12/20/07 Recipes and stock solutions described in Protein Expression and Purification 41: 207-234 (2005)
Protein Production by Auto-Induction in High-Density Shaking Cultures
F. William Studier
Biology Department, Brookhaven National Laboratory, Upton, NY 11973
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Abstract. Inducible expression systems in which T7 RNA polymera transcribes
coding quences cloned under control of a T7lac promoter efficiently produce a wide variety of
proteins in Escherichia coli. Investigation of factors that affect stability, growth and induction of
T7 expression strains in shaking vesls led to the recognition that sporadic, unintended
induction of expression in complex media, previously reported by others, is almost certainly
caud by small amounts of lacto. Gluco prevents induction by lacto by well-studied
mechanisms. Amino acids also inhibit induction by lacto during log-pha growth, and high
rates of aeration inhibit induction at low lacto concentrations. The obrvations, and
metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing
media in which batch cultures grow to high densities. Expression strains grown to saturation in
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non-inducing media retain plasmids and remain fully viable for weeks in the refrigerator, making
it easy to prepare many freezer stocks in parallel and u working stocks for an extended period.
Auto-induction allows efficient screening of many clones in parallel for expression and
solubility, as cultures have only to be inoculated and grown to saturation, and yields of target
protein are typically veral-fold higher than obtained by conventional IPTG induction. Auto-
inducing media have been developed for labeling proteins with lenomethionine, 15N or 13C,
and for production of target proteins by arabino induction of T7 RNA polymera from the
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pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly
滚筒洗衣机使用ud methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
overview 2-5
General
Non-inducing agar plates and stabs for expression strains 6
Non-inducing media currently ud routinely for growing stocks 7
Auto-inducing media currently ud routinely 8
路由器的ipMedia with arabino for auto-induction from T7lac in BL21-AI 8
12,
9,
14
minimal
media
Auto-inducing
Medium for rapid growth of high-density cultures to prepare plasmids 9
Auto-inducing media for labeling with SeMet 10
Auto-inducing media for labeling with 15N and 13C 11
Low-phosphate (25 mM) non-inducing and auto-inducing media 12
High-phosphate (100 mM) non-inducing and auto-inducing media 13-14
solutions 15-20
风住尘香花已尽Stock
Growth media have been developed to control the expression of target genes in E. coli strains such
as BL21(DE3), in which T7 RNA polymera expresd from the inducible lacUV5 promoter in the chromosome directs the expression of target protein from the T7lac promoter in multi-copy pET expression vectors (Studier et al., Methods in Enzymology 185: 60-89 (1990)). The media should also be applicable to other strains where expression is controlled by promoters inducible by lacto or IPTG. Media are also described for auto-induction of promoters inducible by arabino.
Non-inducing media. Fully defined non-inducing media give reliable growth without detectable induction of target protein all the way to saturation. They also support growth to higher cell densities than typical of complex media such as LB (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter) and produce cultures that remain highly viable for weeks in the refrigerator. Even strains expressing target proteins highly toxic to the host remained stable in the non-inducing media, grew sub-cultures with little lag and were fully competent to express target protein. In contrast, unintended spontaneous induction can occur in typical complex media (Grossman et al., Gene 209: 95-103 (1998)), which can stress or kill productive cells and favor poorly expressing derivatives. The common practice of using cultures grown to saturation in LB or other complex media to produce ed cultures for production of target protein may be responsible for many cas of poor expression in IPTG-inducible expression systems. Although addition of gluco can prevent such induction, find
ing a gluco concentration that reliably prevents induction in complex media without cultures becoming very acidic at saturation proved difficult or impossible.
Unintended induction is almost certainly due to small amounts of lacto prent in enzymatic digests of cain, such as tryptone or N-Z-amine, in commonly ud media such as LB. Cain comes from milk, which contains lacto, and different lots of purified cain ud to make growth media may well contain different levels of residual lacto. The high concentrations of amino acids in such media suppress induction by lacto during log-pha growth, but less than 0.001% lacto can cau induction upon approach to saturation in cultures grown at moderate levels of aeration. Such inducing activity appears to be relatively common in commercial growth media made from enzymatic digests of cain. Therefore, isolation of expression strains, growth of freezer stocks for long-term storage, and growth of ed stocks for protein production should all be done in reliable non-inducing media such as MDG or MDAG.
