Pharmabt_report.docx

NATIONAL UNIVERSITY OF HO CHI MINH CITY INTERNATIONAL UNIVERSITY

LABORATORY REPORT PHARMACEUTICAL BIOTECHNOLOGY LECTURER: DR. NGUYỄN HOÀNG KHUÊ TÚ

Group members: Trần Vũ Minh - BTBTIU13372 Phan Chí Thiện - BTBTIU13182 Vũ Nguyễn Thanh Tùng - BTBTIU14269 Lưu Tấn Lợi - BTBTIU13102 Nguyễn Anh Vũ - BTBTIU13240

TABLE OF CONTENTS Lab 1: UPSTREAM PROCESSING (BIOPROCESS TECHNOLOGY) Lab 2: DOWNSTREAM PROCESSING: CELL EXTRACTION Lab 3: DOWNSTREAM PROCESSING: CONCENTRATION AND DIALYSIS Lab 4: SDS - PAGE ELECTROPHORESIS ANALYSIS AND BLOTTING Lab 5: FORMULATION AND PREPARATION FOR PHARMACEUTICAL PRODUCTS Lab 6: PREPARATION OF KILLED AND LIVE ATTENUATED MICROORGANISMS FOR VACCINE

LABORATORY 1: UPSTREAM PROCESSING (BIOPROCESS TECHNOLOGY) I.

AIM After transforming the host cells with target gene and obtaining the positive cells (cells that bare the recombinant construct), it is required that the cells are cultured to an optimal condition and induced to overexpress the recombinant protein; that is the aim of this practical. The host used in this practical is the E. Coli BL21 (DE3) strain which is a strain that utilizes a two-promoter system. In details, the targeted gene is inserted into the region regulated by a T7 (from bacteriophage) promotor that is only recognized by T7 polymerase and T7 polymerase gene is regulated upstream in another region by a Plac promoter. So that, IPTG will induces the transcription and translation of T7 polymerase which in turn, induces expression of the targeted gene. In addition, the BL21(DE3) strain also contains a Ribosomebinding-site in its plasmid to ensure higher expression.

II.

PROCEDURE The principal steps of this practical is as followed, although there are a certain of steps that have been prepared in advance by Ms. Thu (the teacher assistant). All of these steps below are repeated with the control sample, which is the BL21(DE3) strain without the targeted gene insertion. 1. We inoculate the transformed colony with the recombinant construct in 1 ml LB containing 50 µg/ml ampicillin in order to selectively grow only the recombinant strain (BL21(DE3) has AmpR gene; culture is incubated at 37oC overnight for the culture to be saturated. (Prepared in advance) 2. Next, take 100 µl of saturated into 10 ml of LB with 50 µg/ml ampicillin in a 200 ml flask. Incubate at 37oC until reach OD 0.5 – 0.6 at 600 nm. (Prepared in advance) 3. The culture was then incubated at 25-28oC with aeration for 2 hours. After induction, the culture was transferred to 50 ml falcon. 4. It was then centrifuged at 8000 rpm for 25 minutes at 4oC. Pellets were collected because our protein is intracellular protein for later sections. 5. Put 1mL of PBS and mix well by pipette. Transfer the mixture to new eppendorf. Then centrifuge 13000 rpm, 10 minutes, 4oC. 6. NOTICE: pellet and supernatant are separately collected for downstream analysis.

III.

RESULTS Both the sample and the control show no complication during this practical due to the fact that there is no current quality control in between. The results of this practical are discussed in LAB 2 part of this report.

IV.

DISCUSSION This part will be the answers to the questions and comments raised in class.  

First, the ampicillin concentration in LB can vary from 25 µg/ml to 100 µg/ml and the host cells can be selected and grow normally. Here, we decided on 50 µg/ml. Next, the OD of 0.5 – 0.6 at 600nm is equivalent to the culture reaching the log phase. For expression of recombinant proteins, optimal expressions are observed when the cells are at the most bioactive phase of growth, that is, the log phase.



