Report on Lactate Dehydrogenase (LDH)

Abstract of Lactate Dehydrogenase (LDH)

Lactate Dehydrogenase (LDH), is a tetrameric enzyme composed of two subunits LDHA and LDHB. LDH primarily functions to convert pyruvate into lactate and nicotinamide adenine dinucleotide (NADH) to NAD⁺. LDHA is of interest as cancer cells metabolism utilizes LDHA to elevate rate of anaerobic glycolysis even in the presence of oxygen. Thus, LDHA is an attractive enzyme for studies to develop novel cancer specific therapies. In our experiment, it was essential to determine if cloned rLDHA from previous experiment was able to be expressed successfully while retaining its biological function of converting lactate to pyruvate. This was achieved by IPTG induction, enzyme assay, SDS-PAGE and western blotting. Through IPGT induction, it allowed T7 RNA polymerase to be expressed leading to rLDHA overexpression. Presence of rLDH was determined by enzyme assay through its catalytic activity of lactate to pyruvate. Result obtained was positive. To further characterized rLDH, SDS-PAGE was used and result showed a 36 kDa band which was approximately similar to known LDHA molecular weight of 35 kDa. Western blotting further confirmed SDS-PAGE result with signal amplification at 36 kDa band. Overall, expression and functionality of cloned rLDHA was checked for and with high confidence able to undergo downstream experiments, studying of protein structure and development of novel therapeutic agents.

