market value of biopharmaceuticals indicated the rapid

advances in support treating incurable disease. The consistent product quality and cost-

effectiveness are considered as key criteria for manufacturing recombinant protein in

biopharmaceuticals [14]. E. coli is considered as a desirable expression system that

host several recombinant proteins with its characteristics of rapid growth rate and eas-

ier genetic manipulation [13].

The first human-derived insulin recombinant protein for the treatment of diabetes

has gained regulatory approval in 1982. This product was manufactured from E. coli

expression system [13]. Since then, the large number of biopharmaceuticals (Table

2.1) is being produced in microbial expression system. Neumega, an enzyme used in

3

treatment for preventing chemotherapy-induced thrombocytopenia, is derived from E.

coli for clinical use. Fulphila manufactured by Mylan is a recombinant protein used

for treatment of neutropenia and is produced in E. coli [1].

Well characterized with genetics information, the E. coli expression system has

been broadly applied for producing various proteins in both small and large scale pro-

duction [13]. The production of protein in E. coli is preferred in small scale production

due to its inexpensive substrate useful for analyzing functional characteristics of pro-

tein and for screening of new drug [13]. Moreover, with characteristics of fast growth

rate and easy scale-up process, the E. coli expression system has been favored by in-

dustry with large scale production [14]. All the therapeutic products listed in Table 2.1

are produced in E. coli.

4

Table 2.1: Therapeutic recombinant protein produced from E. coli [1]

Product name Therapeutic indication Company Approved year

Humulin Diabetes Eli lilly 1982

Intron A Cancer, hepatitis Schering Plough 1886

Neupogen Neutropenia Amgen 1991

Betaferon Multiple sclerosis Bayer pharma 1995

Rapilysin Myocardial infarction Roche 1996

Neumega Thrombocytopenia Pfizer 1997

Rebetron Chronic hepatitis Schering plough 1999

Beromun Soft-tissue sarcoma Boehringer ingelheim 1999

Lantus Diabetes Sanofi 2000

Kineret Rheumatoid arthritis Amgen 2001

Natrecor Congestive heart failure Johnson& Johnson 2001

Pegasys Hepatitis C Roche/Genentech 2002

Somavert Acromegaly Pfizer 2003

Fortical Osteoporosis Upsher-smith laboratry 2005

Omnitrope Growth disturbance Sandoz 2006

Preos Mascular degeneration Novatis 2006

Accretropin Growth hormone deficiency Emergent biosolution 2008

Gattex Short bowel syndrome NPS pharmaceuticals 2012

Lucentis Osteoporosis NPS pharmaceuticals 2013

Oncaspar Lymphoblastic leukemia Baxalta innovation 2016

Admelog Diabetes melitus Sanofi 2017

Fulphila Neutropenia Mylan 2018

Nivestym Neutropenia Pfizer 2018

5

2.2 Production of soluble recombinant protein in E. coli

The key characteristics of production in E. coli are depicted as in Figure 2.1 by the

SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis. The E. coli is

considered as the simplest and the cheapest expression system that undergoes simple

genetic manipulation and enables quick cultivation of the protein [5]. Moreover, the

opportunities as biosimilar agent reside in recombinant drug produced from E. coli

due to its expiration from patent protection. Compare to the price of drug manufactured

from mammalian cell, the price of producing drug from E. coli is cheaper, and thus,

favored by developing countries. However, the E. coli expression system poses the

major issues associated with formation of IBs where the proteins are aggregated as

inactive forms [6]. The inability for post-translational modifications is considered as

a significant problem associated with production in E. coli [5]. Therefore, production

in E. coli is often being challenged with protein produced from mammalian cell or

yeast that has more capability on purifying protein with ability for post-translation

modification [14].