Harvesting cells later than the log phase may observe low protein yield and high protein degradation. That is also the reason why we must discard and redo if our culture OD passes the OD range of 0.5 – 0.6 at 600nm. IPTG induces the expression of T7 polymerase which expresses targeted protein. Optimal IPTG concentration depends mainly on the vector and the targeted protein. IPTG concentration if too low can lead to under-expression hence low yield; too high can lead to over-expression and kill host cells hence limit the optimal yield. So it is required that the experimenter try with difference concentrations of IPTG (normally ranged from 0.5 mM to 1.0 mM) and optimize for the optimal expression.

LABORATORY 2: CELL EXTRACTION I.

AIM With the right means (incorporation of secreted signal of extracellular proteins), recombinant protein can be extracellularly recovered. However, in the case of our practical, the recombinant proteins are found intracellularly, therefore, it is required that the cells must be broken down to obtain the protein of interest. There are numerous downstream processing steps for the recombinant protein, however, this part will only cover the cell extraction. To be more specific, the method we used is purely mechanical, it involves the use of lysozyme and glass bead in combination with grinding to lyse the cell wall. Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan, which is the major component of gram-positive bacterial cell wall. This hydrolysis in turn compromises the integrity of bacterial cell walls causing lysis of the bacteria. Glass beads is a physical factor that assists lysozyme to effectively break down the host’s cell wall.

II.

PROCEDURES The basic steps are as follow, this is also the same for the sample and the control. 1. Collect the pellets from the first session. 2. Weigh them by weighing the empty eppendorf first, then weigh the eppendorf with pellet and obtain the pellet’s weight. 3. After that, mix the pellet with lysozyme and glass beads with the ratio of 10 : 1 : 1 accordingly. Add a very small amount of PBS and mix well. 4. Grind with the provided grinder for 30 minutes to 1 hour on ice. Note that the color of the grinder may come off and that is not an issue. 5. Add PBS with the ratio of 1/10 (weight/volume) 6. Mix well and centrifuge 13000rpm, 10 minutes at 4oC. 7. Collect supernatant, use 50 µl for SDS-PAGE confirmation of targeted protein and store the rest for downstream processing at -20oC.

III.

RESULTS Sample (0.05g) Control(0.04g) Lysozyme 0.005g Lysozyme 0.004g Glass beads 0.005g Glass beads 0.004g Table. The amount of lysozyme, glass beads for the tubes

IV.

DISCUSSIONS With mechanical methods, it is required that the cells are always on ice. This is to maintain the conformation and activity of the recombinant proteins inside the cell. According to the mentor, mechanical methods have the advantages of improving experimental skills, understanding the underlying mechanisms and in some instances, having higher efficacy compared with commercial kits.

LABORATORY 3: CONCENTRATION AND DIALYSIS

I.

AIM Concentration  To precipitate protein for X-ray crystallization to study its 3D structure and functions, and for later purification  To get high concentration of recombinant protein product Dialysis 

II.

To remove salt out of the extractions so that we can keep the protein for long time

PROCEDURE 1. Intracellular protein recovery  Transfer 10 µL of cell extract from lab 1 to a new labeled Eppendorf for dialysis  Use micropipette to measure the remained cell extract:  Set the micropipette to 100 µL  Transfer 10 µL of cell extract to a new labeled Eppendorf  Keep pipetting until the volume of cell extract in the Eppendorf below 100 µL (air bubble forms when sucking)  After transferring, the volume of used sample and control cell extract is 300 µL and 200 µL, respectively  Go to http://www.encorbio.com/protocols/AM-SO4.htm to calculate the amount of ammonium sulfate needed to get 40% saturated solution  Setting and results are in table 1  Weigh 0.07 g and 0.05 g ammonium sulfate with electrical balance, then add to the corresponding Eppendorf of cell extract  Vortex the Eppendorf gently until ammonium sulfate dissolve completely  Centrifuge at 12,000 rpm for 15 minutes at 4oC  Use micropipette to transfer the supernatant to a new labeled Eppendorf  Calculate the amount of ammonium sulfate needed to get 80% saturated solution  Setting and results are in table 1