1.0 Introduction of Lactate Dehydrogenase (LDH)

Lactate Dehydrogenase (LDH) is a tetrameric enzyme which converts Pyruvate to Lactate by reducing nicotinamide adenine dinucleotide (NADH) to NAD ⁺. LDHA is generally in the form of tetramer. It consists of almost 332 amino acids, which help it in formation of biolab structure. As the main function of LDHA is the conversion of pyruvate to lactate and to transfer NADH to NAD^+.  LDHA activate when substrate and cofactor attach. The activated site is present at the groove in between two domains will get close. Eventually, Arg 105 will grip pyruvate strongly in activated circles. On the other hand, hydrogen anion will get transfer from NADH ring to oxygen atom. LDHA is located in cytoplasm of the cell but sometimes it also been found in nucleus or mitochondria. In cytoplasm it plays its role in glycolysis but it works as DNA binding protein inside the nucleus where it also takes part in DNA duplication and transcription. (1) maintain glycolysis, (2) generate sufficient Adenosine Triphosphate (ATP) for energy when oxidative phosphorylation route is affected. LDH has 5 isoforms composed of LDHA (M subunit) and LDHB (H subunit). Distribution areas include: LDH-1(4H) in red blood cells and muscle, LDH-2 (3H,1M) in white blood cells, LDH-3 (2H,2M) in lungs, LDH-4 (1H,3M) in kidney, pancreas and placenta and LDH-5 (4M) in skeletal muscle and liver. Cancer cells metabolism utilizes LDHA to enhance aerobic glycolysis even in presence of oxygen for rapid cell proliferation (Kim, 2006). The metabolism process in cancer cells is quite different from the non-transformed cells. This help them to get excess of biomaterials and energy for much proliferation. Reprogrammed metabolism helps in resisted cell death and some more malignant characters. If the glycolysis is accelerated it will help in some important aspects of cancer metabolism. Accelerated level of glycolysis can help biomolecules present in cell by supplying them with precursors. Glycolysis involve the production of lactate which contribute on larger scale in the process of malignant progression such as replenishing NAD⁺ the purpose of glycolysis, enhancing immune escape or decrease in pH for invasion. Lactate dehydrogenase A work in converting pyruvate to lactate and vice versa. So, the LDHA activation has a relation with diverse cancer. In this way we can say that LDHA can act as a target for prevention and treatment of cancer. Thus, LDHA is of interest in cancer research to develop cancer therapeutic agents. For example, LDHA can be degraded by acetylation of at Lysine-5 through hypothesized chaperone-mediated autophagy (Zhao, 2013). Previous molecular cloning experiment confirmed rLDHA gene had a silent mutation hence, not impeding protein expression. As eukaryotic rLDHA was expressed in bacteria host, complications may arise, e.g. misfolding. Using the same pET11-H6α vector, the following interest was to express functional rLDHA, required for downstream experiments (e.g. studying of LDH structure to develop novel cancer therapeutic agents). To conclude with confidence that cloned rLDHA were functional, rLDHA overexpression, purification and characterization was done through: (1) IPGT induction, (2) specific enzyme assay, (3) SDS-PAGE and western blotting. If functional, LDH enzyme would convert pyruvate to lactate and vice-versa. rLDHA were first overexpressed to provide ample yield for subsequent experiments. BL21(DE3) E. coli utilized T7 expression system to overexpress rLDHA via strong bacteriophage T7 promotor. T7 promoter in pET11-H6α vector, caused BL21(DE3) E. coli cells to divert its resources to synthesis rLDHA proteins rather than its own endogenous proteins (control to maximize protein field). Log phase was optimum to express rLDHA where cells were the fittest, dividing exponentially and expressed chaperones to remove misfolded proteins (Betts, 2002). Upon IPGT induction, lac repressor was inactivated, allowing expression of T7 RNA polymerase facilitated by E. coli polymerase to occur and overwhelming T7 lysozyme inhibition produced by pLysS plasmid. Binding of T7 RNA polymerase to T7 promotor allowed overexpression of rLDHA (Kang, 2007). OD was measured before and after IPGT induction to ensure cells were at log phase and no cell death observed respectively. To obtained purified rLDH, BL21(DE3) E. coli cells were lysed with repeated cycle of freezing and thawing, disrupting cell wall through ice crystal formation. Lysis was aided by cytoplasmic T7 lysosome via degradation of cell wall. PMSF and Dnase 1 was added to inhibit activated protease and digest nucleic acid to removed viscosity respectively. Cells were centrifuged to pellet proteins to the bottom and cell debris to supernatant. To isolate purified rLDH, Ni-NTA affinity chromatography was used. Choice of correct buffer, ionic strength and pH was necessary to keep proteins soluble, protect integrity and maintained physiological pH for stability. pH 7.4 stock phosphate buffer was chosen to prepare Ni washing, binding and elution buffer. Ni²⁺ ions immobilized on nitrilotriacetic acid (NTA) sepharose resin had high affinity for rLDH due to 6x His-tag allowing rLDH to bind with higher affinity than non-target proteins via dative bonding of nitrogen atom in histidine molecules. Low concentration of Imidazole (washing buffer) was used to removed non-specific proteins followed by high concentration of imidazole (elution buffer) to elute purified rLDH. To test for rLDH successful expression, functionality and purity in purified fractin, protein assay coupled with specific enzyme activity assay were utilized. BSA protein assay allowed detection of protein presence through dye colour change from brown to blue. Protein absorbance were measured at 595nm and BSA protein standard curve was used to estimate unknown protein sample concentration (mg/ml) required for calculation of specific enzyme activity and total enzyme activity. Enzyme assay used NADH product formation as rLDHA identifier as it was distinguishable from other components of the reaction at 340 nm. Product formation (ΔA/min) was measured via spectrophotometer at 340nm over 3 minutes using L-lactate and NAD⁺ as substrate and co-factor respectively. Enzyme activity was calculated by Beer’s law: A = εbc. Specific enzyme activity was calculated to determine purity of rLDH after purification. Purification factor and percent yield were calculated to determine effectiveness of purification step and preservation of enzyme activity after purification respectively. SDS-PAGE was used as a semi-qualitative test to determine successful rLDH expression and purity through relative molecular mass, band intensity/thickness and number of bands in purified sample. If purification was successful, a single band of 35 kDa (known size of LDHA) would be observed. Successful overexpression was determined by comparing LDH band intensity between induced and non-induced sample. Proteins molecules were separated via application of electric field with smaller molecules migrating faster than larger molecules due to restrain from polyacrylamide gel pores. SDS being an anionic detergent function to confer negative charge to proteins and denature proteins to its primary structure. As charge-to-mass ratio are approximately same across SDS denatured polypeptides, separation of proteins was dependent on relative molecular mass (ThermoFisherScientific, 2020). Coomassie blue was used to stain and fixed protein bands. As dye binds stronger to proteins than gel matrix, it allowed removal of dye from protein free areas conferring blue stained protein bands for visualization. Western blotting was used to detect presence of rLDHA using specific antibodies. After SDS-PAGE, protein bands were electro transferred onto PVDF membrane. Milk proteins were added to prevent non-specific primary antibody binding to PVDF membrane, reducing signal-to-noise ratio. Indirect method with secondary antibody conjugated to horseradish peroxidase was used to detect rLDHA through signal amplification. Horseradish peroxidase substrate DAB and 4CN were added in conjunction to enhance visibility of rLDHA band. Overall, enzyme assay, SDS-PAGE and western blotting was used as triple conformational test to provide evidence for presence of LDH enzyme.