6

Figure 2.1: SWOT analysis of recombinant protein production in E. coli

7

The correctly folded protein with higher levels of overall soluble protein is ideal

for production of recombinant proteins. To achieve correctly folded and soluble pro-

tein from the formation of IBs, additional denaturing and refolding steps are required

after the lysis of the cell [5]. The yield after the refolding steps is relatively low and,

therefore, refolding is considered as undesirable due to the increase in production cost

[4]. Sorensen et. al., reported the cause of protein aggregates to be the stress that is in

response to over-expression of recombinant protein in target [6]. Since the E. coli pos-

sess high level expression system, the triggering response from stress on the cell and

maintenance of its expression have widely been suggested [14]. Another reason was

reducing environment of cytoplasm where the formation of di-sulfide bond is inhibited

[4].

In order to prevent mis-folding of protein and formation of IBs, various methods

have been suggested. One of the methods to optimize and to prevent IBs formation is

to use of chaperones. The most widely used chaperones in E. coli are DnaK, DnaJ,

GrpE, GroEL, and GreoE that induce the soluble expression of the protein [14]. In

an aim to lower the metabolic burden associated with recombinant protein expression,

most commonly used method is by mediating environmental condition during the pro-

duction [5]. Previous studies suggested that the reduced temperature improved the

soluble expression of protein [5, 7]. Prenchevicius et. al., reduced the IPTG concentra-

tion for induction and lowered the growth temperature of induced cultures to optimize

the expression of ArtinM lectin recombinant protein [8]. Golotin, et. al., optimized

the culture temperature and inducer concentration for the expression of cold-adapted

alpha-galactosidase [15]. To enhance soluble expression of recombinant protein, the

types of strain used, media, transcription rate, and use of plasmid all were reported to

affect the expression of the protein [4].

8

2.3 Recombinant 30Kc19α-Runx2 protein

The Runt-related transcription factor 2, known as Runx2, is well understood for

mediating their effect to stimulate osteogenic differentiation by binding to osteoblast

specific genes. The Runx2 functions a significant role in mediating various signals

related to the osteogenic differentiation [16]. One of major drawback for up-regulating

recombinant Runx2 is that it cannot be synthesized as biologically active form without

supplementation of cell-penetrating peptide [17].

30Kc19 protein, a member of 30K family, is derived from Bombyx mori hemolymph.

Considering the nature compound from silkworm hemolymph, the 30Kc19 protein is

reported to inhibit apoptosis in human cell system, thereby having potential for ther-

apeutic uses [10, 11]. This protein is composed of α-helix N-terminal and β-sheet

C-terminal. Amongst them, 30Kc19α protein is expressed to have protein stabilizing

properties [18]. In addition, the characteristic of this protein was found to function as

cell penetrating protein by bringing the external protein into the cell [9, 10, 12]. The

properties of 30Kc19α is further reported to simultaneously enhance the expression

of fusion protein in soluble form, stability and enable activity of transcription factors

[11].

The previous studies have reported that fusion of 30Kc19α and Runx2 produces

protein with soluble expression and this fusion protein induces osteogenic differen-

tiation in mesenchymal stem cell by allowing intracellular delivery of Runx2 [12].

However, there are no reports on enhancing soluble expression of the recombinant

30Kc19α-Runx2 for commercial production. Therefore, this study examined effect of

manufacturing condition to determine the optimal temperature and time of production

after induction.

9

Chapter 3

Materials and Methods

3.1 30Kc19α-Runx2 protein expression

The vector, pET-23a/30Kc19α-Runx2, was obtained from a previous study [12],

and transformed into Rosetta2 (DE3, Novagen) E. coli expression system. The cells

were inoculated in LB medium with 100 µg/ml ampicillin for overnight at 37 ◦C with

agitation speed of 160 rpm. The inoculated medium was transferred to 250 mL flask

with 100 mL LB ampicilin medium and cultured at 37 ◦C with 160 rpm. To study

growth profile, 1 mL samples were collected for measuring cell density every 30 min-

utes. The target initial OD600 value was 0.5 where Isopropyl β-D-1-thiogalactopyranoside

(IPTG, 0.1 mM) was induced where the temperature and agitation speed was con-

trolled to provide optimal condition for cell culture growth and protein production.