Sample 40% 80% 0.3 0.34

Control 40% 80% 0.2 0.23

Starting volume of solution in ml Desired percentage saturation Ammonium 40 80 40 Sulfate Starting percentage saturation of 0 40 0 Ammonium Sulfate Amount of ammonium sulfate (g) 0.07 0.1 0.05 Final volume (ml) 0.34 0.39 0.23 Table 1 Setting and results of ammonium sulfate calculation

Setting

Results

    

80 40 0.07 0.27

Weigh 0.39 g and 0.27 g of ammonium sulfate with electrical balance, then added to corresponding Eppendorf Vortex the Eppendorf gently until ammonium sulfate dissolve completely Centrifuge ate 12,000 rpm for 15 minutes at 4oC Discard the supernatant with micropipette Resuspend the obtained pellets with 1 mL of PBS (pH7.4)

2. Extracellular protein recovery  Transfer each 1 mL of culture broth to 2 Eppendorf  Centrifuge the Eppendorf at 12,000 rpm for 10 minutes at 4oC  Collect the supernatant  Add ammonium sulfate for 80% saturation (weighed and given by TA)  Vortex gently until ammonium sulfate dissolve completely  Centrifuge the Eppendorf at 12,000 rpm for 10 minutes at 4oC  Discard all supernatant  Resuspend the obtained pellet in 1 mL of PBS III.

RESULT 1) Evaluate the result of cell extraction step (lab 1) The extracted protein solution should have brown color. If the obtained solution has no color, then it is too diluted or has no protein at all. The cell extract solutions of our group (figure 1) had blur brown color. Since the solutions were nearly transparent, the amount of protein we obtained was too low. Figure 1 Cell extract solution (Left: sample, right: control)

2) Why do we stir or vortex the Eppendorf after adding ammonium sulfate? Because we want ammonium sulfate dissolve completely in the protein solution. The principle of this step is that NH4+ and SO42- ions will compete with water molecules, leaving proteins interact with each other by hydrophobic interaction. The protein molecules will form clumps and become separated from the liquid. If we do not vortex the Eppendorf, then most of salt will settle down to the bottom of the tube and the

remained ammonium sulfate in the solution is not enough to precipitate the protein. 3) Why do we add ammonium sulfate for 40% saturation first? In the cell extract solution, there are other proteins beside the targeted one. Since all proteins are extracted by a same principle, we must first precipitate the unwanted protein by adding ammonium sulfate 40% to get rid of contaminating our products. 4) Why do we collect supernatant after adding AS for 40% but collect pellet after adding AS for 80%? After adding AS for 40%, the unwanted proteins will be precipitated and our target is still in liquid phase, so we need to collect the supernatant. By adding AS for 80%, we transfer the target protein to the solid phase in the pellet which is collected after this step. IV.

DISCUSSION Talking about the result of lab 1, the cell extract quality of our group was not quite good since they had a pale brown color, meaning that the concentration of target protein in the solution was too low. Since the obtained solution from other groups who used the same chemicals, samples, and applied the same protocol had high concentration of intracellular protein, the quality of lysozyme as well as the expression level of bacteria cannot account for our unsuccessful outcome. Therefore, the low protein concentration is probably due to the cell disruption step. By an incorrect calculation, we had weigh the glass beads that was beyond the needed amount. Fortunately, our mistake had been corrected before lysozyme was added, but it more or less affected our result. Besides, we might not grind the cells enough, so they were not disrupted completely and not all proteins were released to the cell lysate. Furthermore, the grinding step took a lot of time, so the protein might degrade or denature in the meantime. In short, all three above reasons contributed to the nearly failed product of our group.