2.0 Results of Lactate Dehydrogenase (LDH)

2.1 OD readings of bacteria culture before and after IPTG induction

Table 1: Optical density reading recorded at 600 nm before and after IPTG inductionTable 1 description: Bacterial culture was allowed to attained OD₆₀₀ at 0.46 before 1mM IPTG was introduced. From table 1, OD₆₀₀ of 0.46 suggested that bacteria cells were in early log phase and ready for induction. Log phase was where cells were most active. From table 1, upon induction for 3 hr, bacteria culture OD₆₀₀ reached 1.78. This suggested that bacteria cells continued to grow and increase in population reaching mid-stationary phase.

2.2 Normalized average absorbance and protein concentration of fractions

Table 2: Normalized average absorbance of protein samples measured at 595 nm using plate reader.

Table 2 description: Sample fractions absorbance were normalised. 50x dilution for uninduced, induced and flow through and 2x dilution for purified fraction were selected to estimation of protein concentration (appendix). From Table 2, it was observed uninduced fraction had the highest absorbance followed by induced, flow through and purified sample. This was proportionate to protein concentration as seen in Table 2. However, induced sample contained lower protein concentration than uninduced sample as seen in Table 2 (discussed in…).

2.3 Purification summary table of fractions

Table 3: Purification summary table of fraction samples to estimate purity and presence of rLDHA.

Table 3 description: Sample fractions NADH product ΔA/min was measured (described in methods). Neat uninduced and flowthrough fraction and 50x dilution induced and purified fraction were chosen for assay. From Table 3, it was observed significant enzyme activity (nM/min) was present for all fraction except, uninduced sample. This suggested: (1) uninduced sample contained low levels of rLDH (2) presence of rLDH, due to NADH product formation observed. To determine rLDHA purity in fractions, specific enzyme activity (umol/min/mg) was calculated. From Table 3, it was observed purified fraction had highest specific activity of 120.3 μmol/min/mg while uninduced fraction had the lowest specific activity followed by flow through fraction. Enzyme activity and specific activity from Table 3 suggested: (1) purified fraction was of highest rLDH purity, with with 9.63 times more rLDH than induced fraction after Ni-NTA affinity chromatography. (2) Flow through fraction mostly contained junk proteins due to low specific activity compared to initial induced and purified fraction thus, column was efficient in isolating rLDHA to purified fraction. (3) Overexpression was successful due to huge increased specific activity in induced fraction compared to uninduced fraction thus, pLysS in BL21(DE3) E. coli bacterial cells was functioning to inhibit leaky expression of T7 RNA polymerase. Percentage yield was calculated to determine how well enzyme activity was preserved after Ni-NTA affinity chromatography. From table 3, it was observed that purified sample had a yield of 20.8 %. This suggested that there was a significant loss of rLDH activity. In addition, purification and flow through fraction did not add up to approximately 100 % yield.

