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Cloning using pET28a and Protein Expression in DH5alpha and BL21

Cloning using pET28a and Protein Expression in DH5alpha and BL21


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Can someone please direct me to an e-resource or a book that will help a newbie like me learn in depth about Cloning using pET28a and Protein Expression in DH5alpha and BL21. Though I have done cloning using TA vectors, pET28a is very new to me. I need to design primers with restriction sites to insert my GOI into pET28a, so would like to learn how to choose these sites too.


Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification

Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.

Keywords: Cloning expression purification recombinant bovine SRY protein sex determinant region Y chromosome.


[Cloning and expression of human interleukin-21 cDNA in E.coli]

Aim: To clone full length cDNA of human interleukin-21 (IL-21) and express it in E.coli.

Methods: Total RNA was isolated from peripheral lymphocyte stimulated with anti-CD3 antibody. 5' and 3' terminal fragments of IL-21 gene (242 bp and 425 bp fragments respectively) were amplified using RT-PCR. The full length IL-21 cDNA was amplified by recombination PCR from the products of RT-PCR. The expression plasmid pET28a(+)-IL21 was constructed by inserting IL-21 cDNA into pET28a(+)and then was transformed into BL21(DE3). Expression of hIL-21 was induced by IPTG at 37 degrees Celsius for 5 h. The target protein was purified through Ni(2+)-chelating Sepharose Fast Flow. Purified rhIL-21 was refolded by using dialysis method. And the bioactivity was detected by MTT on costimulating the T cell proliferation with anti-CD3.

Results: IL-21 was cloned and expressed in E.coli successfully. SDS-PAGE analysis showed the IL-21 was expressed in the form of insoluble inclusion body. The refolded rhIL-21 could stimulate the proliferation of mature human T-cells in the presence of anti-CD3.

Conclusion: The rhIL-21 with bioactivity was obtained, which lays the foundation for study of its function.


II. Expression Troubleshooting

In this section, we present different strategies for optimizing recombinant protein production in E. coli when encountering expression obstacles. Possible reasons and solutions in each case are discussed in the following tables.

When the protein of interest cannot be detected through a sensitive technique (e.g., Westernblot) or it is detected but at very low levels (less than micrograms per liter of culture), the problem often lies in a harmful effect that the heterologous protein exerts on the cell.

  • Lower induction temperature
  • Grow in poor media
  • Use promoters with tighter regulation
  • Lower plasmid copy number
  • Use pLysS/pLysE bearing strains in T7-based systems
  • Use strains that are better for the expression of toxic proteins (C41 or C43)
  • Start induction at high OD
  • Shorten induction time
  • Add glucose when using expression vectors containing lac-based promoters
  • Use defined media with glucose as source of carbon

2) Protein aggregation

The buildups of protein aggregates are known as inclusion bodies (IBs). IB formation results from an unbalanced equilibrium between protein aggregation and solubilization. So, it is possible to obtain a soluble recombinant protein by strategies that ameliorate the factors leading to IB formation.

  • Add fusion partners, including thioredoxin, DsbA, DsbC
  • Clone in a vector containing secretion signal to cell periplasm
  • Lower inducer concentration
  • Lower induction temperature
  • Use a solubilizing partner
  • Co-express with molecular chaperones
  • Supplement media with chemical chaperones and cofactors
  • Remove inducer and add fresh media
  • Lower inducer concentration
  • Lower temperature
  • Add fusion tags, including GST, MBP, SUMO, etc.
  • Generate truncated forms of protein
  • Lower induction temperature
  • Shorten induction time
  • Grow in poor medium
  • Add heat shock chaperones

Sometimes a truncated form of protein is expressed rather than a complete wild protein. Reasons of the phenomenon and possible solutions are given below.