LABORATORY 4: SDS-PAGE ELECTROPHORESIS ANALYSIS 1. INTRODUCTION Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, abbreviated as SDS-PAGE, is an experimental technique utilized for qualitative and quantitative protein concentration. Specifically, the technique measures the presence and concentration of the protein. 2. PRINCIPLE The principle of this type of electrophoresis relies on the denaturation of proteins into primary structure, thereby allowing the proteins to separate according to specific molecular size. Additionally, the molecular weight of the protein is measured based on the protein ladder designed by biotechnology companies. 3. PROCEDURE 3.1. MATERIALS Acrylamide

Bis-acrylamide

Glycerol

Tris 0.75M (pH 8.8)

Tris 0.25M (pH 6.8)

TEMDED

Ammonium per sulfate

Glycine/Tricine

SDS

Trizma

SDS-PAGE electrophoresis apparatus 3.2. METHODS 3.2.1. RESOLVING GEL PREPARATION

3.2.2. STACKING GEL PREPARATION

Tris HCl (pH 8.45) 5ml

Tris HCl (pH 6.8)

1.55ml

Acrylamide

5ml

Bis-Acrylamide

0.8ml

TEMTED

14μl

TEMED

14μl

APS

14μl

APS

14μl

Glycerol

1.5ml

H2O

5ml

H2O

3.5ml

TEMPTED and Acrylamide must be added at last to polymerize the mixture, which turns into the gel. 3.2.3.

SAMPLE PREPARATION

The sample is mixed with sample suffer. Then the mixture is heated for 5 minutes at 95C. Next, the sample is loaded into wells in the following order: Protein Ladder – Crude control – Control 80% - Crude Sample – Sample 80% After loading into the gel, the electricity of 100V and 80A is set up to run the gel.

3.2.4.

STAINING AND DESTAINING

The gel is stained with Coomassive Blue and destained later. 4. RESULT

There are 10 wells in total in this gel. From the bottom to the top, the first well (well 1) is the protein ladder. Our group’s wells are in the following orders: Well 3: Crude Control

Well 4: Control with 80% ammonium sulfate

Well 5: Crude Sample

Well 6: Sample with 80% ammonium sulfate

5. CONCLUSION According to the result, there are no target proteins visualizing in the gel. This issue could be explained by the following points: + The technique is inappropriate during the labwork such as collecting pellet, resuspending cells + Cell extraction is not performed thoroughly, which leads to the loss of proteins.

6. DISCUSSION What is the function of resolving gel and stacking gel? _ Stacking gel allows the proteins to gather in the same line for several minutes at initial stage of electrophoresis prior to entrance into resolving gel. Thus, the bias of some proteins entering the resolving gel faster than the others is decreased maximally, thereby increasing the accuracy of the

experiment. The principle relies on the low concentration of acrylamide (7% yielding large pore size) and low pH (6.8) for ion Glycine and ion Chloride to sandwich the proteins. _ Resolving gel allows the proteins to separate according to their specific molecular weight. The principle relies on the small size pore thanks to Tris HCl pH 8.45. Why is there a difference in pH between stacking gel (6.8) and resolving gel (8.45)? _ The pH 6.8 of Tris HCl makes chloride negatively charged and glycine neutral, thereby the glycine moves slowest and chloride moves fastest. That way, the proteins is held between glycine and chloride and pushed down into the resolving gel. The line with intense blue color at the top indicates the proteins are concentrated in the same line. _ The pH 8.45 of Tris HCl makes both chloride and glycine negatively charged, thereby allowing the glycine to move as fast as chloride and the protein is separated based on different molecular weight and small pore size of the resolving gel. What is the function of Acrylamide? _ Acrylamide conducts polymerization by combining acrylamide molecules into a long single chained polymer, forming linear polymers or polyacrylamide. Acrylamide polymerization is induced by APS. What is the function of Bisacrylamide? _ Bisacrylamide cross links between linear polymers formed from Acrylamide. The more number of cross links, the smaller the pore size. What is the function of Sodium Dodecyl Sulfate (SDS)? _ SDS is used to denature native proteins into poplypeptide by disrupting noncovalent interaction. Alternatively, this detergent linearizes the protein structure so that proteins could be separated by only molecular weight. What is the function of 10% Ammonium persulfate (APS)? _ APS is utilized along with TEMPED to speed up and induce the polymerization of acrylamide and bisacrylamide monomers. What is the function of TEMDED (Tetramethylethylenediamine)? _ Similar to APS, TEMDED induces the polymerization of acrylamide and bisacrylamide.