2.3 SDS-PAGE of fractions for purity and presence of rLDHA

Figure 1 description: In semi-qualitative SDS-PAGE test, purified fraction was compared against other fractions to determine molecular weight, abundance and purity of rLDHA. Protein concentration of 4 μg was standardized among fractions. Results were presented in Fig. (1). It can be noticed from Fig. (1) lane 7 had a thick protein band of 36 kDa (green arrow) and reduction of protein bands after purification of initial induced fraction (lane 5). Removed protein bands can be observed in lane 6. This suggested (1) 36 kDa band was rLDHA due to co-migrating with positive control LDHA, (2) purified fraction was purer in rLDH after Ni-NTA affinity chromatography from induced fraction, (3) Column had effective binding due to minimal rLDHA present in flowthrough fraction as dull 36 kDa band (purple arrow) was observed in lane 6. It also can be noticed from Fig .(1), lane 5 had a brighter 36 kDa band (orange arrow) than lane 4 (blue arrow). This suggested after IPGT induction 36 kDa band protein molecules were overexpressed and inhibition by pLysS in BL21(DE3) E. coli bacterial cells was working. This provided further evidence 36 kDa band was rLDHA as IPGT induction was used to overexpressed rLDHA Positive control in lane 8 contained purified LDHA was used to determine if rLDHA had similar molecular weight. Co-migration of positive control LDHA band to 36 kDa (cyan arrow) suggested that purified sample 36 kDa band was most likely rLDHA. However, unknown protein bands (red arrow) were observed in lane 6 and intensity of lane 4 smearing were not consistent with lane 5 and 6 though, equal proportions of 4 μg proteins were loaded.

2.4 Relative mobility and estimated molecular mass of protein bands from SDS-PAGE

Table 4: Estimated molecular mass from protein ladder standard curve (refer to appendix). 

Table 4 description: Protein molecular weight estimated from standard curve of known protein standards using equation of straight line: y = -1.2449x + 2.4075. Suspected rLDHA molecular mass were computed based on relative mobility. From table 4, suspected rLDHA molecular mass was 36 kDa which was a close approximate to expected LDHA of 35 kDa. This provided further evidence 36 kDa band was highly probable to be of rLDHA. Unknown protein bands of lane 6 in Fig.1 molecular weight observed in purified sample were computed. (discussed in).

2.5 Western blotting of fractions for presence of rLDHA

Figure 2 description: In a semi-qualitative western blotting test, presence of rLDHA was detected using indirect detection method as described in methods section. Protein concentration of 4 μg was standardized among fractions. Intensity of signal amplification was compared between fractions. The results were presented in Fig. (2). It can be noticed from Fig. (2) only a single 36 - 37 kDa band had signal amplification. This confirmed 36 kDa band observed from SDS-PAGE was indeed rLDHA as antibody used were specific to rLDHA. It also indicated primary antibody was specific as no unspecific binding were observed. It can be noticed from Fig. (2), Lane 7 had high signal amplification, lane 4 had moderate signal amplification while lane 3 and 5 had low signal amplification. This indicated: (1) Purified fraction had highest rLDHA concentration as equal proportion of proteins (4 μg) were loaded, (2) rLDH were successfully overexpressed from uninduced to induced fraction after IPGT induction, (3) pLysS plamid in BL21(DE3) E. coli bacterial cells was working, (4) flow through fraction contained low levels of rLDHA due to majority of rLDHA being bounded to column (good binding efficiency). In addition, western blotting results indicated unknown protein bands seen in purified fraction of SDS-PAGE in Fig (1), were junk proteins due to no observed signal amplification at 22, 25 and 79 kDa. Positive control in lane 9 contained purified LDHA and its signal amplification validated 36-37 kDa band were rLDHA. However, partial degradation can be observed in lane 4 and 7 (discussed in).

2.4 Relative mobility and estimated molecular mass of protein bands from SDS-PAGE

Table 5: Estimated molecular mass from protein ladder standard curve (refer to appendix). 

Table 5 description: Protein molecular weight estimated from standard curve of known protein standards using equation of straight line: y = -1.489x + 2.5959. rLDHA molecular weight were computed based on relative mobility. From table 5, signal amplified bands had approximately same molecular mass with expected LDHA of 35 kDa. This indicated that 36 kDa was rLDHA.