The IPTG induced culture were further incubated at different temperature (20, 25, 30,

or 37 ◦C) at 160 rpm, for different incubation period (1, 2, 4, and 6 hours, and overnight

growth) after the induction. At each specific point, the samples were collected (Table

3.1) to evaluate expression of 30Kc19α-Runx2 protein. The collected samples were

further harvested prior to the protein analysis. The samples were centrifuged at 7,000

rpm at 4 ◦C for 10 minutes. The cultured medium was discarded and cells were dis-

solved in His-binding buffer (20 mM imidazole (Sigma-Aldrich), 20 mM Tris-HCl

10

(Sigma, USA), 500 mM NaCl (Junsei, Japan), pH 8.0). Followed by the resuspension,

the sonication was performed at 25% amplitude for 2 minutes, with 5 seconds interval

between each sonication to agitate particles in a sample. The centrifugation at 12,000

rpm at 4 ◦C for 20 minutes was performed on sample to separate the soluble and in-

soluble protein (Figure 3.1). The SDS-PAGE was used to quantify and analyze the

samples. The pelleted samples were either immediately used for analysis or frozen at

-20 ◦C for further use.

11

Figure 3.1: Schematic diagram of the experimental protocol

12

Figure 3.2: Experimental condition : (a) temperature shift and (b) growth profile

13

Table 3.1: Twenty experimental groups for the optimal condition

Time after IPTG induction 20 ◦C 25 ◦C 30 ◦C 37 ◦C

1 hour 20 ◦C, 1 25 ◦C, 1 30 ◦C, 1 37 ◦C, 1

2 hours 20 ◦C, 2 25 ◦C, 2 30 ◦C, 2 37 ◦C, 2

4 hours 20 ◦C, 4 25 ◦C, 4 30 ◦C, 4 37 ◦C, 4

6 hours 20 ◦C, 6 25 ◦C, 6 30 ◦C, 6 37 ◦C, 6

Overnight (O/N) 20 ◦C, O/N 25 ◦C, O/N 30 ◦C, O/N 37 ◦C, O/N

14

3.2 SDS-PAGE Analysis

The samples were compounded with 2X Tris-glycine SDS sample buffer in 1:1

(v/v). The samples were then heated for 2 minutes at 100 ◦C for protein denaturation.

20 µl of each sample were loaded into the wells of 12 % polyacrylamide gels followed

by the electrophoresis. The electrophoresis was run at 80 V for 2 hours. The gel was

stained using coomassie blue solution for 1 hour and immersed in destaining solu-

tion overnight. The stained gels were further quantified for analysis using the ImageJ

software.

3.3 Western blot analysis

After conducting SDS-PAGE of loaded sample, the separated gels were transferred

to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare) for the western

blotting. The blocking solution (5 % skim milk in 0.1 % PBS-T), anti-His tag primary

antibody (Abcam, UK), and anti-mouse IgG-HRP secondary antibody were treated to

the transferred membrane. The G: BOX Chemi XL system (Syngene, UK) was used to

visualize the transferred band on membrane and the images gained from the software

were further quantified using ImageJ software.

15

3.4 Assessment of soluble fraction by SDS-PAGE

The protein expression was analyzed by SDS-PAGE. The protein collected with

soluble and insoluble solution samples were loaded on SDS-PAGE for coomassie blue

staining. 20 μl of standard solutions and samples were loaded to 10-well plate. The

intensity of band was calculated to fractionate soluble and insoluble expression.

3.5 Assessment of protein expression by SDS-PAGE

The standard solution was used 50, 100, 200, 400 µg/ml and was added to each

well. The intensity of band was analyzed comparing with BSA standard for quantifi-

cation of protein.