  • Lower induction temperature
  • Grow in poor media
  • Induce at high OD
  • Induce at low temperature
  • Shorten induction time
  • Use protease inhibitors when breaking cells
  • Change another fusion protein
  • Move fusion protein to C-terminal
  • Induce at low temperature
  • Shorten induction time
  • Change to poor media

Obtaining a nice amount of soluble protein is not the end of the road. The protein may still be of bad quality, i.e., it does not have the activity it should.

  • Use a solubilizing partner
  • Co-express with molecular chaperones
  • Monitor disulfide bond formation and allow further folding in vitro
  • Lower temperature

References:

Fakruddin M et al (2012). Critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant E. coli. ISRN biotechnology, 132013:590587.

Francis DM, Page R (2010). Strategies to optimize protein expression in E. coli. Current Protocols in Protein Science, Chapter 5:Unit 5.24.1-29.


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Cloning and Expression of hGAD65 Gene in E. Coli BL21

https://doi.org/10.22146/ijbiotech.7868

Rista Nikmatu Rohmah (1*) , Soraya Widyasari (2) , A. Aulanni’am (3) , F. Fatchiyah (4)

(1) Departement of Biology, Faculty of Mathematic and Natural Science, Universitas Brawijaya, Malang
(2) Departement of Biology, Faculty of Mathematic and Natural Science, Universitas Brawijaya, Malang
(3) Departement of Chemistry, Faculty of Mathematic and Natural Science, Universitas Brawijaya,Malang
(4) Departement of Biology, Faculty of Mathematic and Natural Science, Universitas Brawijaya, Malang
(*) Corresponding Author

Abstract

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DOI: https://doi.org/10.22146/ijbiotech.7868

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RESULTS

RNA Extraction—

About 20 μg of total RNA was obtained from each preparation. The RNA quality was assured by the presence of bands corresponding to 18S and 28S ribosomal RNA (Fig. 1A).

Isolation and Amplification of ocI ORF—

The ocI ORF was obtained by RT-PCR, resulting in a product with about 300 bp, corresponding to the ocI ORF (only from RNA of germinated seeds). The PCR product is shown in Fig. 1B.

Cloning in pET28a—

After the transformation of E. coli BL21 (DE3), 60 clones were chosen for screening by PCR. Some of them were found to contain the insert with a size of around 300 bp (Fig. 1C). The plasmid DNA of three of these clones was then extracted and sequenced. One clone had the correct sequence, but the other two failed to display good quality sequences.

Induction of Expression, Solubility Test, and Protein Purification—

The protein expression was induced with 0.4 m M IPTG over a 3-h period. As shown in the 15% SDS-PAGE (Fig. 2A), the protein expression was induced after 1 h and did not apparently increase over the subsequent 2 h.

The recombinant OCI was obtained in both soluble and insoluble fractions (Fig. 2A). There was enough protein in the soluble fraction to purify in the native form. The purification was done with a nickel resin, and the protein elution began with 50 m M of imidazole. However, the best elution was achieved in the washing with 100 and 250 m M imidazole (Fig. 2B). The samples thus collected were pooled, and a total of 10 mg of protein per liter of culture was obtained. Dialysis was performed to remove excess imidazole.

Antifungal Activity Assay—

The antifungal activity of the recombinant protein was tested against the filamentous fungus T. reesei. Fungal growth was successfully inhibited in both concentrations tested (50 and 100 μg/ml) (Fig. 3).

Writing of the Manuscript—

The results were compiled and discussed, after which the students wrote this article under the supervision of the course coordinators.


Choice of Vector

The basic architecture of an E. coli expression vector is shown in the figure below and contains the following features:

Selectable marker. In the absence of selective pressure plasmids are lost from the host. Especially in the case of very high copy number plasmids and when plasmid-borne genes are toxic to the host or otherwise significantly reduce its growth rate. The simplest way to address this problem is to express from the same plasmid an antibiotic-resistance marker and supplement the medium with the appropriate antibiotic to kill plasmid-free cells. The most used antibiotics and their effective concentrations are listed in table 1.