3.0 Discussion of Lactate Dehydrogenase (LDH)

3.1 OD readings of bacteria culture before and after IPTG induction

OD readings were monitored prior to IPGT induction to ensure bacteria cells reached log phase. Readings obtained was 0.46 OD as seen in Table 1, where bacterial cells were at early log phase and ready for IPTG induction. Log phase was specifically chosen to induce IPGT as total number of bacteria cells were increasing exponentially and actively producing proteins for replication. Induction of IPGT allowed bacteria cells to diverts its energy and resources to synthesized rLDHA proteins while slowing down bacteria replication (control to maximise protein field). This is due to IPTG activating T7 RNA polymerase promotor which was a stronger promoter than host RNA polymerase promotor for gene expression. Furthermore, T7 RNA polymerase being a highly active enzyme, enhanced transcriptional activity of rLDHA rates several times higher than E.coli RNA polymerase and terminated transcription less frequently (Tabor, 2001). Lag phase was not chosen for induction as total number of bacteria cells were extremely low thus, affecting protein production and yield. Stationary phase was not chosen for induction as nutrients would have been depleted, affecting cells ability to express genes though, it contained highest number of bacteria cells (Zwietering, 1990). OD reading after IPTG induction was 1.78, indicating late stationary phase. Increased in bacteria cells over time provided evidence that 1mM IPGT was not potent enough to kills cells, rLDHA was not toxic and incubation period was not too long (cells were not at death phase). If rLDHA was toxic or 1nM IPTG was too strong for bacteria cells, OD value would not increase much, may not change or decreased from 0.46 OD. If bacteria were in death phase OD reading would decrease from 0.46 OD.

3.2 rLDHA successful expression and functionality of Lactate Dehydrogenase (LDH)

IPTG induction was used to express rLDHA gene located in pET11-H6α plasmid. IPTG induction mechanism was described in 3.1. To determine if rLDH in pET11-H6α plasmid silent mutation had no effect (e.g. no truncation) on protein expression and function, specific enzyme activity was conducted. As LDH is a protein catalyst, assay was based on its known enzymatic reaction of oxidising lactate to pyruvate using NAD⁺ as cofactor. By measuring the distinguishable absorbance property of reduced NADH at 340 nm, served as rLDH identifier. Based on results of Table 2, all fractions excluding uninduced sample had a significant increase in enzyme activity (nM/min). This gave an insight expressed rLDH was functional due catalytic ability to convert lactate substrate (provided in reaction) to pyruvate. Low enzyme activity was present in uninduced fraction due to inhibition of T7 RNA polymerase synthesis by lac repressor, while leaky expression was inhibited by T7 lysozyme of pLysS plasmid. However, this was not sufficient to prove rLDHA presence as unknown enzymes produced by host bacterial cells could utilized lactate as substrate. To further prove enzymatic activity was caused by the effect of rLDH, SDS-PAGE and Western blotting results were investigated. Since SDS-PAGE denatured rLDH to its primary structure, characterization was determined by its subunit LDHA known molecular weight of 35 kDa. Equal proportions of 4 μg of protein was loaded and difference in thickness/brightness of similar band will indicate higher concentration. Firstly, to characterize rLDH, purified fraction was look into. If purification was successful, a single band would be observed. Though, 4 bands were observed, 36 kDa band was of interest due to: (1) being approximately similar to expected LDHA band of 35 kda, (2) positive control containing pure LDHA co-migrated to 36 kDa. Western blotting result provided further confirmation as only 36 kDa band signal amplification were observed. This validated, 36 kDa band was of rLDHA as specific antibodies were used. To further prove, 36 kDa band was indeed rLDHA, uninduced, induced and flow through fraction were investigated. As IPGT was used to overexpressed rLDH by inactivating lac repressor to allow binding of T7 polymerase to T7 promotor, difference in band intensity between two fractions should be observed. Based on results, only 36 kDa band transitioned from dull 36 kDa band in uninduced fraction to bright 36 kDa band in induced fraction. Specific activity and western blotting result were consistent whereby uninduced fraction contained a much lower specific activity than induced fraction by 12.47 (μmol/min/mg) and uninduced fraction had a dull signal amplification compared to moderate signal amplification of induced fraction respectively. Thus, validated 36 kDa was of rLDHA, overexpression was successful and pLysS plasmid in BL21(DE3) E. coli bacterial cells was working. As junk proteins were discarded into flowthrough fraction from initial induced fraction via centrifugation of Ni-NTA column, dull 36 kDa band should be observed. This was due to rLDH being bounded to Ni-NTA column based on 6x histidine tag affinity for Ni²⁺. Based on results, only 36 kDa band transitioned from bight 36 kDa band in induced fraction to dull 36 kDa in flowthrough fraction. Specific activity and western blotting result were consistent whereby flow through fraction contained a much lower specific activity than induced fraction by 12.47 (μmol/min/mg) and uninduced fraction had a dull signal amplification compared to moderate signal amplification of induced fraction respectively. This provided further validated 36 kDa band was rLDH. However, rLDHA degradation was observed in Fig. 3, with severity increased from induced to purified fraction due to longer reaction time. Degradation was likely due to adding insufficient PMSF. This can be rectified by adding stronger protease inhibitor or higher concentration of PMSF to fraction. But degradation was not critical as RLDH was still functional based on enzyme activity of NADH formation. Uninduced and flowthrough fraction were not affected due to adding PMSF separately thus, sufficient concentration could be added and majority of rLDHA was eluted to purified sample respectively. Overall, confirmation of  that 36 kDa band seen in SDS-PAGE by Western blot results proved it expression of rLDHA was successful with no observed truncation and confirmed enzyme activity of lactate to pyruvate was indeed rLDH (Triple confirmation test).