3.6 Protein purification and quantification

After coomassie blue staining on experimental groups, the group with the highest

product quantity and soluble fraction of protein was selected for additional experi-

ments. The expression of recombinant protein was cultured in 4 L shaking flask and

collected after culture. Cell pellet after centrifugation was resuspended with 60 mL

of His-binding buffer (20 mM imidazole (Sigma-Aldrich), 20 mM Tris-HCl (Sigma,

USA), 500 mM NaCl (Junsei, Japan), pH 8.0). The cells were lysed using sonication

(30 % amplitude, pulse on 5 s, pulse off 5 s) and centrifuged at 12,000 rpm for 30

minutes at 4 ◦C. The filtration of supernatants was performed with 0.22 μm bottle top

filter (Jebiofil, Korea). The recombinant protein was purified using fast protein liq-

uid chromatography (FPLC; GE Healthcare, Sweden). Filtered soluble proteins were

loaded onto His-binding buffer-filled HisTrap HP colunm (GE Healthcare). To wash

weakly bounded protein, His-washing buffer (50 mM imidazole, 20 mM Tris-HCl, 0.5

M NaCl, pH 8.0) was flowed through the column. The remained proteins were eluted

with His-elution buffer (350 mM imidazole, 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0).

16

After the elution, the buffer was changed to Dulbecco’s Modified Eagle’s Medium

(DMEM Biowest, France) by using a desalting column (GE Healthcare).

17

Chapter 4

Results

4.1 Effect of temperature shift on growth profile of E. coli

The cell growth profile in E. coli before and after IPTG induction with tempera-

ture shift was studied. Sorenson et. al., has reported that the formation of IBs is largely

contributed by the growth rate after IPTG induction [6]. Since the growth rate of E

coli is related to the cultivation temperature, four different induction temperature con-

ditions were selected for the production of recombinant 30Kc19α-Runx2 protein. The

transformed cells were cultured at 37 ◦C and exposed to temperature shift after IPTG

induction as Table 3.1. The growth profile for each temperature was monitored by

OD600 for every 30 minutes. The temperature shift was completed prior to collection

of first subgroup. As seen from Figure 3.2, the growth rate obtained from 20 ◦C was

significantly lowered than the growth rate observed from 37 ◦C.

4.2 Process optimization for soluble expression of recombi-

nant 30Kc19α-Runx2 protein

The soluble expression of recombinant protein is considered to be an indication

of active functioning protein where the proteins are folded correctly [5]. To examine

18

optimal condition for producing soluble expression of recombinant 30Kc19α-Runx2

protein, experiments were conducted under twenty experimental conditions. Temper-

ature and cultivation time after IPTG induction were differentiated to find optimal

condition. The constructed pET-23a vector containing 30Kc19α and Runx2 were de-

livered to E. coli and recombinant 30Kc19α-Runx2 was expressed in 100 mL flask

scale. The lysates were separated in fraction of soluble and insoluble expression and

analyzed using SDS–PAGE. Figures 4.1 showed representative SDS-PAGE images of

the soluble and insoluble fraction of cell lysates containing recombinant 30Kc19α-

Runx2. From the data, we confirmed the expression of 30Kc19α-Runx2 protein with

71.2 kDa of band size.

19

Figure 4.1: A representative SDS-PAGE image of soluble and insoluble expression of

30Kc19α-Runx2 recombinant protein

20

4.3 SDS-PAGE analysis of soluble fraction of recombinant

30Kc19α-Runx2 protein

Soluble fraction of recombinant protein was analyzed using SDS-PAGE by mea-

suring intensities of soluble supernatant (S) and insoluble pellet (I) bands. The soluble

fraction of protein was calculated by dividing the intensity of soluble band from the

sum of the intensities of soluble and insoluble bands. Figure 4.2 depicts images of

coomassie blue stained gels where the protein was expressed at induction temperature

of 20 ◦C with samples prepped after 1, 2, 4, and 6 hours, and overnight growth after

0.1 mM IPTG induction. The soluble fraction was the highest at the cultivation time of

4 hours at induced culture. The soluble fraction gradually increased until induced cul-

tivation time of 4 hours and decreased soluble fraction of 58.9 % at overnight growth.