The use of ampicillin requires special care. The selectable marker, b -lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture. Some possible solution are:

  • grow overnight cultures at 30°C or less.
  • spin overnight cultures and resuspend the pellet in fresh medium to remove the produced b -lactamase.
  • use the more stable carbenicillin instead of ampicillin.

Regulatory gene (repressor). Many promoters show leakiness in their expression i.e. gene products are expressed at low level before the addition of the inducer. This becomes a problem when the gene product is toxic for the host. This can be prevented by the constitutive expression of a repressor protein.

The lac-derived promoters are especially leaky. These promoters can be controlled by the insertion of a lac-operator sequence downstream the promoter and the expression of the lac-repressor by host strains carrying the lacI q allele (for medium copy number plasmids) or from the same or a helper plasmid (for higher copy number plasmids). Alternatively, repression can be achieved by the addition of 1% glucose to the culture medium.

Origin of replication. The origin of replication controls the plasmid copy number.

Promoter. The promotor initiates transcription and is positioned 10-100 nucleotides upstream of the ribosome binding site. The ideal promoter exhibits several desirable features:

  • It is strong enough to allow product accumulation up to 50% of the total cellular protein.
  • It has a low basal expression level (i.e. it is tightly regulated to prevent product toxicity).
  • It is easy to induce.

An extensive list of possible promoters is given in table 2. The most used promoters are indicated in red .

Transcription terminator. The transcription terminator reduces unwanted transcription and increases plasmid and mRNA stability.

Shine-Delgarno sequence. The Shine-Dalgarno (SD) sequence is required for translation initiation and is complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5'- TAAGGAGG -3'. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues.

Start codon. Initiation point of translation. In E. coli the most used start codon is ATG. GTG is used in 8% of the cases. TTG and TAA are hardly used.

Tags and fusion proteins. N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other proteins (fusion partners) offer several potential advantages:

  • Improved expression. Fusion of the N-terminus of a heterologous protein to the C-terminus of a highly-expressed fusion partner often results in high level expression of the fusion protein.
  • Improved solubility. Fusion of the N-terminus of a heterologous protein to the C-terminus of a soluble fusion partner often improves the solubility of the fusion protein.
  • Improved detection. Fusion of a protein to either terminus of a short peptide (epitope tag) or protein which is recognized by an antibody or a binding protein (Western blot analysis) or by biophysical methods (e.g. GFP by fluorescence) allows for detection of a protein during expression and purification.
  • Improved purification. Simple purification schemes have been developed for proteins fused at either end to tags or proteins which bind specifically to affinity resins (see table 3 and references therein).

Protease cleavage site. Protease cleavage sites are often added to be able to remove a tag or fusion partner from the fusion protein after expression. Most commonly used proteases are listed in table 4. However, cleavage is rarely complete and often additional purification steps are required.

Multiple cloning site. A series of unique restriction sites that enables you to clone your gene of interest into the vector.

Stop codon. Termination of translation. There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.


Cloning, Expression, and Purification of Recombinant Lysostaphin From Staphylococcus simulans

How to Cite: Farhangnia L, Ghaznavi- Rad E, Mollaee N, Abtahi H. Cloning, Expression, and Purification of Recombinant Lysostaphin From Staphylococcus simulans, Jundishapur J Microbiol. 2014 7(5):e10009. doi: 10.5812/jjm.10009.

Abstract

Background: Staphylococcus aureus is one of the most common causes of nosocomial infections and its resistance to antibiotics is a global concern. Lysostaphin is an antimicrobial agent belonging to a major class of antimicrobial peptides and proteins known as the bacteriocins. It exhibits a high degree of anti-staphylococcal bacteriolytic activity.

Objectives: In this study, high level of recombinant mature lysostaphin in Escherichia coli was produced by using pET32a expression vector.