Auto-inducing media. Media such as ZYM-5052 and MDA-5052 have been formulated to grow IPTG-inducible expression strains, initially without induction, and then to induce production of target protein automatically, usually near saturation at high cell density. A limited concentration of gluco is metabolized preferentially during growth, which prevents uptake of lacto until the gluco is depl
eted, usually in mid to late log pha. As the gluco is depleted, lacto can be taken up and converted by β-galactosida to the inducer allolacto. Allolacto caus relea of lac repressor from its specific binding sites in the DNA and thereby induces expression of T7 RNA polymera from the lacUV5 promoter and unblocks T7lac promoters, allowing expression of target proteins by T7 RNA polymera. Depletable gluco can also allow auto-induction of arabino-inducible promoters by arabino in the medium, and the method could also be applied to promoters regulated by other metabolites subject to catabolite repression or inducer exclusion. Even strains that produce target proteins highly toxic to the host cell can grow normally and express their protein by auto-induction. Strains defective in lacZ
(β-galactosida) or lacY (lacto permea) are not likely to be suitable for auto-induction by lacto becau they will be unable to import lacto or convert it to allolacto.
Lacto itlf is not a particularly good carbon and energy source to support continued target protein production after auto-induction, becau production of target protein competes so successfully for resources that proteins of the lacto operon may not accumulate to levels that provide efficient u of lacto. Glycerol is a good carbon and energy source that does not interfere with induction and allows growth to much higher culture densities. As with gluco, metabolism of glycerol generates a
cid, but very high densities of auto-induced cultures can be achieved with sufficient aeration and maintenance of a pH near neutral.
Growth conditions. Reasonably good aeration is important for maintaining pH near neutral and obtaining growth to high culture densities. We typically grow cultures in an incubator shaker at 20°C or 37°C and 300-350 rpm, using vesls and volumes of culture that give approximately equivalent levels of aeration. For auto-induction of many cultures in parallel to test expression and solubility, we grow 0.5 ml of culture in 13x100 mm glass culture tubes. Usually only a few microliters of such cultures is sufficient for all needed analys by gel electrophoresis. Up to 2.5 ml of culture in non-inducing media is grown in 18x150 mm glass culture tubes to make freezer stocks, plasmid preps or ed stocks for moderate-scale auto-induction, although 1.5 ml is probably preferable. Seed stocks for larger-scale auto-induction can be grown in Erlenmeyer flasks, the culture occupying approximately 5-10 % of the flask volume. Moderate-scale auto-induction can u 400-500 ml of culture in 1.8-liter baffled Fernbach flasks (Bellco).
Trace metals. Growth to high density in fully defined media requires the addition of trace metals. A concentration of 0.2x trace metals (recipe for 1000x stock solution on page 19) is sufficient but, if a mixture of trace metals is not available, 100 µM FeCl3 will increa saturation density about as well.
Growth in media containing ZY has not been limited by lack of trace metals but, given the variability of complex media components, it ems prudent to add 0.2x trace metals, if available. For target proteins of unknown metal content, 1x trace metals provides nine different metals in amounts sufficient to saturate substantial production of target protein, and 5x can be tolerated with little effect on growth. Individual metals can be supplied for production of target proteins known to bind specific metals. Trace metal mix at 1x concentration or FeCl3 at concentrations higher than about 10 µM tend to precipitate slowly in growth media. Such precipitation does not em to inhibit growth but might decrea availability to metal-binding target proteins. The prence of 1 mM citrate ems to reduce or prevent such precipitation.
Additives. Fully defined media must be appropriately supplemented for growth of strains with nutritional requirements: at least 150 µg/ml of methionine is needed to support growth of
B834(DE3) to saturation in MDG , and 1 µM thiamine (vitamin B1) is 10-fold more than needed to support high-density growth of XL1Blue-MR, an F-recA1 strain we u as an initial recipient for expression plasmids. I also found that growth of cultures in most batches of complex media such as LB, 2xYT or terrific broth is limited by lack of magnesium. Simply adding 2 mM MgSO4 typically incread saturation densities by 50% to 5-fold.
Kanamycin resistance. Although BL21(DE3) is unable to grow in LB containing as little as 10 µg of kanamycin per ml, it grows well even at ten-fold higher levels of kanamycin in the comparably rich auto-inducing medium ZYP-5052, which contains 100 mM phosphate. However, kanamycin at 100 µg/ml does prevent growth in media reformulated to contain only 50 mM phosphate (or less), such as the auto-inducing medium ZYM-5052 we u currently.
Freezer stocks for long-term storage of expression strains are made by adding 0.1 ml of 100% w/v (80% v/v) glycerol to 1 ml of culture that was grown to saturation in a non-inducing medium such as MDG or MDAG-135, mixing well and placing in a -70°C freezer. Reasonably well aerated cultures in MDG or MDAG at 37°C typically saturate at an OD600 around 7-10. Working stocks are grown from freezer stocks by scraping up a small amount of frozen culture with a sterile plastic pipettor tip and inoculating non-inducing medium. The stability, viability and reliability of protein expression from cultures grown in non-inducing media makes it possible to work with many strains in parallel. Re-transformation or streaking out cultures to obtain a "fresh" single colony each time a protein is to be produced, an unfortunate and tedious practice in some labs, is not necessary for reliable expression of target protein.