The maximum soluble fraction was observed at 4 hours post-induction for 20 ◦C. For

quantitative measures, refer to Table 4.1.

Similar trends in protein expressed at inducted temperature of 25 ◦C was observed

as in Figure 4.3. The graph shows that the cultivation time of 1 hour presents the lowest

soluble fraction, 30.1 %, whereas the highest soluble fraction, 59.5 % was observed at

cultivation time of 4 hours after IPTG induction.

The overall trend of 25 ◦C indicated that the soluble fraction gradually increases

up to 4 hours after IPTG induction and then decreases as the fraction of insoluble

aggregates form increases. When cultivated overnight, the soluble fraction reached to

44.7 % up to 4 hours after IPTG induction and then begans to decline again.

The graph of soluble fraction of induced temperature of 30 ◦C is described in

Figure 4.4. The highest soluble expression was observed at induced cultivation time of

6 hours after IPTG induction. The highest ratio of soluble expression was observed as

73.8 %.

Soluble fraction of induced temperature of 37 ◦C was analyzed as in Figure 4.5.

In contrast to graph observed from 20, 25, and 30 ◦C, the soluble fraction reached

21

highest at initial stage of IPTG induction, 1 hour with soluble fraction of 50.8 %, and

aggregated protein formed rapidly at 37 ◦C. The lowest soluble fraction was observed

at induced culture of 2 hours after the induction. The soluble fraction gradually in-

creased from 23.3 to 43.3 % as cultivation time after IPTG induction increases from 2

hours to overnight. For induced temperature of 37 ◦C, the majority was being produced

as insoluble aggregates.

The summary of soluble fraction depicted for post-induction temperature of 20,

25, 30, and 37 ◦C, respectively, is shown in Figure 4.6. The results indicated that the

post-induction temperature of 20 ◦C with cultivation time of 4 hours produced protein

with highest soluble fraction.

22

Figure 4.2: Soluble fraction of recombinant 30Kc19α-Runx2 produced at 20 ◦C

23

Figure 4.3: Soluble fraction of recombinant 30Kc19α-Runx2 produced at 25 ◦C

24

Figure 4.4: Soluble fraction of recombinant 30Kc19α-Runx2 produced at 30 ◦C

25

Figure 4.5: Soluble fraction of recombinant 30Kc19α-Runx2 produced at 37 ◦C

26

Figure 4.6: Summary of soluble fraction in recombinant 30Kc19α-Runx2

27

4.4 SDS-PAGE analysis of total amount of soluble recombi-

nant 30Kc19α-Runx2 protein

This study examined total amount of soluble recombinant protein using SDS-

PAGE analysis. 20 µl of samples were loaded on each well of the gels and 50 µg/ml

BSA was used for comparing amount of protein expressed among gels. The protein

concentration was calculated by loaded BSA sample on each gel after coomassie blue

staining. The Figure 4.7 and 4.8 depict amount of soluble protein produced at 20, 25,

30, and 37 ◦C.

For protein induced at 20 ◦C, the largest total amount of soluble protein was ob-

served at overnight growth with 445.7 µg/ml. The comparably large amount of soluble

protein was produced at post-induction of 4 hours with 442.7 µg/ml. There was in-

crease in soluble protein concentration by 6-fold from the post-induction time of 1

hour and 3-fold from the post-induction time of 2 hours. Overall, the protein produced

at cultivation temperature of 20 ◦C were consistent after post-induction of 4 hours.

The amount of protein induced at 25 ◦C are examined by SDS-PAGE analysis

in Figure 4.7, and the amount of soluble protein was quantified by ImageJ software

in Figure 4.8. The greatest amount of soluble-expressed protein was observed at 4

hours post-induction with protein concentration of 437.8 µg/ml. Compare to other

post-induction time, significant increase in amount of soluble protein was identified.