Materials and Methods: The S. simulans gene encoding lysostaphin was extracted, amplified by polymerase chain reaction (PCR), and sub-cloned in prokaryotic expression vector pET32a. E. coli BL21 (DE3) plysS were transformed with pET32a-lys and gene expression was induced by IPTG. The expressed protein was purified by affinity-chromatography using (Ni-NTA) resin.

Results: PCR and sequencing results confirmed the successful cloning of the target gene into the vector. The expression of protein was induced by IPTG and high concentration of the recombinant protein was obtained via the purification process by affinity-chromatography.

Conclusions: Our data showed that the recombinant mature lysostaphin protein produced by pET32a vector in E. coli system was very efficient.

1. Background

Staphylococcus aureus is a major cause of both nosocomial and community-acquired infections worldwide that causes a wide range of diseases including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food-poisoning, carbuncles, and boils (1). Increased emergence of multidrug resistance among methicillin-resistant S. aureus (MRSA) strains has become a major concern in the hospital environment as it imposes a tremendous financial burden and increased morbidity and mortality due to hard-to-treat systemic infections (2).

Nowadays vancomycin is the antibiotic of choice for treatment of MRSA however, accumulating mutations in S. aureus have led to intermediate resistance to vancomycin (VISA) (3). It has left us with the spectrum of very few effective antibiotics being available to treat S. aureus infections and with the probability that resistance to the remaining antibiotics would likely occur. Therefore, The issue of drug resistance in this group of pathogens needs to be addressed via appropriate use of existing drug as well as the development of novel agents (4, 5). Staphylococci are potential targets for bacteriocins including lysostaphin.

Lysostaphinis a glycine-glycine endo peptidase produced by S. simulans that specifically cleaves the glycine-glycine bond unique to the inter peptide cross-bridge of S. aureus cell wall. Due to its unique specificity, lysostaphin has a high potential for treating antibiotic-resistant Staphylococcal infections (1). Studies on the secondary protein structure of lysostaphin have revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 amino acids), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 amino acids), and the hydrophobic mature lysostaphin (6). Mature lysostaphin is a single polypeptide chain with molecular weight of 27 KDa (7).

Lysostaphin is unique in possessing extremely high activity against a variety of staphylococcal strains including MRSA. S. aureus is one of the most prevalent microorganisms of skin flora and coding sequence of the mature mostly isolated from wound, skin, and soft tissue infections. Therefore, production of the pure and effective recombinant lysostaphin (r-lysostaphin) protein in vitro could lead to a potential treatment for S. aureus. Previously, lysostaphin has been cloned and expressed heterologously in Escherichia coli (8), in the simian kidney cell line (9), and in Lactococcus lactis (10). At present, several lysostaphin overexpression systems are described (9-11). Although researcher have tried to improve the amount of lysostaphin production in these studies, the yield has remained unsatisfactory and purification methods are complicated.

2. Objectives

In the present study, we described a new expression system for producing r-lysostaphin in E. coli, a safe non-pathogenic host, on a laboratory scale with potentials to industrial scale. In addition, high-capacity Ni-NTA resin purification procedure was used for obtaining large amounts of pure lysostaphin.

3. Materials and Methods

3.1. Bacterial Strains, Vectors and Other Regents

S. simulans PTCC 1442 (Iran) and pET32a vector (Novagene, USA) was purchased. This vector is able to express a fusion protein with a 6-histidine tag at thrombin site and a T7 tag at the N-terminus. These additional amino acids increase the size of expressed protein near 15 KDa. Restriction enzymes, DNA ligase (Fermentas, Lithuania), were obtained. E. coli strain DH5α (f-gyr A96 Nalr, recA1 relA1 Thi-1 hsdR17 r-k m+k, Stratagene, USA) were used for initial cloning. The recombinant pET32a (pET32a- lysostaphin) was transformed into E. coli, BL21 (DE3) pLysS (f–ompthsdB, rB- mB-, dcm gal, DE3, pLYsScmr) as host strain from (Novagene, USA).