BLOTTING 1. INTRODUCTION The Western blotting is conducted in this experiment to identify the target protein, thereby displaying whether the target protein is present on SDS-PAGE electrophoresis in case of blurry protein band. 2. PRINCIPLE The principle relies on the transfer of the protein band to the PVDF membrane through electric current conducted by the buffer.

3. PROCEDURE 3.1.1.

MATERIALS

Gel

Anode buffer I

PVDF membrane

Anode buffer II

Foreceps

Cathode

Gloves

Methanol

Distilled water

Tris base

Glycine

Trans-blot

3.1.2.

METHODS

_ Prepare the membrane and filter paper which the size is the same as that of gel. _ Soak the membrane into methanol _ Drain the excess liquid on the membrane. _ Soak one filter paper in anode buffer I and the other filter in anode buffer II _ Soak two filter paper in cathode buffer _ Air must be eliminated in order not to interrupt the electric current _ Put all the materials in the blot sandwich and run the blot with 50V for 30 minutes. 4. RESULT As the semi-dry Western Blotting failed, there was no picture taken for display. 5. CONCLUSION The reasons leading to the failure of the blotting are as follows: + The filter paper was dry for a long period of time before putting in the Western machine. + Production of bubble causes the interruption of electric current. + The blotting was running for an excessive amount of time + The size of gel and filter is not prepared equivalently to one another. 6. DISCUSSION What is the function of Methanol? _ The methanol is added to induce the binding affinity of the proteins to the membrane. What is the function of Anode buffer I? _ As a number of excessive protons are generated on the surface of anode plate during blotting, Anode buffer I is utilized to neutralize those excess protons.

What is the function of Anode buffer II? _ The anode buffer II has the same composition and function as anode buffer I, except the concentration is reduced to 25mM. What is the function of Cathode buffer? _ The cathode buffer contains e-aminocaproic acid serving as the trailing iron during the transfer and is reduced from the cathode buffer when it migrates through the gel toward the anode. That way, the binding of proteins is increased up. Why do we use Cathode and Anode buffer separately? _ Anode buffer and Cathode buffer facilitate the efficiency of transfer or blotting by having different buffers at the anode and the cathode.

LABORATORY 5: FORMULATION AND PREPARATION FOR PHARMACEUTICAL PRODUCTS I. INTRODUCTION - Pharmaceutical formulation, in pharmaceutics, is the process in which different chemical substances, including the active drug, are combined to produce a final medicinal product. The word formulation is often used in a way that includes dosage form. - Formulation studies involve developing a preparation of the drug, which is both stable and acceptable to the patient. For orally taken drugs, this usually involves incorporating the drug into a tablet or a capsule. It is important to make the distinction that a tablet contains a variety of other potentially inert substances apart from the drug itself, and studies have to be carried out to ensure that the encapsulated drug is compatible with these other substances in a way that does not cause harm, whether direct or indirect. - The aim of this study is to formulate and compounding Cucumin cream as well as understand the pharmaceutical formulation and check the quality of the product. II. MATERIALS and METHODS Cucumin cream is manipulated according to the formulation: Albumin (10 mg/ml)

: 10 µl

Cucumin powder

:1g

Vaseline

: 10 g

Tween 80

:1g

Material: Glass rods, Motar, Bottle, Scissor, Beaker, Distilled water, Vaseline, Tween 80 and Pipette set Methods: Cucumin Cream - Weigh vaseline and tween in the separate containers. - Drop 10 µl albumin solution into motar. - Add tween into the solution slowly. - Add curcumin powder into the mixture in motar and mix well. - Add vaseline into the mixture slowly and mix well. - Check the quality of the preparation. • Check color, appearance, homogeneity • Quantification of protein concentration in the product • Check the stability of protein • Check microbial limitation • Check hypersensitivity III. RESULTS and DISCUSSION

-

For the formation of cream, it was more difficult to mix the cucumin into the Tween 80. Therefore, the mixture was then slightly scurfy until the condensation was observed.

-

After mixed, the final mixture had fresh yellow color from the color of the cucumin.