From post-induction of 1 hour, the protein concentration was reduced by 2-fold at

post-induction time of 2 hours. The amount of soluble protein was increased by 4-fold

thereafter. The significant reduction on protein expressed in soluble form was observed

at 6 hours and overnight growth after the induction. At 30 ◦C, the largest amount of

soluble protein was obtained at post-induction time of 6 hours. The total amount of

soluble recombinant protein were gradually increased from the induction with shifted

temperature.

The patterns of protein produced at 37 ◦C were different from those produced at

28

20 and 25 ◦C. The smallest amount of soluble protein were produced at 37 ◦C after

post-induction time of 1 hour. The concentrations were increased by 6-fold after post-

induction time of 2 hours and 3-fold after post-induction time of 4 hours. Comparably

consistent amount of soluble protein were produced for 6 hours and overnight growth,

respectively. The analyzed results are summarized in Table 4.1.

Overall, the largest soluble protein were produced at induced temperature of 20 ◦C

with cultivation time of 6 and 4 hours, respectively.

29

Figure 4.7: SDS-PAGE images for soluble recombinant 30Kc19α-Runx2

30

Figure 4.8: Total amount of soluble recombinant 30Kc19α-Runx2

31

Table 4.1: Total quantity and soluble fraction of recombinant 30Kc19α-Runx2

Group Soluble fraction (%) Total amount (µg/ml)

20 ◦C, 1h 42.1 ± 2.71 285.8

20 ◦C, 2h 46.3 ± 3.29 167.8

20 ◦C, 4h 76.2 ± 1.29 442.7

20 ◦C, 6h 59.9 ± 0.15 404.3

20 ◦C, O/N 58.9 ± 0.19 445.7

25 ◦C, 1h 30.3 ± 10.9 188.7

25 ◦C, 2h 50.1 ± 1.22 102.8

25 ◦C, 4h 59.5 ± 12.5 437.8

25 ◦C, 6h 53.1 ± 0.18 46.6

25 ◦C, O/N 44.7 ± 1.36 55.3

30 ◦C, 1h 48.4 ± 0.44 149.5

30 ◦C, 2h 52.1 ± 1.19 279.3

30 ◦C, 4h 62.3 ± 0.40 239.3

30 ◦C, 6h 73.8 ± 0.44 418.8

30 ◦C, O/N 65.3 ± 1.29 356.2

37 ◦C, 1h 50.8 ± 11.24 17.6

37 ◦C, 2h 24.3 ± 3.10 98.3

37 ◦C, 4h 28.9 ± 5.16 61.1

37 ◦C, 6h 35.7 ± 2.47 35.8

37 ◦C, O/N 43.3 ± 2.46 54.7

32

4.5 Expression and purification of 30Kc19α-Runx2 protein

Recombinant 30Kc19α-Runx2 protein was expressed by varying the cultivation

temperature and time condition to enhance the soluble expression. The results from

the optimization test indicated that induced cultivation temperature of 20 ◦C at induced

cultivation time of 4 hours shows highest soluble fraction (%) and produces relatively

high amount of soluble protein. Therefore, 30Kc19α-Runx2 was further expressed at

scale of 4 L culture and purified using FPLC (Figure 4.9). After purification, the con-

centration of produced protein was calculated using a series of BSA standard solution

with 400, 200, 100, and 50 µg/ml. Based on the standard solution, 48.64 µg/ml of pro-

tein was produced. Then, western blot analysis was performed to identify 30Kc19α-

Runx2 protein, and the band of 71.2 kDa was observed (Figure 4.9).