Protein Purification Kit (Qiagene, Germany) were provided. MHB (Muller Hinton Broth, Sigma, USA) and LB broth (Luria Bertani Broth, Sigma, USA) were used for routine bacterial culture. The required antibiotics, ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) (Sigma, USA), were added to LB media according to the reference recommendation (12). All chemicals were obtained from Merck (Germany) and the enzymes were obtained from Fermentas (Lithuania) or Cinagene Tehran, (Iran) Companies.

3.2. Isolation of Plasmid DNA

After overnight incubation of S. simulans in MHB at 37°C, bacterial cells were centrifuged at 4000rpm for 5min and the pellet was suspended in 100 μL of SET buffer (sucrose 50 mM, EDTA 10 mM,Tris-HCl 25 mM, pH = 8). Plasmid DNA was prepared according to standard mini-preparation of plasmid Method. Briefly, bacterial pellet was obtained from 1.5 mL overnight bacterial culture re suspended in SET buffer and left at room temperature for 5 min. Afterwards, the bacterial cells were lysed by fresh, cold lyses buffer(NaOH5N, SDS 10%, DDW), then KAC (KAC 5M, Acetic acid, DDW) was added.

The cell debris, proteins, and chromosomal DNA were removed by two times phenol/chloroform/isoamyl alcohol (25:24:1) Mixture. DNA was precipitated by ethanol (100%) and washed in ethanol (70%) then, the pellet was dried on air and resuspended in TE buffer. The quality and quantity of purified plasmid DNA were assayed by 0.8% Agarose gel electrophoresis in 1X TBE buffer and spectrophotometry(260/280 nm), respectively (12).

3.3. Polymerase Chain Reaction and Construction of the Recombinant Plasmid pET-lys

Primers were designed according to the full-length of lysostaphin gene sequence (Gene Bank accession no: X06121). The coding sequence of the mature peptide (738 bp) was amplified by polymerase chain reaction (PCR) using the following primers: 5´-AGA GGA TCC GCT GCA ACA CAT GAA -3´ (forward primer with an endonuclease site BamHI) and 5´-CGC CTC GAG TCA CTT TAT AGT TCC-3´ (reverse primer with an endonuclease site XhoI). Restriction endonuclease sites for BamHI and XhoI were incorporated at the 5´-and 3´-end of the mature gene, respectively, for sub-cloning purposes. Gene amplification of lysostaphin gene was performed in a total volume containing 20 ng of template DNA, 0.5 μM of each primers, 2 mM Mg2+,200 mΜ of each deoxynucleotide triphosphate,1X PCR buffer, and 2.5 unites of Taq polymerase.

The following program (Eppendorf PCR) were used for amplification: Hot start at 94°C for five minutes, followed by 30 cycle of denaturation at 94°C for one minute, annealing at 56°Cfor one minute, and extension at 72°Cfor one minute. The program followed by a final extension at 72°C for five minutes. The PCR products were analyzed in 0.8% agarose gel in 1X TBE buffer and purified from gel by High Pure PCR product purification kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instruction.

The PCR product was digested with BamHI and XhoI and was ligated to pET32a, which were digested by the same restriction enzymes, to generate the recombinant plasmid pET32a-lys (pET-lys) using T4 DNA ligase (12). The E. coli DH5α was used for transformation of pET32a-lys plasmid. The transformed bacteria were selected by screening the colonies on Ampicillin (100 μg/mL) containing media and plasmid purification. Then colonies were further analyzed by restriction enzyme digestion and PCR. The lysostaphin gene of the recombinant plasmid was sequenced by Sanger method.