3.3 Binding Efficiency and specificity of Ni-NTA column of Lactate Dehydrogenase (LDH)

To accessed binding efficiency of Ni-NTA column, induced and flowthrough fraction were investigated. Prior to purification, induced fraction contained specific activity of 12.5 μmol/min/mg and after centrifugation, it dropped to 0.33 μmol/min/mg in flowthrough fraction. This indicated flowthrough sample contained minimal levels of rLDH with 38 times less rLDH compared to induced fraction. This was consistent with SDS-PAGE results, wherby a dull 36 kDa rLDHA band (purple arrow) in flowthrough fraction compared to bright 36 kDa rLDHA band of induced sample (orange arrow) was observed. Bands intensity of SDS-PAGE were similar in western blotting results whereby flowthrough fraction had low amplification signal of 36 kDa rLDHA band compared to moderate amplification signal in induced fraction. This indicated: (1)  majority of rLDH remained bounded to Ni-NTA column with 98% binding efficiency (based on yield), (2) His-tag of rLDH was in right orientation (3) majority of rLDHA were eluted to purified fraction and was consistent with purified fraction having highest specific activity. Though, Ni-NTA column binding to was concluded to be efficient, having 2.05 percentage yield (Table 3) indicated minor loss of rLDHA to flowthroug fraction. Loss of rLDH was most likely column reaching maximum binding capacity due to: (1) competition of binding by junk proteins which conferred similar/higher affinity to rLDH. (2) Too high concentration of rLDH which overloaded column. However, extend of loss was insignificant as purified fraction still contained high amounts of rLDH. This was backed up by purified fraction  containing highest specific activity, thickest and highest amplification of 36 kDa rLDHA band in SDS-PAGE and western blotting respectively (4 μg of protein was loaded for all fraction). To accessed specificity of Ni-NTA column, purified fraction was investigated. Based on results, non-target protein bands was observed (red arrow) in Fig .1. This could be due to: (1) junk proteins having similar/higher number of histidine molecules in an adjacent fashion conferring approximately same affinity as rLDHA, (2) insufficient concentration of imidazole used to washed column. The former explanation was more likely and was consistent with hypothesised reason of loss of rLDH to flowthrough fraction. Thus, indicating Ni-NTA column was not specific to rLDHA due to its limitation to bind any protein molecules with similar number of histidine molecules as rLDH. This can be rectified by using a column containing a specific ligand or antibody to rLDHA

Using a higher concentration of imidazole was not ideal as junk protein band 1 seemed to bind strongly to Ni²⁺ based on similar band intensity with 36 kDa rLDHA. Non-target bands observed in purified fraction were junk proteins produced by bacteria host as similar bands were observed in uninduced, induced and flowthrough fraction. This was further confirmed by western blotting whereby, no signal amplification of non-target bands at 22, 25 and 79 kDa were observed.