33

Figure 4.9: Expression and purification of 30Kc19α-Runx2

34

Chapter 5

Discussion

This study examined an optimized production condition with varying temperature

and time after IPTG induction. The key indicators selected from the result of cultiva-

tion were soluble fraction and amount of soluble recombinant protein produced. The

SDS-PAGE was used to analyze the results. The results from experiments proposed

that the highest soluble fraction and largest amount of soluble protein was expressed

when manufacturing 30Kc19α-Runx2 recombinant protein with reduced temperature

of 20 ◦C and production of 4 hours in 100 mL flask scale. On the other hand, the least

amount of soluble expression and fraction was observed when cultivated at 37 ◦C after

IPTG induction. The soluble fraction was increased in proportion for 4 hours at 20, 25,

and 30 ◦C. Interestingly, the soluble fraction at 37 ◦C was reduced at post-induction

time of 2 hours and gradually increased with post-induction time of 4 hours.

There are multiple contribution factors in producing recombinant protein that have

direct influence on cellular metabolism [19, 20]. To minimize differences between

cultured condition, the media volume, media composition, flask size, shaking speed,

and mixing time were kept constant. The concentration of IPTG used were reduced to

0.1 mM, compare to previous studies on recombinant protein production [12]. Indeed,

IPTG was triggered for induction of protein expression at optical cell density of 0.5.

The target protein is transcribed by active T7 RNA polymerases. Thus, reducing the

35

concentration of IPTG decreases the mRNA expression, causing less probability of

protein aggregation [21]. Islam et. al., reported that induction of IPTG at high con-

centration prevent the cellular growth [22]. Larentis et. al., examined that high IPTG

concentration further imposes negative effect on soluble expression of the protein [23].

The IPTG induction of 0.1 mM is in align with previous studies, in which the reduced

concentration enhanced the expression of protein in soluble form.

The lowered culture temperature to 20 ◦C has resulted in both increase in fraction

and amount of soluble recombinant protein. In previous studies, it was identified that

lowered growth temperature often lead to reduced formation of aggregated IBs protein

by minimizing the rate of the protein synthesis, thereby affecting the stress on the cell

and decreasing the hydrophobic interaction rate between adjacent polypeptide [24,

25]. However, the lowered temperature have potential to cease the protein production

and decrease the total protein expression [26]. On the contrary, the over-production of

protein imposes IBs formation in media, leading to the metabolic burden to the cell

[24]. Ifollia et. al., has reported that the most IBs are observed at fastest growth rate

where the less abundant of IBs are observed at slowest growth rate [27]. Therefore, it

was hypothesized that reduced temperature with longer production will enhance the

soluble expression of recombinant 30Kc19α-Runx2 protein.

Theoretically, and based on the results of this study, the fastest growth rate are ob-

served at induced temperature of 37 ◦C, and the least amount of soluble protein was

observed at 37 ◦C. The main factor contributed with these results are increase of IBs

formation. The amount of soluble protein after 6 hours for 25, 30, and 37 ◦C do not

have significant difference regardless of temperature shift as IBs are formed in propor-

tion to overall cellular growth. The protein produced from 20 ◦C with post induction

of 4 hours provided the most significant results. Although the post-induction time for

overnight growth produces the largest amount of protein, the increase in amount of

soluble protein produced in fraction to total amount of protein produced were high-

est at 4 hours post-induction. Therefore, the production at 4 hours post-induction is

36

favorable at production temperature of 20 ◦C considering the energy consumed with

efficient manufacturing of recombinant protein.

The western blot results showed two protein bands. One band is observed at 71.2

kDa, which is theoretical size of recombinant 30Kc19α-Runx2 protein. Another band

observed was located at 61.1 kDa. Because His-tag antibody was used for this study,

small size of band was considered as degraded forms of protein. Interestingly, the

same sample loaded on gels for coomassie blue staining had single band at 71.2 kDa.

Regardless, the band in 61.1 kDa in coomassie blue staining is negligible considering

the relative intensities between two bands.

The development of large-scale production of recombinant protein requires a un-

derstanding of production condition and key operating variable in bio-pharmaceutical

manufacturing. This problem was investigated in laboratory 100 ml scale in this study.