3.4. Expression and Purification of Recombinant Mature Lysostaphin

The expression host E. coli BL21 (DE3) pLysS was used as transformation host for pET-lys vector. This strain, containing T7 RNA polymerase gene under the control of lacUV5 promoter, was transformed with pET-lys. A single colony of transformed E. coli BL21 (DE3) with pET-lys was incubated overnight on shaking incubator in 2 mL Luria-Bertani broth (LB) medium containing Ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) at 37°C with constant agitation (200 rpm). The next day, 500 μL of cultured materials was removed and inoculated in 25 mL LB broth (per liter: 14 g yeast extract,12 g Bactotryptone, 10 g NaCl, 1 g KCl, 0.5 g MgCl, 0.5 g CaCl).

The culture was grown in an OD600nm of 0.6 with vigorous shaking (200 rpm) at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM for expression of mature lysostaphin in E. coli. The incubation period continued for another four hours at 37°C with shaking at 200 rpm. In order to produce the expression protein, bacterial suspension were tested at two and four-hour intervals and analyzed on 12% SDS-PAGE (13). The expressed protein was purified using Ni-NTA agarose column according to manufacturer’s instruction (Qiagene, Hilden, Germany).The purified protein was dialyzed and refolded with PBS (containing PMSF 0.2mM, pH = 7.2) at 4°C overnight. The quality and quantity of purified recombinant mature lysostaphin was analyzed on a 12% SDS-PAGE gel electrophoresis with Bradford methods (14).

4. Results

4.1. Isolation of Plasmid

The plasmid DNA of S. simulans was extracted and the concentration was adjusted to 4 μg/μL that were used as template for amplification of the gene encoded lysostaphin.

4.2. Construction of the Recombinant Plasmid pET-lys

The sequencing result was confirmed by comparing to database using basic local alignment search tool (BLAST) software. Enzyme digestion procedure, PCR assay, and sequencing result showed that target gene was inserted correctly into the recombinant plasmid pET-lys (data are not show).

4.3. Expression and Purification of Recombinant Mature Lysostaphin

The positive recombinant plasmid was transformed into the host, E. coli BL21 (DE3). The addition of IPTG induced the overexpression of approximately 42 kDa molecular weight recombinant protein. The expressed protein was purified successfully via affinity chromatography using Ni-NTA resin (Figure 1). The purification and dialysis process resulted in the yield of about 30 mg of purified protein from 1 L of E. coli BL21 (DE3) + pET-lys culture.

The SDS-PAGE gels shows uninduced cell extract from E. coli BL21(DE3)+PET-lys for one hour(lane 1) and two hours (lane 2), induced cell extract for two hours(lane 3) and four hours (lane4), and Extracted Proteins after Ni-NTA affinity chromatography (lane 5). A high-range molecular weight Marker is shown on left (lane M).

5. Discussion

In this study, the mature lysostaphin recombinant protein from S. simulans was cloned, expressed under the control of T7 promoter, and purified using Ni-NTA resin. The obtained results showed that pET 32a system was very efficient.

Unlike an antibiotic, which interferes with bacterial growth, lysostaphin is highly effective in lysing S. aureus cells throughout the metabolic stage. Earlier methods for production of lysostaphin endopeptidase aimed to purify it from crude extract of S. simulans (15, 16), which might be contaminated with small amounts of pyogens/allergens. In addition, mature lysostaphin is cleaved off the propeptide again using S. simulans extract (9, 17). However, purification of wild-type lysostaphin is very difficult. Although several methods of lysostaphin production have reported, the yield and purity were very limited (11, 17, 18).

There are a number of reports for expression of lysostaphin endopeptidase in E. coli using the lysostaphin endopeptidase promoter (6, 8). Proendopeptidase was also expressed in eukaryotic system under the transcriptional control of Cytomegalovirus (CMV) promoter (9). The expression of recombinant prolysostaphin in Bacillus subtilis and B.sphaericus was reported that revealed their ability to secret large amount of lysostaphin into the culture medium. B. sphaericus produces about five times more lysostaphin than its natural source (19).