-

For this kind of form, it was easier to test the quality analysis. By eyes checking, our product seemed to be very beautiful and homogenous. By testing onto skin of several students, the results shown that the cream was really good that no one got allergy or any reactions

-

For checking microbial limitation, we test the 2-week sample in agar gel at room temperature. After 24 hours, there are not any microorganism which grown in agar

-

For check the stability of protein and the quantification of protein concentration in the product, we use SDS page and blotting to test. The result seem to be disappointed because there is nothing in membrane That should have been two bands (including cucumin and albumin) in the membrane. The reasons can come from the quality of cucumin and albumin in the sample (they can be too easy to degrade after 2 week) or wrong SDS/blotting technique or problem with probe (loses its activity, resulting in a complete lack of any signal)/membrane/electrical condition (the proteins cannot bind to membrane).

Figure 5.1. The completed mixture

Figure 5.2. The weight of preparation cream after mixing (8.129 g)

Figure 5.3. The membrane after blotting

Figure 5.4. The preparing sample procedure

LABORATORY 6: PREPARATION OF KILLED AND LIVE ATTENUATED MICROORGANISM FOR VACCINE I.

II.

INTRODUCTION  Purpose: This supplies the primary steps for preparation of killed or live attenuated vaccinein. MATERIALS AND METHODS  Materials and Chemical Reagents Pathogen microorganism (Streptococcus); Lysogeny broth (LB); LB agar; Formalin; PBS (pH 7.4); Centrifugator; Test tubes; Incubator; Petri dises; Pipet set.  Methods

Streptococcus was prepared before class. It was grown to nearly stationary phase (optical density = 0.80 at wavelength of 600 nm). Firstly, we pipetted 100µl of the culture into an eppendorf, which contained 900µl of LB medium, to dilute the culture 10 times, mixes well then pipetted 100µl of 10 times diluted culture into an eppendorf (contained 99µl of LB medium) to dilute the culture 10 4 times, mixes well. Following the previous steps we prepared 106 times and 107 times dilute culture then transfered 100µl in diluted eppendorf into a petri disk contaning LB agar and culture overnight in 370C. Finally, we counted the CFU the next day. Next, we pipetted 100µl of the stock culture to three eppendorfs, centrifuged in 5 mins with 1200 rpm at room temperature and collect the pellets then discarded the supernatant. After that, we added 0.07% formalin into each eppendorf and vortexed in 30, 60, and 90 mins for each eppendorf, respectively. Then we centrifuged in 10 mins with 1200 rpm at room temperature, collect pellets and discarded the supernatants. Next, we added PBS into eppendorf to wash the pellets and this step repeated two times. After that, we vortexed and then centrifuged in 10 mín with 1200rpm at room temperature. After that, we added 100µl PBS into each eppendorf and resuspended the pellet with PBS. Finally, we transferred 100µl into a petri disk contaning LB agar and culture overnight in 300C - 370C then counted the CFU the next day. III.

RESULTS

From the left to the right: 1st is 60 mins petri disk, 2nd is 90 mins petri disk, 3rd 30 mins petri disk, and the last one is 10-7 petri disk

CFU formula: 𝐶𝐹𝑈 =

number of colony on agar surface x dilution factor volume of culture plate

Bacterial culturing with 10-7 dilution has 12 colonies Counting CFU of culturing plate with different dilution factor as following: Dilution factor 10-7 has cfu per microliter as: 12∗10^7 = 1,200,000 (bacteria/µL) 100 IV.

DISCUSSION

The meaning of dilution from the stock to 10-7 is to let the growth of colonies separate from each other and easy for counting the number of them on the petri disk. We vortexed three ependorfs after putting formalin to kill all the bacteria in 30, 60, and 90 mins to make sure the bacteria died. In addition, PBS solution was added to wash all the bacterial spores out. The result after spreading the dyed bacteria in the petri disks is not what we expected because of the growth of some bacteria inside. The reason is may be when we transferred the solution into the petri disks, it was contaminated by the bacteria in the atmosphere. Moreover, the table used to transfer was contaminated by some bacteria and chemicals. In the process of adding chemicals like PBS, or time of waiting for Formalin killing bacteria is not enough so they lead to the growth of bacteria.