Auto-induction works well over the entire range of temperatures suitable for growth. Auto-inducing c
ultures are typically inoculated with one-thousandth volume of an MDG or MDAG ed culture and grown to saturation overnight at 37°C. In general, increasing the rate of aeration increas the density at which the culture auto-induces and saturates, and also increas the minimum concentration of lacto needed for good auto-induction. Routine auto-inducing media contain 0.2% lacto, a concentration chon to be well in excess of that needed for good auto-induction over the range of conditions we u.
Cultures grown at 20°C usually auto-induce and saturate at higher culture densities at than at 37°C (probably due to the higher solubility of oxygen at lower temperature). Higher saturation densities combined with slower growth at low temperature means that cultures may become quite den after overnight incubation but may not yet be induced, so care must be taken not to collect low-temperature cultures before they have saturated. The time needed for auto-induction at low temperatures can be shortened by incubating a few hours at 37°C, until cultures become lightly turbid (probably OD600 less than 1), and then transferring to the lower temperature.
Incubation for veral hours at saturation after auto-induction usually has little effect on accumulation or solubility of target protein. Auto-induced cultures typically saturate at an OD600 around 7-10 at 37°C under the conditions we u but can reach 20-30 in favorable cas. Culture d
ensities greater than OD600 ~50 have been attained by using higher concentrations of glycerol, metabolic balancing of pH with aspartate or succinate, and higher levels of aeration. Auto-inducing media should be capable of producing even higher densities in batch culture in fermenters, where high levels of aeration can be maintained for large culture volumes. When the target protein is sufficiently toxic to the host, expression of even small amounts as auto-induction begins may prevent much further increa in density, and such cultures may not achieve densities much higher than OD600 of 1-2. Becau the OD600 is due to light scattering rather than absorption, an accurate reading requires dilution of the culture (in water) to a concentration that gives a reading between about 0.030 and 0.200, typically a 100-fold dilution.
Parallel growth of many non-induced or auto-induced cultures is feasible becau cultures are simply inoculated and grown to saturation. This is a great convenience and simplifies manual or automated induction and analysis of multiple clones compared to conventional IPTG induction, which requires monitoring growth of each culture and adding inducer at the proper stage of growth. Others have been successful using the recipes in the multi-well plates commonly ud in automated systems, but it was important to keep volumes low enough that reasonably good mixing and aeration was obtained.
Stock solutions were designed for convenience and flexibility in asmbling different media, and to avoid combining components that are incompatible upon autoclaving. Media are usually asmbled from autoclaved or filter-sterilized stock solutions immediately before u, but most appear to be stable for extended periods in the refrigerator if contamination by mold spores is avoided. Stock solutions are stored at room temperature, except as noted in the recipes.
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Fully defined, non-inducing media we currently u routinely: pages 6-7
MDAG-11 is a fully defined, non-inducing medium for agar plates and stabs to lect, titer and distribute expression strains, and for liquid suspension of colonies to be retained temporarily as standing cultures at room temperature when purifying strains. MDG and MDAG-135 are fully defined, non-inducing media for growing shaking cultures of expression strains for freezer stocks, working stocks (which remain highly viable for weeks in the refrigerator), and for preparing plasmids from BL21(DE3) or other host strains susceptible to unintended induction.
Cultures that grow slowly becau they are stresd by basal expression of a highly toxic target protein should be grown in MDAG-135 rather than MDG. Such cultures are likely to contain a mixture of newly-divided growing cells that have not yet produced a transcript of the toxic gene and c
ells that are dead or dying becau they have produced a burst of toxic target protein, perhaps as little as that generated from a single transcript. The faster rate of division in MDAG-135 should increa the fraction live cells in the population and may allow the production of cultures where esntially all of the live cells are capable of producing target protein rather than consisting primarily of poorly expressing mutants that have overgrown the culture.
Auto-inducing media we currently u routinely: page 8
ZYM-5052 is a complex auto-inducing medium and MDA-5052 is a fully defined auto-inducing medium, both of which can support growth to relatively high densities and produce substantial amounts of a wide range of target proteins. Addition of arabino to ZYM-5052 or MDA-5052 at a final concentration of 0.05% produces media for auto-induction of target genes in BL21-AI (Invitrogen), where production of T7 RNA polymera is under control of the pBAD promoter and the target gene is expresd from the T7lac promoter.
Complex medium we currently u for rapid growth of high-density cultures: page 9
虚的成语ZYM-505 is a complex medium that produces den cultures of commonly ud lab strains, satisfies complex nutritional requirements, and is uful for rapid growth of high-density cultures for preparing
plasmids. The possibility of unintended induction may make ZYM-505 poorly suited for growing some expression strains.
Auto-inducing media for labeling target proteins with SeMet, 15N or 13C: pages 10-11
Low-phosphate (25 mM) non-inducing and auto-inducing minimal media: page 12
High-phosphate (100 mM) non-inducing and auto-inducing media ud earlier: page 13-14
Stock solutions: pages 15-20
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