A New Method for Protein Coexpression in Escherichia coli Using Two Incompatible Plasmids

Yang, Wei; Zhang, Lan; Lu, Zhigang; Tao, Wei; Zhai, Zhonghe

doi:10.1006/prep.2001.1453

Cited by 35 publications

(16 citation statements)

References 22 publications

(5 reference statements)

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“…1B, lower panel) antibodies. It is worth mentioning here that both of the proteins co-expressed (although at different levels) despite using two incompatible plasmids carrying the same replicon, and this was consistent with earlier reports (24).…”

Section: Pkna Expression In E Coli Exhibited Elongated Cells With Sesupporting

confidence: 92%

GTPase Activity of Mycobacterial FtsZ Is Impaired Due to Its Transphosphorylation by the Eukaryotic-type Ser/Thr Kinase, PknA

Thakur¹,

Chakraborti

2006

Journal of Biological Chemistry

109

101

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FtsZ, a homolog of eukaryotic tubulin, is involved in the process of cell division, particularly in septum formation in bacteria. The primary amino acid sequences of this protein are fairly conserved in prokaryotes. We observed that a eukaryotic-type Ser/ Thr protein kinase, PknA from Mycobacterium tuberculosis, when expressed in Escherichia coli exhibited cell elongation due to a defect in septum formation. We found that FtsZ either from Escherichia coli (eFtsZ) or from M. tuberculosis (mFtsZ) was phosphorylated on co-expression with PknA. Consistent with these observations, solid phase binding and in vitro kinase assays revealed the ability of PknA to interact with mFtsZ protein and also to phosphorylate it. We, therefore, ascertained mFtsZ as a substrate of PknA. Furthermore, the phosphorylated mFtsZ exhibited impairment in its GTP hydrolysis and polymerization abilities. Thus, our results highlighted the ability of PknA to phosphorylate as well as to regulate the functionality of FtsZ, the protein central to cell division throughout the bacterial lineage.

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Section: Pkna Expression In E Coli Exhibited Elongated Cells With Sesupporting

confidence: 92%

GTPase Activity of Mycobacterial FtsZ Is Impaired Due to Its Transphosphorylation by the Eukaryotic-type Ser/Thr Kinase, PknA

Thakur¹,

Chakraborti

2006

Journal of Biological Chemistry

109

101

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“…E. coli BL21(DE3) cells (Novagen) cotransformed with the pRARE plasmid were used for heterologous expression. The possible expression of incompatible plasmids over short times has been reported in the past (27,28). Plasmid compatibility for the duration of culture expression was confirmed by the identification of soluble protein products expressed from both coexpressed plasmids (see the supplemental material).…”

Section: Methodsmentioning

confidence: 79%

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum

D’Agostino

Javalkote

Mazmouz

et al. 2016

Appl Environ Microbiol

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The mycosporine-like amino acids (MAAs) are a group of small molecules with a diverse ecological distribution among microorganisms. MAAs have a range of physiological functions, including protection against UV radiation, making them important from a biotechnological perspective. In the present study, we identified a putative MAA (mys) gene cluster in two New Zealand isolates of Scytonema cf. crispum (UCFS10 and UCFS15). Homology to "Anabaena-type" mys clusters suggested that this cluster was likely to be involved in shinorine biosynthesis. Surprisingly, high-performance liquid chromatography analysis of S. cf. crispum cell extracts revealed a complex MAA profile, including shinorine, palythine-serine, and their hexose-bound variants. It was hypothesized that a short-chain dehydrogenase (UCFS15_00405) encoded by a gene adjacent to the S. cf. crispum mys cluster was responsible for the conversion of shinorine to palythine-serine. Heterologous expression of MysABCE and UCFS15_00405 in Escherichia coli resulted in the exclusive production of the parent compound shinorine. Taken together, these results suggest that shinorine biosynthesis in S. cf. crispum proceeds via an Anabaena-type mechanism and that the genes responsible for the production of other MAA analogues, including palythine-serine and glycosylated analogues, may be located elsewhere in the genome. IMPORTANCERecently, New Zealand isolates of S. cf. crispum were linked to the production of paralytic shellfish toxins for the first time, but no other natural products from this species have been reported. Thus, the species was screened for important natural product biosynthesis. The mycosporine-like amino acids (MAAs) are among the strongest absorbers of UV radiation produced in nature. The identification of novel MAAs is important from a biotechnology perspective, as these molecules are able to be utilized as sunscreens. This study has identified two novel MAAs that have provided several new avenues of future research related to MAA genetics and biosynthesis. Further, we have revealed that the genetic basis of MAA biosynthesis may not be clustered on the genome. The identification of the genes responsible for MAA biosynthesis is vital for future genetic engineering.

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“…The presence of different antibiotic selections (ampicillin in pMAL/ pGEX and hygromycin in p19Kpro) facilitated the co-expression of foreign proteins in E. coli using these incompatible plasmids (21,23). The E. coli cells harboring pMAL or pGEX with gene(s) of interest were grown in the presence of ampicillin (100 g/ml) and made competent using standard methods (24).…”

Section: Methodsmentioning

confidence: 99%

Interdomain Interaction Reconstitutes the Functionality of PknA, a Eukaryotic Type Ser/Thr Kinase from Mycobacterium tuberculosis

Thakur¹,

Chaba²,

Mondal

et al. 2008

Journal of Biological Chemistry

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Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality.

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A New Method for Protein Coexpression in Escherichia coli Using Two Incompatible Plasmids

Cited by 35 publications

References 22 publications

GTPase Activity of Mycobacterial FtsZ Is Impaired Due to Its Transphosphorylation by the Eukaryotic-type Ser/Thr Kinase, PknA

GTPase Activity of Mycobacterial FtsZ Is Impaired Due to Its Transphosphorylation by the Eukaryotic-type Ser/Thr Kinase, PknA

Comparative Profiling and Discovery of Novel Glycosylated Mycosporine-Like Amino Acids in Two Strains of the Cyanobacterium Scytonema cf. crispum

Interdomain Interaction Reconstitutes the Functionality of PknA, a Eukaryotic Type Ser/Thr Kinase from Mycobacterium tuberculosis

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