Lysostaphin was also expressed in mice, in which the 5′-flanking region of the Bovine β-lactoglobulin gene directed the secretion of lysostaphin into milk (20). As far as pharmaceutics/therapeutics are concerned, E. coli is considered a safe expression host. Numerous proteins have been expressed in E. coli therefore, E. coli is widely used as an expression host in both research and industry.

In several study, r-lysostaphin were produced through different pET vectors including pET28a with the yield of 22 mg, pET 23b with the yield of 20 mg, and pET15b with the yield of 11 mg of purified protein from 1 L of E. coli BL21(DE3) + pET-lys culture (7, 21, 22). The lysostaphin was also overexpressed and purified using the intein–chitin-binding domain (intein–CBD) as a fusion protein with the yield of 6 mg/L (11). A r-lysostaphin expressed in E. coli is sold commercially by Sigma-Aldrich and is indispensable for Staphylococcal genetic studies it is used for DNA isolation (23), formation of protoplasts, and differentiation of Staphylococcal strains (24).

Further evaluation of the anti-Staphylococcal potential of lysostaphin as a therapeutic agent and its use as a laboratory reagent depends on the availability of large amounts of highly purified protein from a safe and nonpathogenic source. Therefore, the low yield obtained in lysostaphin production (7, 21, 22), pathogenesis, and multi-drug resistance properties of S. aureus (2) as well as high-cost industrial product of lysostaphin have been the principal reason to search for a recombinant source for this therapeutic agent. This is the first report of recombinant mature lysostaphin from Iran.

In the present study, pET32a system was used to express the r-lysostaphin in E. coli. Using this purification method, we obtained about 30 mg of r-lysostaphin per liter of the growth medium in the pET 32a system. In this assay, the r-lysostaphin was purified using Ni-NTA column according to manufacturer’s instruction (Qiagene, Germany). This purification method is very simple and was performed in laboratories that had neither the expertise nor the equipment necessary for traditional protein purification schemes. The procedure for producing r-lysostaphin is quite convenient and efficient and would allow a laboratory to produce large amounts of r-lysostaphin. In this study, in order to obtain high level expression of fusion proteins, E. coli BL21(DE3) plys S was used as a expressed host that is deficient in the known cytoplasmic protease gene products (25). Therefore, the highest expression of lysostaphin in E. coli BL21 (DE3) plys S might be due to protease deficiency in this strain. The pET system has been recognized as one of the most powerful methods for producing recombinant proteins in E. coli and the significant advantages of this system have been widely discussed.

Therefore, we produced mature r-lysostaphin with the presented procedure from E. coli for preparation of large quantity of r-lysostaphin for structure function studies and evaluation of its clinical potential in therapy as well as prophylaxis against staphylococcal infections. Our data showed that mature lysostaphin region of lysostaphin gene can be expressed by pET32a vector in E. coli, and T7 lac promoter might be stronger than other promoters in inducingr-lysostaphin production.

Acknowledgements

This study was conducted with financial assistance from Arak University of medical sciences, Iran, and we are grateful to for their invaluable contribution to this study. This study was the thesis (no: 772) by Mrs. Leila Farhangnia, the master student of Biotechnology at Arak university of medical sciences, Iran.

Footnotes

  • Implication for health policy/practice/research/medical education: Recombinant lysostaphin protein was produced in this study through cloning and expression method can be further implemented for clinical and molecular biology works.
  • Authors’ Contribution: All authors had equal contribution.
  • Financial Disclosure: There is no Financial Disclosure.
  • Funding/Support: Funding for this work was provided by the Arak University of Medical Sciences.

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Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody

Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni + -NTA affinity chromatography (95–98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax.


Trouble transforming pET28a in E. coli BL21(DE3) - (Feb/04/2020 )

We  are  facing  issues  while  transforming  pET28a  vectors  in  expression  strains.  Please  refer  to  the  image  attached  and  suggest  improvements.