3.4 rLDHA purity and yield of Lactate Dehydrogenase (LDH)

To accessed purity of rLHDA after purification, induced and purified fraction were investigated. Results indicated after Ni-NTA chromatography, number of bands in induced fraction drastically reduced to four observable bands in purified fraction. As discussed in 3.3, 36 kDa band was of rLDHA while remaining bands were junk proteins. This was consistent with specific activity result whereby purified fraction specific activity increased by 107.8 μmol/min/mg after purification of initial induced fraction. This indicated purified fraction contained 9.6 times more rLDHA than induced fraction thus, purified fraction became higher in rLDHA purity. In addition, purified fraction had thickest 36 kDa band and highest specific activity indicating it contained the highest purity of rLDH compared to other fractions. Removed junk protein bands from induced fraction was observed in followthrough fraction and consistent with flowthrough fraction containing lower specific activity than purified fraction.  However, overall purified fraction was not pure due to : (1) Junk protein bands observed in purifed fraction, (2  partial degradation of rLDHA observed in western blotting. Junk proteins can be removed by size exclusion chromatography due to difference in size seen in Table 5 compared to rLDH. Additional Enzyme assay and SDS-PAGE would then be implemented to determine  if specific activity increased and a single 36 kDa band obtained respectively. Rectification to rLDHA degradation was described above in 3.2. To access yield, percentage yield was looked into. Result indicated purified fraction had yield of 20.8 % which was relatively low for affinity chromatography. This posed a challenged as subsequent yield would be lowered if another purification method was implemented. But purity was deemed of higher priority for downstream experiments. There was an unexpected tabulation of yield as purified and flowthrough fraction yield did not add up to ~ 100%. The major loss of yield in purified fraction was most likely contributed by rLDH degradation seen in western blotting. Other minor factors that may contributed includes (1) strong ligand-ligate binding of rLDH to Ni²⁺ thus, was not eluted, (2) loss of rLDH during washing step, (3) not storing rLDH in ice thus, loss of functional enzyme activity. To improve yield, we can use a stronger protease inhibitor to prevent degradation, use a molecule stronger than imidazole to compete for binding site and storing rLDH in enzyme in ice box immediately.

3.5 Other anomalous data of Lactate Dehydrogenase (LDH)

Unexpected induced fraction smearing was noted in Fig.1 whereby it does not have equal smearing intensity with induced and flow through fraction though equal proportions of proteins were loaded. This was likely due to: (1) difficulty experienced during loading of sample into well due to formations of cross-link webs. Thus, not all sample was loaded. (2) spillage due to improper loading. This can be rectified by preforming multiple cleaning of well prior to sample loading. Another unexpected result was induced fraction in Table 2 contained lower protein concentration than uninduced fraction though theoretically it should be containing more. Induced fraction being more diluted were ruled out as volume of bacterial culture to lysis buffer was added in proportion. Likely hypothesis includes: (1) Small sample size of cells (uninduced) of the same volume has better lysis efficiency releasing more proteins. (2) Insufficient PMSF added leading to degradation of proteins.

4.0 Conclusion, improvements & Future work of Lactate Dehydrogenase (LDH)

Based on experimental results, silent mutation in rLHDA of previous experiment did not affect expression and functionality. LDHA has a high activated status and much level of profile in many of the novel cancers which include a large mechanism which show every step of gene expression regulation. LDHA enhance proliferation of cancer cells and still manage the survival of cell. It helps in invasion and metastasis of cancer cells. It assists cancer cell in immune escape. This was confirmed with ability to convert lactate into pyruvate, detected by specific enzyme assay via NADH absorbance at 340 nm. rLDH presence was further confirmed with SDS-PAGE and Western blot with 36 kDa band having signal amplification. Ni-NTA column had a high binding efficiency of 98% but not specific. Loss of 0.02% and unspecific binding was concluded to be maximum column binding capacity due to binding of non-target proteins with similar number of histidine molecules to rLDH. Purity of purified fraction did not achieved single 36 kDa band but can be rectified with size exclusion chromatography. Yield was deemed to be acceptable and major loss of yield was concluded to be rLDH partial degradation. There were no major anomalies throughout the experiments and other anomalies described can be rectified. Further improvements to current framework induces: (1) further characterization of rLDHA via total protein estimation (e.g. calorimetry), (2) use a purification technique that confers higher purification factor, (3) perform two-step purification methods to determine if constant specific activity is  achieved, (4) perform additional layer of purity check such as mass spectrophotometry or N-terminal sequencing.

With validation that rLDHA silent mutation in pET11-H6α did not affect overall protein expression and function, further downstream experiments can be implemented. The next phase would be to study its structure to develop potential novel therapeutic agents in cancer, e.g. antagonistic inhibitors.

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