The lab-scale experiment, flask culture or shaking bioreactors, are widely acquired

in bio-industry as a representative screening system for developing new biopharma-

ceuticals or bio-technical process due to its great simplicity and cost effective nature,

with ability to provide high volume of experimental data [26]. On the contrary, the

large scale experiment is performed to produce drug product for industrial purpose

[28, 29]. This study examined the potential application to the production in large scale

for industrial purpose by enhancing the amount of soluble protein produced. Within

the manufacturing process, there are various process parameter that can effect produc-

tion quality and attributes. The most well-known process parameters are temperature,

pH, dissolved oxygen, volume of media, buffer, agitation speed, air, oxygen, nitrogen,

and carbon di-oxygen [24, 29]. Amongst these process control parameters, cultiva-

tion temperature and time after induction were optimized for production of 30Kc19α-

Runx2 protein. The further studies will be needed to examine the roles and ranges of

process controlled parameters, by applying regulated fed batch to reduce the impact

from environmental factors [28]. Finding optimal condition for recombinant protein in

large scale production takes usually months to a year [29]. Hence, the Optimization

37

of production condition has potential to reduce optimization period when applying for

industrial production.

38

Chapter 6

Conclusion

This study was designed to suggest the best cultivation time and temperature af-

ter IPTG induction for producing soluble recombinant 30Kc19α-Runx2 protein. The

soluble fraction was proportion to growth period at cultivation temperature of 20, 25,

and 30 ◦C for 4 hours post-induction. The production temperature at 37 ◦C provided

large amount of inclusion body where the least soluble recombinant protein were pro-

duced. The cultivation temperature at 20 ◦C with cultivation time of 4 hours after

IPTG induction provided the most amount of soluble protein considering the total

amount of protein produced. This suggests that temperature shift to 20 ◦C after IPTG

induction and harvest after 4 hours provided sufficient amount of soluble protein, and

a prolonged cultivation time is meaningless. Finally, 30Kc19α-Runx2 proteins were

expressed with optimal condition, and purified proteins were observed the theoretical

size of protein in western blot analysis. Therefore, we concluded that the optimized

process to express 30Kc19α-Runx2 in E. coli provided increased amount of soluble

recombinant protein.

39

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초록

치료용 단백질을 포함한 바이오 제약 시장이 빠르게 성장함에 따라 효율적인

치료용 의약품에 대한 생산 수요가 크게 성장하고 있다. 본 연구에서는 30Kc19α-

Runx2재조합단백질의수용성발현을위한생산공정을최적화하였다.총 20개의

실험그룹이지정된생산프로토콜에따라분석되었다.제조된 pET-23a / 30Kc19a-

Runx2 plasmid 를 E. coli 에 도입 후 37 ◦C 에서 배양하였다. 수용성 단백질 발현

조건을조사하기위해, IPTG induction후온도 (20, 25, 30, 37 ◦C)와배양시간 (1, 2,

4, 6시간,그리고하루)을조절하여 E. coli에서생산하였다.각조건은 SDS-PAGE

와 Coomassie blue staining 을 통해 분석되었다. 수용성 단백질량은 20, 25, 30 ◦C

조건에서 4시간 발현했을 때 증가하는 추세를 보였다. 전체 샘플에 포함된 불용성

단백질과비교했을때 20 ◦C온도조건의 4시간배양한그룹에서가장높은비율로

수용성단백질이발현되었으며,단백질량또한 20 ◦C에서 4시간배양한그룹에서

가장높게발현되었다.결론적으로수용성단백질발현은 20 ◦C에서 4시간에서발

현했을때가장높은비율과양으로발현되었다.산업에서해당단백질을생산할때

다양한조건중일부를최적화하였으며,이러한최적화과정을통해수용성단백질

의생산량을늘릴수있을것으로기대된다.

주요어: 치료용 약품, 바이오의약품, 30Kc19α-Runx2 재조합 단백질, 수용성 발현,

공정최적화

학번: 2018-27514

44