The quality of vector DNA and transformation efficiency in Top10 is good. All the suggestions will be appreciated

How many times have you observed this phenomenon and which plasmid extraction system are you using? If you are doing manual minipreps (i.e. not kit) it is possible that the DNA is not clean enough to get a good transformation and resulting in no plasmids coming out the final step.

bob1 on Wed Feb 5 02:10:39 2020 said:

How many times have you observed this phenomenon and which plasmid extraction system are you using? If you are doing manual minipreps (i.e. not kit) it is possible that the DNA is not clean enough to get a good transformation and resulting in no plasmids coming out the final step.

we have observed this particular phenomenon 3 times. The plasmid isolation method is manual and the plasmid quantity and quality is good (Verified by nanodrop and quantus). The same plasmid can be transformed in Top10 but not in expression strain. Other plasmids are also isolated by this same method and they are getting transformed properly. This problem is only seen in case of pET28a vector.

Based on your flowchart it seems you have done most of the appropriate experimental controls to eliminate bad plasmid preps and “incompetent” BL21(DE3) competent cells as the problem.  When you say transformation failed, do you mean that you get no colonies at all?  Or you get colonies but they have incorrect (deleted) plasmids?

I’m not an expert with this Novagen plasmid/host system.  I’d revisit the Novagen manual and make sure you have the correct Vector/Expression host pairing.  I took a quick look and BL21(DE3) is indeed the most commonly used strain, but there is one called BLR (DE3) that is a recA- derivative of BL21(DE3) that may stabilize some target genes with repeats.  You also have to consider the possible toxicity of the expressed protein on the bacterial cells. Though that would not explain why you can’t get empty vector into the expression host. Also, when you get stuff from other labs (part A of your chart), you do have to consider the possibility something was not labeled correctly and what you got was not what you think it is.  So I’d do some RE digests and make sure the plasmid you are using is what it is supposed to be.

I am baffled, as are you, at experiment B with the commercial construct- 1 st generation transformation successful with both hosts, 2 nd gen transformation not (but only for the DE3 strain).  Did you try the TOP10 cells for that 2 nd transformation? That would have been an important comparison.

I hope you figure this out and if you do, let us know!

OldCloner on Thu Feb 6 19:59:20 2020 said:

Based on your flowchart it seems you have done most of the appropriate experimental controls to eliminate bad plasmid preps and “incompetent” BL21(DE3) competent cells as the problem.  When you say transformation failed, do you mean that you get no colonies at all?  Or you get colonies but they have incorrect (deleted) plasmids?

 

I’m not an expert with this Novagen plasmid/host system.  I’d revisit the Novagen manual and make sure you have the correct Vector/Expression host pairing.  I took a quick look and BL21(DE3) is indeed the most commonly used strain, but there is one called BLR (DE3) that is a recA- derivative of BL21(DE3) that may stabilize some target genes with repeats.  You also have to consider the possible toxicity of the expressed protein on the bacterial cells. Though that would not explain why you can’t get empty vector into the expression host. Also, when you get stuff from other labs (part A of your chart), you do have to consider the possibility something was not labeled correctly and what you got was not what you think it is.  So I’d do some RE digests and make sure the plasmid you are using is what it is supposed to be.

 

I am baffled, as are you, at experiment B with the commercial construct- 1 st generation transformation successful with both hosts, 2 nd gen transformation not (but only for the DE3 strain).  Did you try the TOP10 cells for that 2 nd transformation? That would have been an important comparison.

 

I hope you figure this out and if you do, let us know!

Failed transformation means no colonies observed. Also, I tried TOP10 cells for the 2 nd transformation and it was successful. I am also puzzled on how to proceed with this problem. I will keep posting on this thread regarding the progress.


Watch the video: 12. Κεντρικό Δόγμα Βιολογίας. Έκφραση γονιδίων. Είδη RNA 2 2ο κεφ. - Βιολογία Γ λυκείου. (May 2022).


Comments:

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